DIAGNOSIS OF COCOA SWOLLEN SHOOT 
VIRUS DISEASE BY POLYMERASE CHAIN 

REACTION (PCR).

A THESIS SUBMITTED

BY

RITA NANA DARKOAH OSEI

TO THE DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE, 
UNIVERSITY OF GHANA IN PARTIAL FULFILMENT OF THE 

REQUIREMENTS FOR THE AWARD OF MASTER OF 
PHILOSOPHY (M. PHIL.) DEGREE

DECEMBER 2000



6 & R - C I 7

' M ' t x

C . |

£

f ' L,
3 7 0 3 8 2

j h & iS  t  r ; -  •



Declaration

I, RITA NANA DARKOAH OSEI HEREBY DECLARE THAT THE 

EXPERIMENTAL WORK DESCRIBED IN THIS PROJECT WAS CARRIED OUT 

BY ME EXCEPT FOR THE REFERENCES TO THE WORK OF OTHER 

RESEARCHERS WHICH HAVE BEEN DULY CITED.

RITA NANA DARKOAH OSEI 

(CANDIDATE)

(SUPERVISOR)

DR Y. D. OSEI 

(SUPERVISOR)

ii



Dedication

TO MY MUM 

MBS AFUA SAFOAA BUSIA

iii



Acknowledgements

My sincere thanks go to my supervisors Dr. Yaa Difie Osei and Dr. Sammy Tawiah 

Sackey for their guidance, advice and support throughout this project. I would also like 

to acknowledge with gratitude the assistance given to me by Professors Neil Olszweski 

and Ben Lockhart of the University of Minnesota.

My gratitude goes to the Ghana Cocoa Growers’ Research Association (GCGRA) and 

Biscuits, Chocolate and Confectionery Companies Alliance (BCCCA), who jointly 

funded this research through Cocoa Research Institute of Ghana (CRIG).

I am sincerely grateful to the authority of CRIG, especially Dr. G. KL Owusu (Director) 

for making it possible for me to cany out this research at CRIG. My appreciation goes 

to Doctors Ollennu, Opoku-Ameyaw, Boye Frimpong, Takrama, Ackonor and Mr. 

Prempeh for their support. I am also grateful to all the staff of the 

Physiology/Biochemistry and Pathology Divisions, especially Miss Evelyn Kwame, 

Messrs Francis Osae-Awuku, Ben Owusu-Ant wi, Fiifi Adu-Amankwa and Sammy 

Affram for their tremendous help.

I would also like to acknowledge all the lecturers, students and staff of the Biochemistry 

Department, University of Ghana especially, Prof. Gyang and my mates John, Kwamina, 

and Nii Kpani. I am grateful to Lynn and all working in Lab 659 (Department of Plant 

Biology, University of Minnesota) for their support. I am also grateful to Doctors M  

Wilson, K. M  Bosompem and G Armah, Mr. C. Brown, Mr. Ayim, Michael, Harry and

iv



Anita, and all staff of Noguchi Memorial Institute for Medical Research (NMIMR), 

Accra, especially members of Parasitolgy Unit Room 133, for their various contributions 

to this work.

To all my friends, especially Lydia and Kwame (Minnesota), Francis, Kwabena, Ato, 

Kojo, Peace, Christabel and Evelyn, I am very grateful for your friendship and support.

My utmost gratitude goes to my Mum, my sisters Mercy and Joyce, my brothers Ernest, 

Charles, Ishmael, Nat and Robert, my special cousin Afua, my nephews Paa Kwesi, 

Papa, Fiifi, Paa Kow and my niece Menee for their immense love and support.

I will finally thank the Lord God Almighty for His abundant love and grace, and for 

making things possible for me.

v



Abstract

Cocoa swollen shoot virus disease causes severe damage to cocoa farms leading to 

substantial losses in crop yield and therefore the country’s revenue from cocoa. This 

study set out to design new PCR primers for the detection of the virus that causes the 

disease.

Thirty-six cocoa swollen shoot virus (CSSV) isolates, randomly selected from 5 main 

groups based on serological and biological properties from the CRIG museum at Tafo 

were used Viral DNA extracted and purified from the infected leaves were used for 

PCR using two sets of universal badna primers 2+T and 3+T. The multiple 

amplification bands produced were cut out, gel purified and hybridised against full 

length cloned PCR DNA of CSSV New Juaben probes to detect the amplification 

products of virus origin. These were then cloned, sequenced and new primers designed 

based on consensus sequences derived from the alignment of the CSSV sequences with 

sequences from other closely related badna viruses.

The new pair of primers (badna primers 1+4) gave a single PCR amplification product 

of 600 base pairs. The thirty-six CSSV isolates from the CRIG museum were screened 

with the new primers to test the efficacy of these new primers. Out of the 36 isolates 

screened, 28 gave the expected amplification product and 8 did not give any 

amplification products. The new primers in comparison with the old primers can be said 

to be better at detecting CSSV.



Table of Contents

DECLARATION 11
DEDICATION 111
ACKNOWLEDGEMENTS IV
ABSTRACT VI
TABLE OF CONTENTS V,i
LIST OF FIGURES x
LIST OF TABLES X1

CHAPTER ONE 1
INTRODUCTION 1
1.0 Introduction 1

CHAPTER TWO 5
2.0 Literature Review 5
2.1 Cocoa swollen shoot virus diseasse 5
2.2 Symptoms 5
2.3 Economic impact and control of CSSVD 5
2.4 The virus morphology and genetics 6
2.5 Virus extraction and purification 9
2.6 DNA extraction and purification 10
2.7 Polymerase chain reaction 11

2.7.1 Uses of PCR 15
2.8 DNA Hybridisation analysis 16
2.9 Cloning of PCR amplification products 17

2.9.1 Ligation 17
2.9.2 Transformation, cell growth and screening of recombinants 18
2.9.3 Plasmid harvesting 18
2.9.4 Caesium chloride (CsCl) -  ethidium bromide purification 19

2.10 DNA sequencing 19
2.10.1 Sanger’s method 20

v i i



2.11 Design of PCR primers 22

CHAPTER THREE 23
3.0 MATERIALS AND METHODS 23
3.1 Materials 23

3.1.1 Chemical and Reagents 23
3.2 Methods 24

3.2.1 Virus isolates 24
3.2.2 Extraction and purification of virus 28
3.2.2.1 Extraction of virus 28
3 2.2.2 Sucrose cushion centrifugation 29
3.2.3 Extraction and purification of virus DNA 29
3.2.4 DNA purification for PCR 30
3.2.4.1 QIAGEN purification 30
32.4.2 2-butoxyethanol purification 31
3.2.5 Agarose gel electrophoresis 32
3.2.6 Polymerase chain reaction (PCR) 32
3.2.7 Gel-purification of PCR products and clones 35
3.2.8. Hybridisation analysis 35
3.2.8.1 Southern DNA transfer 35
3.2.8.2 Dot blots 36
3.2.8.3 Synthesis of DNA probes 36
3.2.8 4 Hybridisation reaction 37
3.2.8.5 Washing and enzyme reaction 37
3.2.8.6 Removal of colour and probe 38
3.2.9 Cloning 39
3.2.9.1 Ligation reaction 39
3.2.9.2 Preparation of competent cells 40
3.2.93 Transformation 41
3.2.9.4 Cell culture and screening 41
3.2.10 Isolation of plasmids 42
3.2.11 Restriction digests 42

v i i i



3.2.12 Large scale plasmid isolation

3.2.13 Caesium chloride purification
3.2.14 DNA sequencing

3.2.14.1 Sequencing gel

3.2.14.2 Primer radio-labelling

3.2.14.3 Sequencing gel electrophoresis
3.2.15 Primer design
3.2.16 PCR annealing temperature optimisation and primer

testing
CHAPTER FOUR
4.0 RESULTS
4.1 Selection and grouping of isolates
4.2 Extraction and purification of virus and virus DNA
4.3 Polymerase chain reaction analysis
4.4 Gel purification of PCR amplification products
4.5 Hybridisation analysis of PCR products
4.6 Cloning of PCR DNA
4.7 Primer design
4.8 Results of annealing temperature optimisation
4.9 Testing of primers 1+4
4.10 Dot blot hybridisation analysis of viral DNA with 1+4

PCR DNA probes
4.11 CSSV sequences from clones from new primers 1+4
CHAPTER FIVE

5.0 DISCUSSION AND CONCLUSION
REFERENCES

43
43
44
44
44
45
46
46

47
47
47
47
48
51
51
54
57
59
59
65

68

70
70
76

ix



List of Figures

Figure 1 Schematic diagram of the PCR process 12
Figure 2 Map of Ghana showing the regional distribution of the 27

selected isolates
Figure 3 The CSSV Genome 34
Figure 4 PCR amplification products with primers 2+T and 3+T 49
Figure 5 Gel-purified PCR amplification bands using primers 52

badna 3+T and 2+T
Figure 6 Hybridised membrane of gel-purified PCR products 53
Figure 7 Screening of PCR clones by restriction digestion of 56

plasmids
Figure 8 Alignment of conserved region of CSSV sequence with 58

that of the other badna viruses 
Figure 9 Optimisation of PCR annealing temperature for primers 60

1+4 using CSSV 1A DNA 
Figure 10 PCR with DNA from isolates from CRIG museum 61

using primers 1+4
Figure 11 CSSV sequences from new primers 69

x



Table 1 Selection of isolates 25

Table 2: Analysis of CSSV CRIG museum isolates by PCR using 50

universal badna primers 2+T and 3+T.

Table 3 Summary of results of experiment to identify virus-coded 55

PCR amplification products.

Table 4 Analysis of CSSV CRIG museum isolates by PCR using new 63

badna primers 1+4.

Table 5 Dot blot hybridisation analysis of virus DNA from CSSV 66

isolates with probes using 1+4 PCR products

List of Tables

xi



CHAPTER ONE

1.0 INTRODUCTION

Cocoa production in Ghana has over the years faced many challenges, but perhaps the 

most enduring has been cocoa swollen shoot disease. Initially, this was thought to be an 

agronomic problem, as the disease manifested itself by severe systemic leaf mosaic 

symptoms. Its name, swollen shoot disease, refers to the swelling of the stem during 

cancerous proliferation of phloem cells, which process contributes to the apical death of 

the plant. Swollen shoot disease was first reported in the Eastern region of the then 

Gold Coast in 1936 (Dale, 1962).

Swollen shoot disease control strategies were based on a zero tolerance philosophy and 

involved cutting down of infected trees and those in contact with them. Alternative 

control measures such as the use of barrier crops to restrict the spread of the disease, use 

of resistant/tolerant cocoa varieties, and cross-protection using serologically closely 

related mild isolates of the Cocoa Swollen Shoot Virus (CSSV) are still being 

investigated (Hughes et al., 1995). Also, breeding programmes are pursued to develop 

resistant breeds, and cultural methods are used to control cocoa swollen shoot disease. 

For example, the Cocoa Services Division (CSD) in Ghana has a sustained program to 

advise fanners on methods of limiting the spread of the disease before new farms are 

cultivated. However, cutting down of infected trees remains the only effective method 

of controlling the disease in Ghana.

Studies into cocoa swollen shoot disease at the Cocoa Research Institute of Ghana have 

focused on the biochemical, biophysical, genomic and biological characterization of the 

disease causal agent. Methods have thus been developed for the extraction and

l



purification of the virus isolates and these in turn have facilitated the development of a 

host of diagnostic tools for their detection and identification in plant tissue extracts.

More recently, polymerase chain reaction (PCR) and immunocapture PCR (ICPCR) 

have been used for the detection of CSSV using three sets of primers (Sackey, 1995; 

Sackey et a l, 1995; Hoffman et al., 1997). The first set of primers, based on nucleotide 

sequences derived from the severe CSSV 1 A, gave single DNA products, but these were 

specific for CSSV 1A and closely related isolates and therefore could not detect many 

isolates that fell outside the 1A group (Sackey and Hull, 1991). The other two pairs of 

primers were designed by Lockhart and Olszewski (1993) based on the nucleotide 

sequence of three conserved bacilliform DNA virus (badna virus) genomic regions, the 

tRNAmct binding site, reverse transcriptase and ribonuclease H genes. The nucleotide 

sequences used were those from the badnaviruses (commelina yellow mottle virus 

(CoYMV), sugar cane bacilliform virus (SCBV), Kalanchoe top spotting virus (KTSV) 

and rice tungro bacilliform virus (RTBV). These, however, were degenerate and most 

CSSV isolates gave many amplification products, most of which were found to be 

artefacts (Sackey etal., 1995).

Breeding programs in producing countries rely on new introductions from the South 

American origin of the Theobroma species, as well as exchanges of improved materials 

from member countries of the Cocoa Producers’ Alliance (CPA). These programs 

therefore rely on the establishment of quarantine facilities in non-cocoa producing 

countries in North America and Europe, where material from different parts of the 

cocoa producing world are assembled and multiplied for distribution. This requires that 

material received is screened for the major pests and diseases including the debilitating

2



witches broom disease of South America and cocoa swollen shoot disease of West 

Africa. Ghana as a major cocoa producing country contributes towards this 

international effort at conservation of cocoa germplasm. As one of the sources of the 

disease, Ghana also contributes towards sustaining the program by providing access to 

diagnostic tools developed for the detection of the various isolates in the country. Even 

though the tools currently available are adequate for Ghana’s needs, they do not entirely 

address the requirements of the international quarantine programmes which have to 

screen for isolates from all of the West African sub-region. This initiative therefore 

aims at developing a new universal diagnostic system for the detection and 

identification of the badnaviruses (Bacilliform DNA viruses) responsible for the disease 

in cocoa.

The project was therefore carried out to identify and design new universal PCR primers 

for the detection of CSSV. The existing primers designed by Lockhart and Olszewski 

(1993) and previously described do not include sequences from cocoa swollen shoot 

virus but those from other key badnaviruses, namely BSV, CoYMV, ScBV, KTSV and 

RTBV. The first three are known in Ghana, as well as Discorea alata bacilliform virus 

(DaBV). Therefore, it was proposed to retain the contribution of BSV, CoYMV, ScBV 

particularly as these bananas, plantains, and sugarcane are often found in cocoa farms, 

while commelina is common as a weed. The nucleotide sequences of CSSV and DaBV 

were to be introduced into the new set of primers.

3



The general objectives of the study were to:

a. develop nucleotide sequences covering a defined segment of several CSSV 

isolates

b. use these sequences to design oligonucleotide primers for the rapid 

detection of bacilliform DNA viruses, and

c. test the specificity and sensitivity of the new primers.

Specific objectives

The specific objectives were:

i. collection of CSSV infected leaves from the Cocoa Research Institute of

Ghana (CRIG) museum

ii. extraction and purification of virus isolates from infected leaves

iii. extraction and purification of virus DNA

iv. amplification of DNA by PCR using the universal badna primers 2+T and

3+T.

v. identification of virus coded PCR amplification products by hybridisation of 

Southern blots

vi. cloning of the virus coded DNA species

vii. sequencing of cloned PCR DNA to develop nucleotide sequence database 

using representatives of virus groups determined by serological methods and 

other badna viruses closely related to the CSSV

viii. design of new primers based on nucleotide sequence database

ix. verification of the efficacy of the new primers.

4



CHAPTER TWO

2 .0  LITERATURE R EVIEW

2.1 Cocoa swollen shoot virus disease

Cocoa Swollen Shoot Virus Disease (CSSVD) was first reported in Ghana in 1936 

(Dale, 1962) and in 1938, Posnette (1940) suggested that the disease was caused by 

several isolates of cocoa swollen shoot virus (CSSV) and transmitted by several species 

of mealy bugs in a semi- persistent manner.

2.2 Host symptoms

CSSVD is characterised by root and stem swellings, mosaic leaf chlorosis, red vein- 

banding of flush leaves, rounding of pods containing fewer, smaller beans in some 

infected trees and in some severe cases, premature death of the plant (Posnette, 1947). 

Various isolates show a range of combinations of these symptoms. Healthy cocoa trees 

that become infected may not show any symptoms for a considerable time. This latent 

period varies according to the type of strain of the virus, the age and condition of the 

tree. The virulent strains may produce severe symptoms within five months on 

sensitive Amelanado, but the mild strains may not express themselves for two years or 

more (Wood and Lass, 1985).

2.3 Economic impact and control of CSSVD

Swollen shoot disease causes severe damage to cocoa farms and therefore substantial 

losses in crop yield and revenue. The strategies for disease control were based on a zero 

tolerance philosophy and involved cutting down of infected trees and those in contact 

with them. At the beginning of the cutting out campaign in 1956, over a million 

affected and contact trees were removed. By 1982, it was estimated that 185.5 million

5



trees had already been removed in the Eastern Region alone and there were still 31.2 

million trees to be removed (Owusu and Thresh, 1983). According to Owusu and 

Thresh (1983), only 23% of all infected trees in a new outbreak were identified because 

many of the infected trees were not noticed or were in the latent phase of infection. It 

was also realised that new infections were occurring faster than the removal of the 

infected trees (Legg, 1982). However, cutting down of infected trees still remains the 

only effective method of controlling the disease in Ghana. Alternative control measures 

such as the use of barrier crops to restrict the spread of the disease; breeding for 

resistant/tolerant cocoa varieties and cross-protection using serologically closely related 

mild isolates are still being investigated (Hughes et al., 1995). There were also cultural 

methods where the Cocoa Services Division (CSD), Ghana, advised farmers before new 

farms were cultivated.

2.4 Virus morphology and genetics

Electron microscopy data have shown that cocoa swollen shoot virus is a bullet shaped 

particle of about 142x 27mn in size (Agrios, 1997). Lot et al. (1991) showed that 

CSSV contained a double stranded DNA genome of molecular weight 7100 base pairs. 

It was proposed as a member of the Bacilliform DNA (Badna) virus (Lockhart, 1990) 

on account of its unenveloped bacilliform particles (Brunt et al., 1964) and circular 

double stranded DNA genome (Lot et al., 1991). Hagen et al. (1993) also provided the 

complete nucleotide sequence of the CSSV Agou 1 strain from the Republic of Togo, 

which partially defined the badnavirus genome organisation.

Classification of the physico-chemical properties of CSSV has been hampered by a 

number of factors. These include the absence of a local lesion host for the biological

6



purification of the components of what may be mixed infections (Paine, 1945; Posnette, 

1947; Posnette and Todd, 1955), and efficient methods of purifying the virus. Other 

difficulties were due to the feet that some of the isolates induced only stem swellings or 

transient leaf symptoms, and did not provide reliable sources of infected leaf material 

for virus purification and analysis. Consequently in Ghana, classification of viruses has 

been based on criteria like the location from which they were first isolated (Posnette, 

1947) host range (Tinsley and Wharton, 1958; Legg and Bonney, 1967) and 

transmission vector (Roivainen, 1976). In some cases, classification was based on the 

indigenous or alternate host plants like Adcmsonia digitata (AD) and Cola 

chlamydantha (CC) from which they were isolated (Attafuah and Tinsley, 1958; Legg 

and Bonney, 1967).

Lesemann et al. (1980), used immunosorbent electron microscopy (ISEM) to detect the 

virus particles. This method, however, required expensive inputs. With the 

development of more effective methods for the purification of the CSSV from infected 

cocoa leaves by Adomako el al. (1983), monoclonal and polyclonal antisera against 

severe 1A were produced. These have been used for the detection of the virus in 

infected tissue extracts and to establish relationship between isolates.

Adomako et al. (1983) described the use of ISEM for the rapid detection of the CSSV in 

virus extract. Sagemann et al. (1983), used enzyme-linked immunosorbent assay 

(ELISA) and in 1985 used ELISA and immunoelectron microscopy (IEM) for the 

detection of the CSSV, where the serological activity of the virus was evaluated. 

Sackey et al. (1990), also raised polyclonal antibodies which differentiated between 

severe isolates, 1A and Nsaba. These methods were good for the determination of the

7



serological relationships between CSSV isolates in the laboratory. They could also be 

used in the field only on trees with the symptoms. The disadvantages here were that, 

symptomless or transient infections could not be detected and the assay produced high 

background values when the antisera had high antibody content.

In the virobacterial agglutination (VBA) method used by Hughes and Ollennu (1993), 

the test successfully detected CSSV in infected sap diluted 1/2560. The test was based 

on the affinity of protein A to the iron (Fe) region of IgG. The advantages here were 

that, the method was simple with rapid results and the IgG required was easy to prepare 

in the laboratory. However, the method was useful only in the detection of CSSV in 

freshly harvested leaf samples.

Further developments in the diagnosis of CSSV include DNA hybridization and 

amplification methods. Polymerase chain reaction (PCR) was first used for CSSV 

diagnosis by Sackey et al. (1995) and immunocapture PCR (ICPCR) by Hoffman et al. 

(1997). Two different sets of primers were used in these studies.

Sagemann et al. (1985) differentiated the CSSV isolates into five groups, (A-E) on the 

basis of leaf, stem and root symptoms, as well as serological properties by enzyme 

linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM). 

When these groups were compared with those based on area of first isolation and 

symptoms expressed, it was found that geographical location had no apparent 

relationship with the serologically characterised groups Sagemann et al. (1985).



2.5 Virus Extraction and Purification

The extraction and concentration of CSSV have been made difficult by the presence of 

large amounts of tannins and mucilage in the cocoa leaves. Purification is however 

achieved by the addition of pectinase, polyethylene glycol (PEG), and standard sodium 

citrate (SSC), alternating high speed centrifugation and low speed centrifugation to 

remove cellular debris and filtration through celite and sepharose. The high speed 

centrifugation is carried out to pellet the virus while the low speed centrifugation is 

carried out to clarify the virus extract (Lot et al., 1991).

More effective extraction, concentration and purification of CSSV from the infected 

leaves to give higher yields, was facilitated by the addition of the enzyme pectinase to 

the extraction buffer to degrade mucilage in the cocoa leaf extracts (Adomako et al., 

1974). The mucilage and particulate host plant materials were effectively removed by 

the pectinase and the release of the virus particles from the leaves was enhanced. 

Polyethylene glycol (PEG 6000) in the extraction buffer enhanced precipitation of the 

virus particles. Once freed from the mucilage and plant materials, filtration and 

concentration were possible without losing much of the virus.

Further purification of the extracts was necessary to get rid of more of the host plant and 

other unwanted materials. Filtration of crude preparations through Celite 545 

(Adomako, 1974) was also found to be very beneficial but the yield of virus was 

compromised by the filtration step.

9



2.6 DNA Extraction and Purification

The quality of the virus DNA extracted is very crucial for PCR and depends on the 

source material (tissue), presence or absence of contaminants, method of extraction and 

purification and the precautions taken to maintain the integrity of the DNA. The plant 

tissues are known to contain large quantities of proteins, cell wall materials, and 

phenolic compounds which tend to contaminate the virus extract. The extraction buffer 

contains ethylene diamine tetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), 

Tris-HCl and MgCfe. The EDTA inhibits the activities of naturally occurring DNase, 

which will degrade the virus DNA once it is released from its protective walls. The 

SDS is an ionic detergent that dissolves away lipid membranes to free the DNA (Roe et 

al., 1996). Proteinase K is also added to break down proteins.

Extraction with either Tris-EDTA (TE) saturated phenol and phenol: chloroform 

solution removes the SDS, proteins and other related substances from the nucleic acid- 

buffer mixture (Newton and Graham, 1997). Addition of sodium acetate and ethanol or 

NaCl and isopropanol to the aqueous upper phase precipitate the virus DNA.

To further purify the virus DNA, the enzyme RNase may be used to digest 

contaminating RNA. Other purification methods like the use of 2- butoxyethanol can 

be used to remove more polysaccharides and phenolic compounds. There are also spin 

column chromatography methods that can be used to clean up the DNA sample and to 

get rid of very small single stranded and double stranded molecules.

10



2.7 Polymerase Chain Reaction

The polymerase chain reaction (PCR) is an in-vitro process used for amplifying specific 

regions of the genome of DNA by a factor of 106 (Mullis et al., 1986; Mullis and 

Faloona, 1987). The amplification is achieved in the presence of suitable primers, 

deoxynucleoside tri-phosphates (dNTPs), magnesium chloride (MgCl2), the DNA 

template to be amplified, the enzyme Taq DNA Polymerase and the enzyme buffer.

The double stranded template DNA is first heat denatured to obtain single strands. The 

primer anneals to the strands and the Taq polymerase uses the dNTPs to extend the 

strand thus, producing another complementary strand (figure 1).

PCR involves the repetitive cycles of heat denaturation of the double stranded DNA to 

be amplified at a high temperature (92-95°C), annealing of primers to template 

(complementary) DNA strands at a lower temperature (37-55°C) and extension of the 

new strands at 72°C. According to Saiki et al. (1988), the temperature chosen for 

annealing of the primer is a compromise. This is because at lower temperatures (37°C), 

annealing is more efficient but there is a significantly increased amount of mis-priming. 

At higher temperatures (55°C) however, there is increased amplification specificity but 

the overall efficiency is decreased.

11



Target Region ■

Unamplified DlslA %

Denature and 
anneal primers

Extend primers

Denature and 
anneal primers

Extend primers

Denature and 
anneal primers

Extend primers

H
yc

|  Cycles 4-30

-3'

*3'

=Shgrt "target’ product

|  =Long product

Amplification of short ’target' product

Figure 1. Schematic diagram of the PCR process

12



Normally, a series of reactions are set up to determine the optimal annealing 

temperature. Extension of the annealed primers by the Taq DNA polymerase uses the 

dNTPs at an increased temperature (72°C) for 3 minutes. The cycle then restarts. The 

result is an exponential increase of the DNA with the generation of over a million 

copies of the DNA of interest in several hours.

The PCR process is directed by sequence-specific oligonucleotide primers of between 

20-24 nucleotides in length. The primers are designed with the knowledge of the 

sequence to be amplified. The primers thus locate and anneal to complementary 

sequence on the template DNA. The annealing step is an important parameter in 

optimising the specificity of a PCR.

The starting material, the template DNA may be single stranded or double stranded 

DNA. The DNA to be amplified should be free of contaminants or non-target materials 

to avoid their amplification rather than the target DNA. Normally, sub-nanogram 

quantities of the template DNA are used for PCR.

Taq DNA Polymerase, the enzyme used in PCR is a thermostable polymerase isolated 

from the thermophilic bacterium Thermus aquaticus (Taq). Earlier reports on PCR by 

Saiki et al. (1985) and Mullis et al. (1987) involved the use of Klenow fragment of E. 

coli polymerase I. This enzyme is heat labile, therefore, fresh enzyme had to be added 

at the beginning of each cycle of the PCR. The results were the generation of a 

heterogeneous set of products in addition to the DNA of interest. This is due to the non­

specific binding of primers and non-processivity of the enzyme under those conditions. 

The discovery of Taq DNA polymerase therefore solved these problems because the

13



enzyme was added only once at the beginning of the reaction (Erlich, 1989). Apart 

from Taq DNA polymerase, its cloned and modified version, Tth DNA polymerase 

from Themtus thermophihis, Pfu DNA polymerase from Pyrococcus furiosus and a few 

others can also be used for PCR (Newton and Graham, 1997).

Taq DNA polymerase has an optimal extension rate (polymerisation rate) of 35-100 

nucleotides per second at between 70-80°C (Newton and Graham, 1997). Synthesis of 

DNA at higher temperatures was advantageous because non-specific binding was 

greatly reduced (Horn, 1988).

The dNTPs are the building blocks of the DNA. They are made up of the four bases of 

the DNA (purine, pyrimidine, thymine and adenine) that are used by the Taq DNA 

polymerase to synthesize the new DNA strand. High purity dNTPs are used, normally 

at concentrations between 200-400|iM. However, Taq DNA polymerase has a higher 

fidelity even at lower dNTP concentrations (10-100(j.M). An optimal concentration here 

depends on the concentrations of MgCl2 and the primer, the number of PCR cycles, the 

length of the amplified product and the stringency of the reaction (Newton and Graham, 

1997).

The presence of divalent cations is critical in PCR. According to Chein et al. (1976), 

Mg2+ ions form a soluble complex with dNTPs, which is essential for dNTP 

incorporation. They also stimulate the polymerase activity and increase the Tm of the 

double stranded DNA and primer/template interaction. The optimal concentration of 

magnesium ions is quite low (1.0-1.5mM) thus, the template DNA should not contain 

high concentrations of chelating agents like ethylene diamine tetraacetic acid (EDTA).

14



Any change in the volumes of any of the components of PCR will affect the 

concentration of the magnesium ions available. A change in the concentration of the 

MgCk can have a dramatic effect on the specificity and yield of the PCR products. 

Generally, the insufficiency of the ions will lead to low yields whiles excess will result 

in the accumulation of non-specific products (Newton and Graham, 1997).

There are several buffers available for PCR. Most common buffers used with Taq DNA 

polymerase and its modified versions come in a lOx concentration and contain lOOmM 

Tris-HCl, pH 8.3 at room temperature; 500mM KC1; 15mM MgClz and 0.1% (w/v) 

gelatin.

2.7.1 Uses of PCR

PCR has been used in the diagnosis of genetic disorders (SaiM et al., 1985; Saiki et al.,

1988), the analysis of allelic sequence variation (Sambrook et al., 1989), and analysis of 

mutation or any research that involves the rapid cloning and sequencing of homologous 

DNA fragments (Howe and Ward, 1989). The PCR has also been used for the analysis 

of individual identity in forensic samples by the amplification of highly polymorphic 

DNA regions (Paabo et al., 1988; Higuchi et al., 1988), and the examination of 

nucleotide sequences from ancient preserved specimens (Paabo, 1990). Recently, PCR 

has been applied to difficult problems in developmental biology. For example, PCR of 

cDNA has been used to study V-J region combinations in the T-cell receptor a-chain 

(Loh et al., 1989) and the examination of the mRNAs for growth factors in small 

numbers of macrophages isolated from wounds actively undergoing healing (Rappolee 

etal., 1988).

15



2.8 DNA Hybridisation analysis.

Hybridisation involves the use of a radioactively- or fluorescently-labelled probe made 

up of a piece of single stranded DNA to detect and bind to its complementary base pairs 

to form duplexes on a solid phase. A cloned virus DNA probe was used to identify 

virus coded PCR products. The DNA species to be hybridised are first separated 

according to size by agarose gel electrophoresis and then transferred onto a solid 

support/phase (normally a nitrocellulose filter or nylon membrane) through capillary 

action by the technique known as Southern transfer (Southern, 1975). Before the 

transfer, the DNA is denatured in situ to separate the duplex strand by causing breakage 

of the hydrogen bonds linking the complementary base pairs (Sambrook et al., 1989). 

The separation is achieved by either heating to a temperature (Tm) between 60-80°C or 

by soaking the gel in high salt concentrations. The nucleic acid species are neutralized 

in situ to prevent the separated strands from re-annealing.

The relative positions of the nucleic adds fragments are maintained during the transfer 

(Sambrook et al., 1989). The DNA fragments are then immobilized onto the membrane 

by baking in a vacuum oven or cross-iinking in an Ultra Violet (UV) cross-linker before 

the hybridisation can take place (Khandjian, 1986).

After hybridisation, the membrane is washed. A radioactively-labelled probe bound to 

its complementary sequence can be detected by autoradiography. The non-radioactively 

labelled probe can be detected by enzyme-linked immunoassay using antibody 

conjugate. A subsequent colour reaction is initiated at basic pH by the addition of a 

phosphate and a salt. A blue colour precipitate starts to form from within a few minutes 

up to three days. After the reaction, the membrane is washed in water and the

16



hybridised fragments can be seen visibly as blue bands. For re-use, the colour of the 

membrane can be removed with dimethyl formamide (DMF) and the probe removed 

with N ad  and SDS.

2.9 Cloning of PCR amplification products

Cloning of PCR products allows generation of relatively large amounts of the amplified 

region to be available whenever needed without having to repeat the reaction. PCR 

products are used in various other studies, such as for sequencing and hybridisation, 

thus the need to clone the amplicon (Newton and Graham, 1997).

The cloning process involves the use of ligase to ligate the amplified DNA into a 

suitable enzyme digested vector to form a recombinant molecule. This is followed by 

transformation of the recombinant molecule into a suitable bacterial cell line, growth 

and screening of colonies by isolation of the plasmid DNA, enzyme digest and 

hybridisation with cloned virus DNA probe.

2.9.1 Ligation

Ligation reaction involves the use of DNA ligase to introduce the amplified DNA 

template into the genome of a plasmid vector to form the recombinant molecule. The 

DNA ligase catalyses the reaction, where there is a formation of a 3’, 5’ phosphodiester 

bond that links two adjacent nucleotides of the amplified DNA and the plasmid vector.

There is a wide range of plasmid vectors commercially available. They can be acquired 

already digested or they can be developed in the laboratory for cloning. There are the 

pUC series, the pGEM series, the pBluescript (Mezei and Storts, 1994) series and a lot

17



more For PCR cloning, dT overhang vectors can be used due to the dA-overhang in 

the PCR products. There are also commercially available blunt-ended cloning vectors 

like pCR-Script™ from Stratagene. To use the blunt-ended vectors for PCR the 

products will have to be polished to remove the dA- overhang.

2.9.2 Transformation, cell growth and screening for recombinants.

Transformation involves the introduction of the recombinant DNA molecule into 

suitable competent bacteria cells for replication and multiplication. This can be 

achieved either by electroporation method where the competent cells are prepared and 

stored in glycerol. There is also the heat shock method where the cells are stored in 

calcium chloride. Here, the transformation mixture is given conditions of consecutive 

cold, hot and cold to enhance the uptake of the recombinant DNA molecule into the 

bacteria cells.

The transformed cells are grown on agar plates containing the appropriate antibiotic, 5- 

bromo-4- chloro-3- indolyl-|3- D- galactoside (X-gal) and isopropylthio-P-D- 

galactoside (IPTG). IPTG induces the amino-terminal fragment of P- galactosidase and 

is capable of intra-allelic complementation with a defective form of p- galactosidase 

encoded by the bacterial cells. Bacteria exposed to IPTG thus form blue colonies in the 

presence of X-gal (Horwitz et al., 1964).

2.9.3 Plasmid harvesting

Plasmids are isolated from single bacterial cell cultures. The plasmids can be harvested 

either by alkaline lysis, a modification of two methods (Bimboim and Doly, 1979; Ish- 

Horowicz and Burke, 1981) or by the heat lysis method adopted from Holmes and

18



Quigley (1981). In both cases, the cells are exposed to detergents and then lysed by 

boiling or treatment with alkali to release the recombinant molecule. The boiling 

method is however not normally recommended because endonuclease A is not 

completely inactivated and the bacteria release relatively large amounts of carbohydrate 

in this method (Sambrook et al., 1989).

2.9.4 Caesium chloride (CsCl) - ethidium bromide purification

Equilibrium centrifugation in CsCl - ethidium bromide gradients of the plasmids has for 

many years been the method for the purification of plasmids, especially for large-scale 

preparations. According to Cantor and Schimmel (1980), the ethidium bromide causes 

the double helix of the DNA to unwind and bind to the dye at the rate of 1 ethidium 

bromide molecule to 2 base pairs. This differential binding of dye gives different 

buoyant densities to the linear and closed circular DNA molecules in CsCl- ethidium 

bromide gradients.

2.10 DNA Sequencing

DNA sequencing enables the determination of the base sequence in a nucleic acid. The 

object of DNA sequencing in this project was to develop a sequence database from 

which a primer could be designed. There are two main methods, the chemical reactions 

of Maxam and Gilbert (1977) and the enzymatic reactions of Sanger et al. (1977). Both 

methods produce a series of DNA molecules differing in length by a single nucleotide. 

These molecules can then be separated by gel electrophoresis (Maxam and Gilbert 

1977; Sanger et al., 1977).

19



Sequencing can be done using both single and double stranded DNA as well as cloned 

DNA. DNA species subjected to PCR can also be used. There are however, 

considerable advantages in sequencing double stranded cloned DNA than in single 

stranded and uncloned DNA (Howe and Ward, 1989). In the earlier years of the 

introduction of sequencing, single stranded and or circular DNA were mostly used 

(Gronenbom and Messing, 1978; Sanger et al., 1980; Messing et al., 1981). However, 

sequencing of plasmid DNA has become a powerful technique being used increasingly 

as the methods were improved. This is because very long inserts have been found to be 

unstable in Ml 3 phages but stable in plasmid vectors. Short inserts were sequenced 

completely and easily (Hong, 1981). Double stranded DNA has also been found to be 

amenable to directed deletion sequencing methods, which allowed easy and accurate 

screening for size and deletions.

2.10.1 Sanger’s method

Sanger’s method, also known as the chain termination method, was evolved from the 

positive/negative (+/-) sequencing technique by Sanger and Coulson in 1975. This 

technique first described the use of a specific primer for extension by DNA polymerase 

base specific chain termination and the use of polyacrylamide gel electrophoresis 

(PAGE) to separate single stranded DNA differing by a single nucleotide. The 

technique was however inaccurate and clumsy hence, the introduction of the chain 

terminating dideoxynucleoside triphosphates (ddNTPs) method (Sanger et a l, 1977).

The ddNTPs involved in this method lack their 3’-hydroxyl group on the deoxyribose 

and thus prevent the formation of phosphodiester bond with the subsequent dNTP.

2 0



These ddNTPs, which are randomly selected, when present in the reaction mixture get 

incorporated into the growing chain and terminate the chain.

The reaction products are a mixture of oligonucleotide chains and their lengths are 

determined by the distance between the terminus of the primer used and the point of 

premature termination (Sambrook et a l 1989). Oligonucleotide populations are 

generated from small amounts of four different ddNTPs (ddATP, ddCTP, ddGTP and 

ddTTP) in four separate enzymatic reactions. The sequence reaction mixture includes 

the template DNA, labelled primer, the ddNTPs, dNTPs, sequencing buffer, DNA 

polymerase and sterile distilled water. The primer anneals to its complementary 

sequence on the denatured template DNA strands. The DNA polymerase then extends 

the chain from the 3’ end of the primer using dNTPs as precursors (Bankier and Barrell, 

1983).

Universal primers are normally used, where they anneal to vector sequences that flank 

the target DNA The primer is first either radio-labelled or fluorescently labelled. The 

radio-labelling can be done using either [y or a-32P] dATPs as well as [35S] dATPs 

(Biggin et al., 1983) with the enzyme polynucleotide kinase. The (3-particles emitted by 

the 32P were found to cause problems including the fact that the bands on the 

autoradiograph were far larger and more diffused than the bands in the DNA gel. Also, 

the decay of 32P caused hydrolysis of the DNA. These make reading of the sequences, 

especially from the top part of the autoradiograph particularly difficult resulting in very 

few nucleotides being read (Sambrook et al., 1989). The 35S dATP is safer to use 

hence, preferred.

21



Two types of DNA templates could be used in Sanger’s method, either single or 

denatured double-stranded DNA. The quality of the template DNA and the type of 

DNA polymerase used are very critical for sequencing. Plasmid minipreparations are 

found to be always contaminated, thus, they are not recommended for sequencing. 

DNA purified by CsCl gradient is recommended for sequencing.

Several different enzymes can be used for the chain termination method of sequencing. 

These include the Klenow fragment of E. coli DNA polymerase I (Sanger et al., 1977), 

reverse transcriptase (Mierendorf and Pfeffer, 1987), modified bacteriophage T7 DNA 

polymerases (Tabor and Richardson, 1987) and Taq DNA polymerase.

2.11 Design of PCR primers

A prime consideration when designing a primer is that it should be complex enough to 

reduce the likelihood of annealing to sequences other than the chosen target (Saiki,

1989). The length of the probe depends on the A+T content. The annealing 

temperature (Tm) for a PCR depends directly on the length and G+C composition of the 

primers. The higher these parameters are the higher the Tm and therefore the 

stringency. The possibility of 3’ overlaps in primer design should be avoided to reduce 

the incidence of primer dimer formation. Stretches of polypurines and/ or 

polypyrimidines should also be avoided.

22



CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemical and Reagents

Disodium hydrogen phosphate (NaJIPCU), sodium dihydrogen orthophosphate 

(NaHjPO^, bacto-tryptone, yeast extract, agar granules, chloroform, isopropanol and 

dimethylformamide (DMF) were obtained from Fisher Biotech (Pittsburg, USA).

Sodium chloride (NaCl), polyethylene glycol (PEG), sodium citrate, sucrose, Tris base, 

sodium dodecyl sulphate (SDS), ethylene diamine tetraacetic acid (EDTA), agarose 

powder, sodium acetate (NaOAc), maleic acid, lauroyl sarcosine, ampicillin, glycerol, 

sodium hydroxide (NaOH), phenol and 2-butoxyethanol were obtained from Sigma 

Chemical Company (St. Louis, Missouri, USA).

Blocking powder, 5-bromo-4-chloro-P-D-galactopyranoside (X-gal) and isopropyl-p-D- 

thiogalactopyranoside (£PTG) were obtained from Boehringer Mannheim™ 

(Mannheim, Germany).

Sodium diethyldithiocarbamate (DIECA), magnesium chloride (MgCl2), magnesium 

sulphate (MgS04), glucose, p-mercaptoethanol, glacial acetic acid and hydrochloric 

acid were obtained from BDH Limited, UK.

Ethanol was obtained from Hayman Limited (UK).

23



Mineral (paraffin) oil was obtained from Fluka Company (Sigma-Aldrich), St. Louis, 

Missouri, USA.

The restriction and modifying enzymes used were obtained from various manufacturers 

and suppliers including Promega Corporation (Madison, USA), Stratagene™ (Austin, 

Texas, USA) and Boehringer Mannheim™.

3.2 METHODS

3.2.1 Virus Isolates

Thirty-six (36) CSSV isolates from the CRIG virus museum were randomly selected 

from each of the 5 groups (A, B. C, D and E) in the report by Sagemann et al. (1985) 

and Hughes and Ollennu (1993) (Table 1). The main group A had 4 subgroups. The 

selection was such that all the cocoa growing regions in Ghana were represented (Figure 

2). The geographical distribution of the isolates thus indicates the various locations of 

the isolates in the cocoa growing regions in Ghana since the isolates were named 

according to the locations from which they were first isolated.

24



Table 1: Selection of Isolates (Modified after Sagemann et al. (1985) and Hughes and 
Ollennu (1993).
Isolate name and 
number Geographic

location
(Region)

Type of leave 
symptoms

ELISA
reactions

ISEM Decoration at 
antiserum 
dilution 1:1600

Group A
Group Ai: Mild Strains
1. SS 167 Eastern vm, t + ++ +
2. SS 365B Eastern vm,t ++ ++ +
3. Worawora Volta vm, t I I l-l- +
4. Koben Ashanti m, t + ++ +
Group B: Mild Strains
5. Bisa Eastern vm,t + +
6.0nyimso-Agogo Ashanti s,t +++ ++ -
7. Donkokrom Brong vm, t + -
Group C: Severe Strains
8. AD 14 Eastern s,t,mo +++ +(-)
9. AD 75 Eastern s, mo +++ +
10. Mampong Eastern s, mo I 1 1 I +(-)
Group D: Severe Strains
11. AD 191 Eastern s, mo + + -
12. AD 7 Eastern s, mo + + -
13. AD 135 Eastern s, mo ++ +
14. Kpeve Volta s, mo + + -
Group £: Mild Strains
15. Peki Volta vm, t + - nt
16. Bobiriso Ashanti - - - nt
Group A2; Severe Strains
17. Bechem Brong s,t
18. Anibil Western s , t
19. Nkawkaw Eastern s,t
20. Nkrankwanta Brong s,t
21. Tediimantia Brong s,t
22. Amafie Western s,t
Group A3; Severe Strains
23 .New Juabeng-IA Eastern s,t
24. Kofi Pare -  1A Eastern s,t nt
25. Tafo Yellows Eastern s,t nt
26. Agyapoinaa Eastern s, t nt
27.Bosomtwe Ashanti s,t nt
28. Bosoratwe Ashanti
Group Aji Severe Strains
29. Onyimso-Agogo
30. Sankore

Ashanti
Brong

s,t

s,t
s,t

nt

nt
nt

31. Miaso Ashanti s,t nt
32 Nsaba Central s.t nt
^  Okerikrom Brong s.t nt
34 Diinn Volta s.t nt
VI Kwakoko-Juansa. Ashanti s. t nt
•36 Enchi Western s. t nt

25



Key:

s = severe; m = mild; vm = very mild; mo = mottled leaf symptom; — = without 

symptom; t = symptoms resemble those induced by strain 1 A; nt = not tested. 
Reactivity in ELISA indicate values obtained after 18 hours substrate incubation: 
++++ = A 405nm>1.0; +++ = 0.300 to 1.0; ++ = 0.150 to 0.300; + = <0.150;

-  = values not distinguishable from the controls.

Reference: Sagemann et al., 1985 and Hughes and Ollennu, 1993.

2 6



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•21 ? 
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AR

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KUMASI

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8/9/11/12/13/25/26 
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CAPE COAST
‘"TAKORADI

Figure 2. Map of Ghana showing the regional distribution of the selected isolates. 
(Modified after Thorold, 1975).

Numbers refer to isolate name and number as in Table 1



3.2.2 Extraction and purification of Virus:

3.2.2.1 Extraction of Virus: -

The virus was isolated and purified from the leaves by the method described by 

Adomako et a l, (1983) and modified by Sackey (1998, unpublished)

Infected cocoa leaves of the selected isolates were harvested from the CRIG museum. 

Twenty grains each of these were weighed and washed thoroughly in mild detergent and 

running tap water to get rid of surface micro-organisms The leaves were homogenised 

in 200ml of extraction buffer A consisting of 0.5M P O ^ buffer (0.5M NaHjPO^ 0.5M 

Na2HP04) pH 6.1, 5mM sodium diethyldithiocarbamate (DIECA), 10% polyethylene 

glycol (PEG) 10,000, 0.2% pectinase, 5mM p-mercaptoethanol). The homogenised 

leaves were transferred into a beaker and shaken for 3 hours at room temperature (25°C) 

and sieved through a cotton cloth. The cellular debris was discarded and the extract was 

then clarified by centrifugation at 15,000g for 20 minutes in a Sorvall centrifuge. 

Sodium chloride (0.2M) and 10% (w/v) of PEG 6000 were added to the supernatant in 

small amounts with 20 minutes of stirring and left at room temperature for 30 minutes.

The extract was centrifuged at 25,000g for 20 minutes to pellet the virus. The pellet 

was then resuspended in 30ml of extraction buffer B consisting of 0.05M PO/tiuffer. 

pH 6.8, 5mM ethylene diamine tetraacetic acid (EDTA), 0.2M NaCl and 5mM 

mercaptoethanol. The extract was clarified by centrifugation at 10,000g for 20 minutes 

and the supernatant transferred into a fresh tube. The pellet was resuspended in another 

30ml of buffer B and re-centrifuged at the same speed and time. The two supernatants 

of the extract were pooled and clarified by re-centrifuged as before.

28



3.2.2.2 Sucrose Cushion Centrifugation

A solution of 30% sucrose in extraction buffer B was prepared and 5ml transferred into 

type 65 centrifuge tubes. The virus extract was then layered onto the sucrose and the 

virus concentrated by centrifugation at 65,000g for 3 hours at 4°C in a Beckman L2-65 

ultra centrifuge. The supernatant was thus discarded and the pellet resuspended in 3 ml 

of buffer B. The extract was then centrifuged again at 4,000g for 10 minutes to clarify. 

The supernatant was transferred into 3ml centrifuge tubes and centrifuged at 90,000g 

for 45 minutes at 4°C in a Beckman L2-65 ultra centrifuge to pellet the virus. The final 

pellet was then resuspended in 1ml of buffer B and aliquoted into eppendorf tubes and 

stored at -20°C until needed.

3.2.3 Extraction and Purification of Virus DNA

To 500|Ld aliquot of the virus extract was added Tris-HCl, EDTA, sodium dodecyl 

sulphate (SDS) and Proteinase K to the final concentrations of 25mM, lOrnM, 1% and 

5 |-ig/ml, respectively. This was made up to a final reaction volume of700^1 with sterile 

distilled water, mixed by vortexing and incubated at 37°C for 30 minutes. The mixture 

was extracted once with an equal volume of phenol by vortexing for 5 minutes. The 

buffer phase was separated by centrifugation and transferred into a fresh sterile tube. 

The extraction was repeated with 1 : i phenol: chloroform. Sodium chloride (5M) to a 

final concentration of SOOrnM and an equal volume of buffered cold isopropanol were 

added to the aqueous phase, vortexed to mix thoroughly and incubated at room 

temperature for 1 hour.

The mixture was centrifuged to pellet the DNA at top speed of microcentrifuge for 10 

minutes. The pellet was then washed with 70% ethanol by adding the ethanol and

29



centrifuging at top speed of microcentrifuge for 10 minutes. This was then drained and

dried in-vacuo. The dried pellet was then dissolved in 80pl of TE buffer, pH 8.0. A 1%
i

agarose gel electrophoresis was used to determine the amount and approximate 

molecular weight of DNA present using 1 OjjJ of the DNA extract. The rest of the DNA 

was frozen for future use.

3.2.4 DNA Purification for PCR
The DNA samples to be used as templates for PCR were further purified by passing 

them through columns from QIAGEN™ or prepared in microcentrifiige tubes using a 

modification of the 2-butoxyethanol purification method described by Hagen et al., 

(1993).

3.2.4.1 QIAGEN Purification

The DNA was purified following the manufacturer’s protocol as described below

Two hundred microlitres of ethanol (96-100%) was added to 400ml of DNA sample, 

and mixed thoroughly by vortexing. The mixture was pipetted into the DNeasy spin 

column, placed in the 2 ml collection tube provided and centrifuge at 6000g for 1 min. 

The flow-through and collection tube were discarded. The DNeasy spin column was 

place in a new 2 ml collection tube, 500 (il Buffer AW1 added, and centrifuged for 1 

min at 6000#. The flow-through and collection tube were discarded. Next, the DNeasy 

spin column was placed in another 2 ml collection tube, 500 pi Buffer AW2 added, and 

centrifuged for 3 min at full speed to dry the DNeasy membrane. The flow-through and 

collection tube were discarded. Following the centrifugation step, the DNeasy spin 

column was carefully removed, placed in a clean 1.5 ml microcentrifiige tube, and 200 

pi Buffer AE pipetted directly onto the DNeasy membrane. The Dneasy spin column

30



was incubated at room temperature for 1 min, and then centrifuge for 1 min at 6000g to 

collect the purified DNA solution.

3.2A.2 2- butoxyethanol purification

In this method, 500^1 of the DNA extract were transferred into fresh 1.5ml 

micro centrifuge tube. If 1he extract available was less than 500[xl, the volume was made 

up to 500^1 with TE buffer. Sodium acetate (3M) was added to a final concentration of 

0.3M followed by 200|jJ of 2-butoxyethanol and mixed thoroughly. The mixture was 

kept on ice for 1 hour to precipitate the polysaccharides and centrifuged at top speed of 

microcentrifuge for 10 minutes to form a gelatinous pellet. The supernatant was 

carefully transferred into fresh tube and the gelatinous pellets discarded. To the 

supernatant, a further 300pl of 2-butoxyethanol were added, mixed thoroughly and kept 

on ice for another hour. It was again centrifuged at the same speed and time to pellet 

the DNA. The pellet was washed in 80% ethanol, drained completely and dried in- 

vacuo for 20 minutes.

The pellet was resuspended in 100|ol of TE buffer and sodium chloride (5M) was added 

to a final concentration of 500mM. An equal volume of cold isopropanol was also 

added, mixed thoroughly and kept in the freezer for 1 hour to precipitate the DNA, The 

mixture was then centrifuged at 12,000g for 10 minutes to pellet the DNA The pellet 

was washed in 80% ethanol and dried in-vacuo. The pellet was re-dissolved in a final 

volume of 50 to 100fd TE buffer pH 8.0. This was kept in the freezer for gel 

electrophoresis, PCR and other future use.

31



3.2.5 Agarose Gel Electrophoresis:

Depending on the number of samples to be run, the appropriate gel tray was chosen. 

Agarose gels were used at 1% and 0.8% concentration. The appropriate amount of 

agarose powder was weighed into a beaker and the appropriate volume of IX TAE 

(8mM Tris-Acetate, pH 8.3; 0.4mM EDTA) containing ethidium bromide to a final 

concentration of 0.5 ng/ml was added. The agar was melted by heating in a microwave 

oven and allowed to cool to approximately 60°C before pouring into the tray with sealed 

ends and well-forming comb inserted. This was allowed to set and transferred into the 

gel tank containing IX TAE after taking out the comb and the seals. Between 5-lOjj.l of 

the DNA samples with appropriate amount of running dye (consisting of glycerol, TE 

and bromophenol blue (BPB) were then loaded and run at 65 volts. The first well was 

always filled with DNA kilobase marker {Hind III lambda marker) to help determine the 

approximate molecular weight of the DNAs. The gel stained with ethidium bromide 

was visualized under ultra violet (UV) light. A photograph of the gel was then taken 

with a Polaroid camera or a gel documentation system. -V

3.2.6 Polymerase Chain Reaction (PCR):

Two pairs of primers namely Universal Badna Primers 2+T and 3+T derived from the 

nucleotide sequences of three (3) conserved regions of some badna viruses were used 

for the amplification of the DNA. The primers had sequences as follows:

Badna T: 5’-CACCCCCGGG(AC)(CT)(AC)(AT)(AGCT)GCTCTGATACCA-3’

Badna 2: 5’-AAATGCGGCCGCTA(CT)AT(ACT)GA(CT)AT(ACT)(CT)T-3’

Badna 3: 5’-AATAGCCGCAT(ACT)AT(ACT)AT(ACT)GA(AG)AC(AGCT)GA-3’

The primers Badna 2+T were expected to give amplification bands of molecular 

weights 1700 and 500 base pairs, while the Badna 3+T were expected to give a single

32



1000 base pairs amplification band (Lockhart and Olszewski, 1983; Sackey, 1995) 

(Figure 3).

The amplification was carried out using a PHC-3 programmable dry block thermal 

cycler from Techne™ The reaction mix of 50 (j.1 contained 1-lOng/j.il of QIA or 2- 

butoxyethanol purified template DNA, IX PCR buffer supplied by the manufacturer 

(Promega, USA), 200|j.M of each of the 4 deoxyribonucleoside triphosphates (dNTPs),

1.5 mM ofMgCl2; 2.0|nM of each oligonucleotide primer (2 + T or 3 + T), 2.5 units of 

Taq polymerase enzyme (Promega, USA). Sterile double distilled water (sddw) was 

used to make the volume up to 50jal. The reaction mix was spun down briefly and 

overlaid with 50 pi of mineral oil to avoid evaporation and refluxing during 

thermocycling.

Amplification conditions were three cycles of 94°C for 30 seconds (template 

denaturation), 37°C for 30 seconds for primer to anneal and 72°C for 180 seconds for 

primer extension. This was followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 

seconds and 72°C for 180 seconds for 35 cycles.

After the amplification reaction, the paraffin oil was removed by extraction with 

chloroform. The reaction product was analysed by using 10p.l aliquot of it in a 1% 

agarose gel electrophoresis and viewed under UV light. A photograph was then taken if 

required.

33



* •  P-Badna T

Figure 3 CSSV genome showing the location of universal Badna primers 2,3 and T

34



3.2.7 Gel purification of PCR products and clones:

When required, the remainder of the PCR product obtained from section 3.2.6, was 

applied to a 0.8% preparative agarose gel electrophoresis, and the various DNA bands 

of interest cut out into separate fresh tubes and 100jj.l of TE buffer pH 8.0 added. The 

tubes were centrifuged briefly and the contents frozen.

The PCR product bands were gel purified by centrifuging the frozen bands at top speed 

for 10 minutes. The aqueous phase was transferred into a fresh tube and phenol 

extracted. The aqueous phase was transferred into fresh tube and precipitated by 

addition of 5M NaCl to a final concentration of 500mM and cold isopropanol. This was 

mixed thoroughly, kept in the freezer for 2 hours and centrifuged at top speed of 

microcentrifuge for 10 minutes to pellet the DNA. The pellet was washed in 70% 

ethanol, drained, dried and the final pellet dissolved in lOjxl of TE buffer pH 8.0 

Aliquot of 5[il was analysed by gel electrophoresis and photograph taken. The bands in 

the gel were transferred onto nitrocellulose membrane for hybridisation. The rest of the 

samples were kept in the freezer for future use.

3.2.8 Hybridisation analysis

3.2.8.1 Southern DNA transfer

For Southern transfer, the gel was trimmed after photography and transferred into a 

container, 0.5M NaOH/1 5M NaCl buffer added enough to just cover the gel and left on 

a shaker at room temperature for about 45 minutes to denature the DNA in the gel. The 

gel was neutralized then by adding 1.5M NaCl/ 1.0M Tris pH 7.5 buffer to the gel and 

shaken for 30 minutes. The DNA in the gel was transferred onto the nitrocellulose 

membrane by a modification of the Southern transfer method to give two mirror images

35



of the gel on two membranes labelled top and bottom. Two sheets of membranes 

(Hybond-N) and four (4) filter papers were cut to the size of the gel, labelled and soaked 

in 20X SSC. The gel was placed in between the two nitrocellulose membranes and 

these were in turn sandwiched by two filter papers on either side of the membranes, 

followed by a stack of paper towels. A weight was placed on the set-up and left for a 

m inim um  of 2 hours. The membranes were removed and the DNA immobilized onto 

the membrane by cross-linking in an UV Stratalinker™ chamber. The membrane was 

either used immediately or kept for future use.

3.2.8.2 Dot Blots

The DNA samples were diluted with the manufacturer's (Boehringer Mannheim™) 

dilution buffer for dot blots and sterile distilled water in the ratio 2.5 to 1.5 respectively 

and heat denatured at 95°C for 10 minutes and immediately cooled on ice. The samples 

were then spotted onto ruled and labelled nitrocellulose membrane using up to 1.5^1 of 

DNA per spot depending on the DNA concentration. The membrane was made damp 

by placing on wet filter paper and the DNA fixed onto the membrane by cross-linking in 

an UV chamber. The membranes were then kept for future use.

3.2.8.3 Synthesis of DNA hybridisation probes

The probe to be used for hybridisation was prepared from a cloned virus DNA and 

labelled with non-radioactive digoxigenin (DIG) labelling kit from Boehringer 

Mannheim™. The hapten DIG was linked through a spacer arm to the corresponding 

nucleotide and the probe was generated with a Klenow polymerase. The template DNA 

was first gel purified, denatured by heating for 10 minutes at 95°C and cooled 

immediately on ice for another 10 minutes. This was used in a 1 Ojal reaction mix

3 6



containing lOng/jj.1 of the template DNA, 0.1 pM of dNTPs, lpM of hexanucleotide and 

0.5(jM of Klenow fragment. These were mixed thoroughly and incubated at 37°C for a 

minimum of 6 hours. The reaction was stopped by addition of 2|il of 0.2M EDTA. The 

synthesized DNA was precipitated by adding 2.5jj,1 of 4M LiCl and 75(xl of pre-chilled 

absolute ethanol and mixed thoroughly. The mixture was then kept in the freezer at -  

20°C for two hours before centrifuging to pellet the DNA at top speed of 

microcentrifuge for 10 minutes. The pellet was washed in 70% ethanol, dried in-vacuo 

and dissolved in 20^1 of TE pH 8.0. Prior to use, the probe DNA was denatured by 

heating in boiling water for 10 minutes and rapidly cooled on ice. Ten microlitres of 

hybridisation solution were added to the probe before adding to the membrane for 

hybridisation. For subsequent hybridisations, the probe was heat-denatured and cooled 

rapidly before using for every hybridisation.

3.2.8.4 Hybridization reaction

Hybridisation was carried out according to the manufacturer’s (Boehringer 

Mannheim™) protocol. The membrane was transferred into a hybridisation bottle and 

10-15 ml of hybridisation solution were added to the content of the bottle and incubated 

in the Hybaid™ mini oven at 60°C for 1-2 hours. The hybridisation solution was 

poured off and the denatured, appropriately diluted probe added. The bottle was again 

incubated in the oven for not less than 6 hours (normally overnight).

3.2.8.5 Washing and enzyme reaction

Hybrid detection was done by the protocol provided by the manufacturer of the 

labelling and detection kit. After hybridization, the probe was poured from the 

hybridization tube into an eppendorf tube and stored in the freezer at -40°C. The

37



membrane was washed twice for 5 minutes each with hybridisation wash buffer 1 (2X 

SSC, 0.1% SDS) at room temperature. The membrane was washed further with wash 

buffer 2 (0.2X SSC, 0.1% SDS) twice at 60°C for 15 minutes each. The membrane was 

then transferred into a container and IX maleic buffer (0.1M maleic acid, 0.14M NaCl 

pH 7.5) added to wash the membrane in preparation for the enzyme reaction.

This was washed on a shaker for about 3-5 minutes after which the buffer was decanted. 

Anti-DIG alkaline phosphatase conjugate was diluted (4pl in 10) with IX maleic buffer 

and added to the membrane. This buffer was decanted and the membrane washed twice 

with IX maleic buffer, first rapidly and then slowly for about 20 minutes. The maleic 

buffer was decanted and digoxigenin substrate buffer (0.1M Tris, 0.1M NaCl, 50mM 

MgCl2, pH 9.5) added to wash the membrane. This was also decanted and 10ml of the 

same digoxigenin buffer containing 45|J of nitroblue tetrazolium salt (NTB) in 

dimethyl formamide (DMF) and 35jil of 5-bromo-4-chloro-3-indolyl phosphate (X- 

phosphate) in DMF was added to the membrane to initiate the colour formation. The 

container was then covered and kept in a dark place for 3 or more hours. The 

membrane was washed, dried, analysed and photographed.

3.2.8.6 Removal of colour and probe

About 150-200ml of DMF was poured into a 2,000ml conical flask and the hybridised 

membrane added. The membrane in the DMF was heated to about 60°C with periodic 

shaking till the colour of the precipitate just disappeared. The membrane was removed 

from the DMF into a bowl containing 100-200ml of 0.2M NaOH and 0.1% SDS to 

remove the probe. This was then incubated for 30 minutes at 37°C. After this, the 

membrane was removed, rinsed with 2X SSC and dried for re-use.

38



3.2.9 Cloning

3.2.9.1 Ligation Reaction

Three types of vectors were used for this reaction. Two (pGEM-T and pCR-Script) 

were commercially acquired from Promega and Strategene™ respectively and the third 

one (KS+dt), was produced in the laboratory with a 3’dt overhang. The KS+dt was used 

for only three of the isolates (Worawora, Donkokrom, and Kofi Pare). pGEM-T was 

acquired with a dT overhang and was used for almost all the other DNA samples. pCR- 

Script was commercially acquired from Strategene without an overhang and was used 

for 2 isolates selected from each of the 8 groups. In this case, the PCR products used 

were first purified by column chromatography and then polished to remove the dA 

overhang before being used for the ligation reaction.

For the pGEM-T and pCR-Script vectors, the manufacturers’ protocols were used. For 

the KS+dt vector, a protocol used at the CRIG Molecular Biology Laboratory was used. 

Each ligation reaction mix consisted of 50-150ng of PCR ampKcon, 0.5-lunit of vector, 

IX of ligase buffer, and l-5units of T4 DNA ligase. For the pCR-Script, ImM of rATP 

and lunit of the enzyme Srf I were added. Each reaction mix was then made up to lOp.1 

with sterile distilled water, vortexed and centrifuged briefly.

The KS+dt reactions were kept in a water bath at 14°C overnight, the pGEM-T reactions 

were kept in the fridge at 4°C overnight or room temperature for 1 hour and the pCR- 

Script reactions were kept at room temperature for 1 hour. After the ligation reaction 

with the KS+dt and the pGEM-T vectors, one tenth (1/10) of the ligation reaction 

volume of 5M NaCl was added to the ligation reaction mix to a final concentration of 

500mM. An equal ligation reaction volume of cold isopropanol was also added. These

39



were vortexed to mix, centrifuged briefly and kept in the freezer for 2-3 hours to 

precipitate the DNA. The mixture was then centrifuged at top speed of microcentrifiige 

for 10 minutes to pellet the DNA. The supernatant was discarded and the pellet washed 

in 70% ethanol. The pellet was then dried in-vacuo and dissolved in lOmM TE buffer 

(pH 6.8). This was used for transformation or kept in the fridge to be used later. The 

pCR-Script reactions after the incubation at room temperature were heat denatured at 

65°C, cooled rapidly on ice and used for transformation or kept in the fridge for future

3.2.9.2 Preparation of competent cells

The cell lines used were XL1 blue and JM83. A sterile tooth pick or pipette tip was 

dipped into a frozen cell culture in glycerol and transferred into a culture tube 

containing 10ml of sterile liquid Luria Bertani (LB) medium (lOg Bacto-tryptone; 5g 

yeast extract; 170mM NaCl). This was incubated in a shaker at 37°C overnight. A 

fresh LB medium was re-inoculated with the bacteria culture in the ratio 100:1 and 

again incubated with shaking for 4V2 to 5 hours. The culture was then chilled briefly on 

ice, transferred into a 250ml centrifuge tube and centrifuged at 4,000g for 10 minutes. 

The supernatant was discarded and the pellet resuspended in cold sterile distilled water 

of equal original culture volume. Centrifugation at the same time and speed was done 

to re-peflet the cells. The pellet was again resuspended in distilled water but half (0.5x) 

of the initial volume of cold sterile distilled water used. This was followed by repeated 

centrifugations and re-suspension of the pellet each time in decreasing volumes (0.2, 

0.02 and then 0.003 times the initial volume of cold sterile distilled water used) of cold 

10% glycerol The final pellet was resuspended in 500^1 of the 10% glycerol and 60|fl

40



which was incubated at 50°C for 1 hour. The digested clones were analysed by a 1% 

agarose gel electrophoresis and a photograph of the gel was taken.

3.2.12 Large scale Plasmid Isolation

From the results of the mini-plasmid isolation, the two best clones based on the insert, 

from each ligation reaction were selected. A 2ml aliquot of one of the two selected 

clones was used to re-inoculate 200ml of LB medium containing ampidllin for large- 

scale plasmid isolation which was done in the same way as the mini-plasmid isolation 

except that 200ml of culture was used and the DNA was re-suspended in 8ml of TE 

buffer. The culture of the other one was used to re-inoculate 5 ml LB medium and the 

plasmid DNA compared with those from the large scale. A 500|j.l aliquot of both 

cultures was stored each, separately, in 700^1 of glycerol at -35°C for future use.

3.2.13 Caesium Chloride Purification

Solid CsCl was dissolved in the plasmid DNA solution at lg CsCl/lml of plasmid DNA 

suspension. The solution was transferred into a centrifuge tube of the Beckman L2-65 

ultra-centrifuge and ethidium bromide added. The tube was balanced with TE and heat- 

sealed. The tube was turned upside down a few times to mix the contents and to check 

for any leakage. The sample was centrifuged at 45,000g for 16 hours at 20°C in a 

Beckman L2-65 ultra centrifuge. First, a hypodermic needle was used to pierce the top 

of the tube. The upper band, which contained linearized and nicked plasmid DNA was 

ignored. The lower band, which contained the closed circular plasmid DNA, was 

carefully taken with another hypodermic needle and transferred into fresh 

microcentrifuge tubes. The ethidium bromide was removed by extraction with water- 

saturated butanol. The CsCl was removed by dialysing against very dilute TE buffer.

43



3.2.10 Isolation of Plasmids

The cultures were taken out of the shaker and 1.4ml transferred into sterile 1.5ml 

microcentrifuge tubes. These were centrifuged at top speed of microcentrifuge for 2 

minutes to pellet the cells. The pellets were thoroughly drained and resuspended in 

200(il of solution A (50mM glucose; 25mM Tris-HCl pH 8.0; 25mM EDTA). After 

incubation at room temperature for 5 minutes in 300^1 of solution B (0.2M NaOH; 1% 

SDS) were added and the tube inverted gently a couple of times to mix and kept at room 

temperature for 10 minutes. Then 400^1 of solution C (cold 3M sodium acetate 

(NaOAc)) were added, mixed thoroughly and kept on ice for 10 minutes. The samples 

were centrifuged at top speed of microcentrifuge for 10 minutes to pellet the cell debris. 

The supernatants were transferred into fresh tubes and 600^1 of cold isopropanol added. 

These were vortexed to mix and left at room temperature for 10 minutes to precipitate 

the recombinant DNA. The recombinant DNA was centrifuged at top speed of 

microcentrifiige for 10 minutes to pellet. The pellets were rinsed in 70% ethanol, re- 

centrifuged and dried in vacuo and dissolved in 70-100^1 of TE-RNase buffer pH 8.0.

3.2.11 Restriction Digest

The 20(j.i reaction mix included 10 |_ig/(al of the recombinant DNA IX restriction 

enzyme buffer, sterile distilled water and lunit each of the enzymes in the double­

enzyme reaction. For the pGEM-T, the enzymes used were A pal and Psil together 

while 2 units of BstZl were used for the single enzyme digests. For the KS+dt, the 

enzymes EcoKX and Hindftl were used together and for the pCR-Script, Sacl and Kpnl 

were used together. Each reaction was mixed thoroughly by vortexing and incubated at 

37°C for a minimum of 1 hour for all the enzymes with the exception of the BstZl,

42



aliquots were transferred into 500ul microcentrifiige tubes and frozen at 70 C in liquid 

nitrogen before storage in a freezer at —35°C.

3.2.9.3 Transformation

Transformation was done using an electroporator. The competent cells and the DNA 

sample of CSSV isolate were mixed thoroughly and transferred into a pre-chilled 

cuvette. The cuvette with the content was then kept on an aluminium foil on ice for 45 

seconds. The cuvette was then wiped dry with a tissue paper, loaded into the 

transporator, which was set at 0.82KV. At this voltage, the DNA was transformed into 

the bacterial cells. The transformed cells were then washed out of the cuvette into a 

1.5ml tube with the LB medium containing glucose, MgCh and MgS04 to final 

concentrations of Q.04mM, O.OlmM and O.OlmM respectively. This was kept in the 

incubator at 37°C for about 20 minutes to initiate cell growth.

3.2.9.4 Cell culture and screening

Sterile LB agar plates containing lOOpg/ml isopropyl B-D-thiogalactopyranoside 

(IPTG), lOOpg/ml 5-bromo-4-chloro-B-D-galactopyranoside (X-gal) and 50pg/ml 

ampidllin were prepared. A sterile bent glass rod was used to spread 100(jl of the 

transformed cells on the LB agar plates. The plates were then labelled, inverted and 

incubated at 37°C overnight to allow cells to grow.

The plates were removed from the incubator and sterile tooth picks were used to 

transfer single white colonies (transformed cells) into culture tubes containing 3 ml of 

sterile LB medium with ampicillin to a final concentration of 20 ug/ml. These were 

incubated at 37°C overnight with vigorous shaking.

41



The contents of bag were transferred into fresh microcentrifuge tube and the DNA 

precipitated with 500mM NaCl and cold isopropanol. The mixture was centrifuged at 

top speed of microcentrifuge for 10 minutes to pellet the DNA, which was later washed 

in 70% ethanol, drained and dried in vacuo. The DNA pellets were then dissolved in 

TE buffer pH 8.0 and used for sequencing.

3.2.14 DNA Sequencing

3.2.14.1 Sequencing gel

The glass slabs for the gel were thoroughly washed with mild detergent, rinsed and 

wiped thoroughly. They were cleaned with 95% ethanol and again wiped dry. The 

smaller slab supposed to be the upper was silanised with 750p.l of Sigmacote from 

Sigma Chemical™ to make it non-sticky. The glass slabs for the gel were prepared and 

clamped together. The 75ml of 5% Long Ranger gel solution containing 7M urea, 1.2X 

TBE from 10X Tris -Borate-EDTA (TBE-890mM Tris-base, 890mM boric acid, 20mM 

EDTA), 5% Long Ranger from 50% Long Ranger from HydroLink™, 10% (w/v) 

ammonium persulphate (APS), 0.5mM N,N,N’,N’ tetramethylethylenediamine 

(TEMED) and distilled water was prepared and filtered. The gel solution was poured 

into the slabs gently and carefully to avoid formation of air bubbles. Sharktooth combs 

were inserted into the gel and clamped. The gel was allowed to set at room temperature 

overnight.

3.2.14.2 Primer Radio-labelling

The pUC/M13 forward primer was end labelled with [y 32P] ATP. A lOpl labelling 

reaction mix was prepared containing 80|j.g of the primer (dried in vacuum to reduce 

volume), lOpmol of [y 32P] ATP, 10X T4 polynucleotide kinase buffer to a IX final

44



concentration, 4units of T4 polynucleotide kinase and sterile distilled water. The mix 

was incubated at 37°C for 30 minutes and the kinase later inactivated at 90°C for 2 

minutes in a thermal cycler.

Two micro litres each of the four different d/ddNTPs (d/ddATP, d/ddGTP, d/ddCTP 

and d/ddTTP) were placed into four correspondingly labelled 500(j.l microcentrifuge 

tubes. Seventeen micro litres of 6\ig of template DNA, 5X sequencing buffer, 1.2|jg of 

the labelled primer, 0.3 unit of sequencing grade Taq DNA polymerase and sterile 

distilled water were prepared. This mixture was briefly vortexed and distributed equally 

into the tubes containing the d/ddNTPs and mixed thoroughly by pipetting. A drop of 

mineral oil was layered on, centrifuged briefly and put into a thermal cycler pre-heated 

at 95°€ for 2-3 minutes. Sequencing was carried out for 30 cycles at 95°C for 30 

seconds for denaturation and 70°C for 30 seconds for both annealing and extension. 

Two micro litres of stop solution containing bromophenol blue and xylene cyanol dye 

was added to stop the reactions and the tubes were then stored at 4°C.

3.2.14.3 Sequencing gel electrophoresis

The electrophoresis system was set up with 0.6X TBE. The system was pre-run at 

80watts for 10 minutes. The gel wells were washed to remove excess urea and the 

sequencing reaction heated at 70°C for 2 minutes to denature the DNA before loading 

into gel. The gel was run at 65 watts for 2 hours. The glass slabs were taken out, 

washed and the smaller upper one removed carefully. The sequencing gel was 

transferred onto Whatman 3mm filter paper cut to the size of the gel. The sequencing 

gel was then covered with saran wrap avoiding air bubbles and dried at 80°C for 2-3 

hours. The sequencing gel was transferred onto a film in dark room and put into an

45



envelope. A weight was placed on the envelope in a cabinet and left overnight. The X- 

ray film was processed and the sequence read on an illuminator.

3.2.15 Primer design

The design of the new primers was based on consensus nucleotide sequences from 

CSSV (severe 1A New Juaben and Nsaba), commelina yellow mottle virus (CoYMV), 

sugar cane bacillifonn virus (SCBV), banana streak virus (BSV), Dioscorea alata 

bacilliform virus (DaBV), and kalanchoe top spotting virus (KTSV). The sequences 

from rice tungro bacilliform virus (RTBV) were excluded in part because though a 

badnavirus, its genome size and organization differs significantly from the others.

3.2.16 PCR annealing temperature optimisation and primer testing

The annealing temperature for the new PCR primers (Badna 1A and Badna 4) was 

optimised using a temperature range between 50°C and 65°C. The PCR was carried out 

for 35 cycles at 94°C for 30 seconds, 55°C to 60°C for 30 seconds and 72°C for 3 

minutes. The primers were optimised using DNA from the New Juaben isolate from 

source (mechanically inoculated and quarantined) as a standard. After the optimisation, 

the primers were used to screen all the 36 isolates in Table 3.1 as well as 24 others from 

the CRIG museum. Uninfected cocoa leaf DNA was used as negative control. Five of 

the 1+4 PCR amplification products (Kofi Pare-1 A, Enchi, SS 167) were randomly 

selected, cloned and sequenced.

46



CHAPTER FOUR

4 .0  RESULTS

4.1 Selection and grouping of isolates

Thirty-six (36) CSSV isolates randomly selected from 5 main groups based on 

serological and biological properties are shown in Table 1. The largest group (A) 

comprised 24 isolates that are closely related to CSSV 1A  Isolates of groups Ai, B and 

E were mild strains whilst those in the other groups, A2, A3, A4 C and D were severe 

strains.

Figure 2 shows the geographical distribution of the isolates selected. As illustrated, all 

the cocoa growing areas (Brong Ahafo, Ashanti, Volta, Western, Eastern and Central 

regions) in Ghana were represented. Most of the isolates (41.7%) are located in the 

Eastern Region of Ghana.

4.2 Extraction and purification of virus and virus DNA

The PEG precipitation method used for the extraction and purification of the CSSV from 

the infected leaves successfully yielded virus for extraction of DNA and development of 

DNA sequences. In all, DNA was extracted and purified from 36 CSSV isolates from 

the CRIG museum Cleaner DNA preparations were obtained by column purification 

compared to the 2-butoxyethanol method of purification

47



4.3 Polymerase chain reaction analysis

Figure 4 shows a typical preparative gel photograph of PCR amplification products 

obtained from DNA of three CSSV isolates (AD7, Peki and Anibil) using badna primers 

2+T and 3+T, Each isolate was analysed in adjacent lanes using the two different sets of 

primers. The odd numbered lanes (1, 3, 5) show PCR amplification products using 

primers 2+T while the even numbered lanes (2, 4, 6) show products from primers 3+T.

For each viral D N A  the PCR reaction yielded between 2 to 5 amplification products 

irrespective of the primer pair used. For the primers 2+T the amplification products 

ranged between 1,700 and 400 base pairs whiles that for primers 3+T, the range was 

from 1,000 to 400 base pairs. As illustrated in Figure 4, the 500 and 400 base pair 

products were common lands appearing in all isolates with the 500 base pair being the 

most prominent. Table 2 summarizes the results of PCR analysis of all the 36 CSSV 

isolates using universal badna primers. As shown, the minimum number of 

amplification bands obtained with badna 2+T primers was one as in CSSV SS365B and 

the maximum was five as in Kwakoko Juansa. On the other hand, badna 3+T gave a 

minimum of two amplification bands for isolates from Bisa and Amafie and a maximum 

of 5 for those from Nkawkaw and Kwakoko Juansa. Generally, most of the CSSV 

isolates gave 3 amplification bands for both 2+T and 3+T primers (Table 2).

48



M 1 2 3 4 5 6

9,416 bp 
6,557 bp

2,027 bp

1,700 bp 

1,000 bp

500 bp 
400 bp

Figure. 4: PCR amplification products w ith  primers 2+T and 3+T

Lane M = X—Hind in molecular weight marker 

Lane 1 = AD7 with 2+T;

Lane 2 = AD7 with 3+T;

Lane 3 = Peki with 2+T;

Lane 4 = Peki with 3+T;

Lane 5 = Anibil with 2+T;

Lane 6 = Anibil with 3+T.

49



Table 2: Analysis of CSSV CRIG museum isolates by PCR using universal badna 
primers 2+T and 3+T.

PCR bands using different primers
Isolate
number

Isolate Name Badna 2+T Badna 3+T

1 SS167 ++++ -H-+
2 SS365B + +-H-
3 Worawora ++++ -H-+
4 Koben +++ +-H-
5 Bisa +++ -H-
6 Qnyimso Agogo +++ +++
7 Donkorkrom +-H- +++
8 AD14 +++ +-H-
9 AD75 +++ +-H-
10 Mampong H-H H-H-
11 AD191 ++ -H-+
12 AD7 +++ +++
13 AD135 +++ +++
14 Kpeve ++++ +-H-
15 Peki -H-f -H-+
16 Bobiriso +++ +++
17 Beoham +4-H- +++
18 Aiiibil 1111 -H-+
19 Nkawkaw ++++ -t-H-H-
20 Nkrankwanta ++++ +-H-
21 Tedumantia 1 III ++++
22 Amafie +++ -H-
23 New Juaben +++ +++
24 Kofi Pare ++++ 4+4-
25 Tafo yellows +++ -H-+
26 Agyapomaa nil 4++
27 Bosomtwe Amakom +++ 44+
28 Bosomtwe +++ 4-H-
29 Agogo -H-+ 4+-f
30 Sankore +++ 4-44-
31 Miaso 11 11 1 1 1 i
32 Nsaba +++ -H+
33 Okerikrom ++++ +4+
34 Djinji -1 H-+ H  11
35 Kwakoko Juansa 1 1 1 1 H Mill
36 Enchi ++++ ++-H-

Healthy Amelonado 
DNA

“

Key
+ = Number of amplification products obtained; 
- = no amplification product

50



4.4 Gel-puritied PCR amplification products

Figure 5 shows a photograph of agarose gel-purified PCR amplification products from 3 

CSSV (Mampong, AD7 and Bobiriso) isolates using badna primers 2+T and 3+T. The 

individual amplification products with molecular weights ranging between 1700 and 400 

base pairs were cut out, gel purified and electrophoresed on agarose gel. The bands 

appeared in ladders as shown in the figure (lanes 2 and 3 and 4 to 6). Bands in lanes 1, 

and 7 to 10, were too faint for photographic reproduction The largest product had a 

molecular weight of 1700 base pairs whilst the smallest band was 400 base pairs.

4.5 Hybridisation analysis of PCR products

Figure 6 shows results of Southern hybridisation analysis of gel-purified PCR 

amplification products with DNA probe made from full-length infectious clone of severe 

CSSV 1A (New Juaben) to detect the virus coded DNA species. The molecular weights 

of the PCR amplification products that were found to be virus coded ranged between 

1700 and 400 base pairs. Amplification products in lanes 3, 4 and 6 did not hybridise 

with the probe whilst the rest all did hybridise.

51



M 1 2  3 4  5 6 7  8 9 10

Figure. 5: Gel-purified PCR amplification products using universal badna primers.

Lane M =. X-Hind HI molecular weight marker;

Lanes 1 to 3 = Mampong with 3+T, bands 1-3 * respectively.

Lane 4 = AD 7 with 2+T, band 1 

Lanes 5 and 7 = AD 7 with 3+T, bands 1 to 3 respectively 

Lanes 8 to 10 = Bobiriso with 2+T, bands 1 and 3 respectively 

* bands were named 1, 2, 3, etc in decreasing molecular weights.

52



1,700 bp 
1,000 bp 
500 bp 
400 bp

Figure. 6: Hybridised membrane of gel purified PCR products

Lanes 1 and 2 = PCR DNAs from AD 14 with 2+T, bands land 2 respectively.

Lanes 3 and 4 = PCR DNAs from AD 14 with 3+T, bands 1 and 2 respectively.

Lane 5 = PCR DNAs from AD 75 with. 2+T, band 1.

Lane 6 = PCR DNAs from AD 75 with 3+T, band 1.

Lanes 7 to 10 = PCR DNAs from Mampong with 2+T, bands 1 to 4 respectively.

Lanes 11 to 14 = PCR DNAs from Mampong with 3+T, bands 1 to 4 respectively.

53



Table 3 summarises results of such hybridisation experiments to identify the virus-coded 

PCR amplification products. Out of the 240 PCR amplified DNA species from all the 

36 isolates, 140 hybridised with the probe (virus coded). One hundred of the PCR 

amplification products did not hybridise with the probe, indicating that they are not of 

virus origin

4.6 Cloning of PCR DNA

Figure 7 shows a sample gel photograph of the Pvu II digested 2+T and 3+T PCR 

clones. The molecular weights of the inserts were compared with that of the PCR 

amplification products before they were cloned to confirm their correct sizes. The sizes 

of the inserts ranged between 1700 and 100 base pairs.

54



Table 3: Summary of results of experiment to identify virus-coded PCR 

amplification products from 36 isolates

Total number of DNA species from PCR 240

Total number of PCR products analysed by 

hybridisation

240

Number of virus coded PCR products 140

Number of PCR products tested but were 

negative

100

Number of virus coded DNA species cloned 68

55



3.000 bp 
(plasmid) 
1,700 bp 
1,500 bp
1.000 bp

Figure 7: Screening of 2+T and 3+T PCR clones by Pvu II digestion.

Lane M =  "K~Hind HI molecular weight marker;

Lane 1 = ADM clone (2+T);

Lane 2 = AD14 clone (3+T);

Lane 3 = AD135 clone (2+T);

Lane 4 = Bechem clone (3+T);

Lane 5 = Kpeve clone (2+T).

56



4.7 Primer design
Nucleotide sequences from PCR DNA clones using the primer pairs badna 2+T and 3+T 

were used along with those from the same region of other badna viruses in the design of 

new primers. From the several CSSV isolates used, only sequences from CSSV 1A (2T) 

and CSSV Nsaba (Sackey 2000) were selected for inclusion in the new primers because 

hybridisation analysis along with serological and host effect had shown that they were 

representative of the Ghanaian isolates of CSSV (Sackey, 2000). The sequences were 

compared with those published and deposited in DNA databanks such as the 

GENEBANK to check if they were CSSV.

A new pair of primers was designed based on consensus sequence from sequences of 

these two CSSV isolates (lA-Kofi Pare and Nsaba) and some selected closely related 

badna viruses (commelina yellow mottle virus (CoYMV); banana streak virus (BSV); 

kalanchoe top spot virus (KTSV); sugar cane bacilliform virus (ScBV); Dioscorea alata 

bacilliform virus (DaBV).

Figure 8 shows the alignment of the sequences of the viruses used in the design of the 

primers. Two oligonucleotide primers (below) were then designed, and named badna 

primers 1 and 4. Badna primer 1 has 29 nucleotides as follows 

Sequence: 5’- CTN TAY GAR TGG YTN GIN ATG CCN TTY GG -  3’

Badna primer 4 has 25 nucleotides as follows Sequence: 5’- TCC AYT TRC ANA

YNA CNC CCC ANC C -  3’

where N — A, T, G or C; Y — C or T; R — A or G

57



BADNA 1 PRIMER
DaBV CTC TAC GAA TGG CTT GTA ATG CCG

CoYMV CTT TAT GAA TGG CTT GTA ATG CCA
BSV CTG TAT GAG TGG CTA GTT ATG CCC
KTSV CTC TAC GAA TGG TTA GTA ATG CCA
DaBV TTC GGA CTG AAG AAT GCT CCT GCC

CoYMV TTT GGT CTC AAA AAT GCG CCC GCT
BSV TTT GGA CTT AAA AAT GCT CCT GCC
KTSV TTC GGT CTT AAA AAT GCC CCT GCT

BADNA 4 PRIMER

CoYMV ACT GGC TGG GGA GCC GTC TGC AAA TGG AAA
BSV GAA GGC TGG GGA GGG GTA TGT AAG TGG AAA
ScBV ACT GGA TGG GGA GCA GTA TGC AAG TGG AAG

KTSV GAA GGT TGG GGT GGC ATC TGC AAA TGG AAG
DaBV GAA GGC TGG GGT GGT GTA TGC AAA TGG AAA

Figure 8: Alignment of conserved region of CSSV sequence with that of the other 

badna viruses.

CSSV: cocoa swollen shoot virus; CoYMV: commelina yellow mottle 

virus; BSV: banana streak virus; KTSV: kalanchoe top spot virus; ScBV: 

sugar cane bacilliform virus; DaBV: Dioscorea alata baciflifbrm virus

58



4.8 Results of annealing temperature optimisation

Figure 9 shows results of amplification of CSSV 1A DNA under different cycling 

conditions. Denaturation in all cases was at 94 for 30 seconds, and primer extension at 

72°C for 3 minutes. Annealing temperatures were 50, 55, 58, 60, 62 and 65°C. There 

were no amplification products at temperatures 50°C, 62°C and 65°C whilst 

amplification products were obtained at 55, 58 and 60°C. At these annealing 

temperatures the expected PCR product of 600 base pairs was obtained. The PCR 

product at 58°C was however of lower intensity than the others. The annealing 

temperature of 60°C was chosen for the screening of isolates with the new primers 

because it had the highest intensity.

4.9 Testing of primers 1+4

Using the annealing temperature of 60°C, 36 isolates from the CRIG museum were 

screened in PCR reactions. DNA from 28 isolates yielded 600 base pair amplification 

products and 8 isolates gave no products.

Figure 10 shows a sample gel photograph of PCR amplification products using the 

badna primers 1+4. The uninfected Amelonado DNA in lane 1 served as a negative 

control. The isolate in lane 3 (Onyimso-Agogo) had a 600 base pair band with low 

intensity as compared to the others which all had relatively high intensities.

59



M 1 2 3 4 5 6

23,130 bp 

4,361 bp 

2,322 bp

  600 bp

Figure 9: Optimisation of PCR annealing temperature for primers 1+4 using CSSV 

1ADNA.

Lane M = \-H ind  HI molecular weight marker;

Lane 1 = annealing temperature at 50°C;

Lane 2 = annealing temperature at 55°C;

Lane 3 = annealing temperature at 58°C;

Lane 4 = annealing temperature at 60°C;

Lane 5= annealing temperature at 62°C;

Lane 6 = annealing temperature at 65°C.

60



600

Figure 10: PCR with DNA from isolates from CRIG museum using primers 1+4

Lanes M = X Hind III molecular weight marker; 

Lane 1 = uninfected Amelonado DNA;

Lane 2 = Worawora;

Lane 3 = Onyimso- Agogo;

Lane 4 = Donkokrom;

Lane 5 = AD 14;

Lane 6 = Mampong;

Lane 7 = AD 7;

Lane 8 = AD 135;

Lane 9 = Peki.

61



Table 4 shows the suxnmaiy of comparison of the PCR results for primers 2+T, 3+T and 

1+4 for all the isolates used. PCR with badna primers 2+T and 3+T for all the initial 36 

isolates gave 2 to 5 amplification lands with the exception of SS 365B which gave only 

one amplification band. PCR with the new badna primers 1+4 however, gave only one 

amplification band for all the 28 isolates that tested positive. Eight of the 36 isolates that 

tested positive with both primers 2+T and 3+T, for example the ADI 91 and AD75 

isolates, tested negative with primers 1+4. The Enchi isolate was not hybridised even 

though both the 2+T and the 3+T had 4 amplification bands each. PCR amplification 

products from primers 2+T and 3+T for AD 191 tested negative in the hybridisation 

reactions. AD 191 also tested negative with the new primers 1+4.

62



Table:4: Analysis of CSSV CRIG museum isolates by PCR using new badna 

primers 1+4.

Isolate num ber Isolate Name PC R  bands using different primers
B adna2+T B adna 3+T B a d n a 1+4

1 SS167 ++++“ +++(1)° +
2 SS365B + +++(3) +
3 Worawora ++++(3) + f+ (l) +
4 Koben ++-K2) +++(3) -
5 Bisa +++ ++(1) -
6 Onyimso Agogo +++(2) +++(3) +
7 Donkorkrom ++-K3) -H-K3) +
8 AD14 ++H2) +++(2) +
9 AD75 -H-f-(l) +++<1) -
10 Mampong ++++(4) ++++(4) +
11 AD191 ++ +++ -

12 AD7 ++-K1) +++<2) +
13 AD135 +++(1) ++-K1) +
14 Kpeve ++++(2) +++ +
15 Peki +++(1) +++(1) +
16 Bobiriso +++(3) +++(2) +
17 Becliem ++++(2) +++(3) -

18 Anibil ++++(2) ++H2) +
19 Nkawkaw ■++++(4) +++++(5) +
20 Nkrankwanta ++++(!) +++(1) +
21 Techimantia ++++<1) ++++(1) -

22 Auiafie +++ ++(1) +
23 New Juaben ++H2) +++(2) +
24 Kofi Pare ++++(4) +++(2) +
25 Tafo yellows +++m +W<2) -

26 Agyapomaa ++++(2) +++(1) -

27 Bosomtwe Amakom ++-H2) +++(2) +
28 Bosomtwe +++(3) 4+H3) +
29 Agogo +++{3) +++(3) +
30 Sankorc +++(2) ++HD +
31 Miaso ++++(3) +++ +
32 Nsaba +++(2) +++<2) +
33 Okerikrom ++++(3) ++-H3) +
34 Djinji ++++(4) ++++(4) +
35 Kwakoko Jnansa +++++(5) ■l+ n + (5) +
36 Enchi ++++(3) ++++(3) -

Healthy Amelonado 
DNA

-ve -ve -ve

63



Key

Expected molecular weights of amplification products 

Badna 2T =1700bp; Badna 3T =1000bp; Badna 1+4 =600bp;

+ = Number of amplification products obtained;

- = no amplification product

Numbers in parenthesis are those shown by hybridisation analysis to be virus coded, 

using cloned DNA from infectious full length clone New Juaben as hybridisation 

probe.

64



4.10 Dot blot hybridisation analysis of viral DNA with 1+4 PCR probes

Table 5 shows summary of the results of dot blot hybridisation of the native virus DNA 

templates of the initial 36 CSSV isolates, using probes made from PCR DNA from the 

new primers 1+4. The isolates have been grouped on the basis of serology. The SS167 

and Nkawkaw probes hybridised very strongly with almost all the DNA of the CSSV 

isolates used whilst ADM and AD135 probes hybridized weakly with only a few of the 

DNA of the isolates used. The Worawora probe also hybridized mildly with some of the 

isolates whilst the Enchi probe hybridised strongly but with a few of the isolates used. 

The healthy Amelonado DNA as expected did not hybridise with any of the probes. The 

Kofi Pare and Bosomtwe Amakom, both of which are of group A3 isolates hybridised 

with all the probes. All the isolates hybridised with at least one of the probes.

65



Table 5: Dot blot hybridisation analysis of virus DNA from CSSV isolates with

probes using 1+4 PCR products.

Test DNA Group Geographical Probes
distribution
(region)

SS
167

Wora­
wora

AD
14

AD
135

Nkaw-
kaw

Enchi

l.SS 167 Ai Eastern + - - + -
2. SS 365B Ai Eastern - - - - + -
3. Worawora Ai Volta + + - - + -
4. Koben Ai Ashanti + + - - ++ -
5-Bisa B Eastern - - - - + -
6. Onynnso-Agogo B Ashanti ++ - - - + -
7. Donkokram B Brong Ahafo + + - - - ++ _
8. AD 14 C Eastern ++ - + - ++ -
9. AD 75 C Eastern + + - - +++ +
10. Mampong C Eastern ++ - - - ++ ++
11.AD 191 D Eastern ++ + - - +++ +
12.AD7 D Eastern ++ - - - -H- -
13. AD 135 D Eastern + - - - ++ -
14.Kpeve D Volta _ - - - ++ -
15.Peki E Volta + - - - + -
16.Bobiiiso E Ashanti +++ - - - +++ -
17. Rechem a2 Brong Ahafo + - - - ++ -
18. AnM A2 Western ++++ + - - +++ -
19.Nkawkaw A2 Eastern - - - - + -
20. Nkrankwanta a2 Brong Ahafo + - - - +++ -
21 .Teehimantia A2 Brong Ahafo ++ + - - -H- ++
22. Amafis a2 Western + + - - + ++ -
23. New Juaben A3 Eastern 1 1 l 1 + - - +++ ++
24. Kofi Pare A3 Eastern 1 1 1 1 + ++ + ++ -H -f
25. Tafo Yellows A3 Eastern ++ + - - ++++ ++
26. Agyapomaa A3 Eastern ++ + - - 1111 +
27. Bosotwe 
Amakom

A3 Ashanti +++ + + + -H- ++

28. Bosomtwe A3 Ashanti +++ - + + ++ +
29. Onyimso- 
Agogo

A4 Ashanti ++++ + - - +++ +

30. Sankore Al Brong Ahafo +++ + ++ - +++ +++
31.Miaso A4 Ashanti +++ + - + +++ ++
32.Nsaha A4 Central ++ - - - -H- —
33. Okerikrom Ai Western +++ - + - -H-+ -
34. Djinji A, Volla +++ + + - -H-f -
35. Kwakoko- 
Juansa

A, Ashanti +++ + + - +++ ++

36. Enchi A, Western ++ - - - -H- ++
Uninfected
Amelonado

- - - - - - - -

66



Key: + = weak hybridisation reaction with probe; ++ = strong hybridisation reaction 
with probe; +++ = very strong hybridisation reaction with probe; 1 I = very, very 
strong hybridisation reaction with probe; -  = no reaction.

67



4.11 CSSV sequences from clones from new primers 1+4

Figure 11 shows sequences of CSSV isolates Kofi Pare (A) and Enchi (B), derived from 

clones using the new primers 1+4. Samples A and B had 853 and 597 base pairs, 

respectively. These sequences were compared with those deposited in DNA databanks 

and were shown to have significant alignment with the CSSV polyprotein gene. Two 

out of the 5 that were sequenced were shown to be virus sequences.

68



A

ccacttacaa atagcacccc agcccacacc aaaaacgttg aagcgtaaga catacataag 60
acgacgtaac tccctctaac tctctctctc attctttcca tttagttatc tctcaagtga 120
tcctgtgggg caggcacaat atacttctgt atcattactt cattatattc accattgctc 180
aaacttagta aaacagcaac aaaactagtg acctaatctc ttcttcaaaa actactgacc 240
tagtttattc ttcattacgt ggctggggtg gtatgtgcaa atggactcaa aaatgctcct 300
gcagtatttc aaagaaaaat ggaccaatgt ttcaaaggca cagaagaatt catagctgtg 360
tatattgatg acatcttggt cttcagcgag actatggcgg aacacaccaa gcatattgga 420
atcatgctaa caatctgcca agaaaatggg ctggtcctaa gcccaaataa aatatgtctt 480
gctcaacgag agattgaatt tttgggcaca atcatctctc aaggtcaaat gaagcttcag 540
cctcatgtca taaagaagat agtcaacaag gcagatatgg agctcgaaac aactaggggc 600
ctaagatcat ttttgggcct cctgaactat gcccgaatct acatacccaa tctggggaag 660
aagctaagtc cactatatgc caaaaccagt cccaccggag aaaagaagtt taatcgacag 720
gattggcatc tgataaaaga aattaaagat atggtccaaa agctcccaaa cctcgctatc 780
ccaccagcaa
gtttgcaagt

gatgctgcat
gga

tatcatagaa agcgatggtt gtatggaagg gtggggtgca 840
853

B
ctatacgagt ggttagtaat gccgttcggg ctcaaaaatg cccctgcagt atttcaaaga 60
aaaatggacc aatgtttcaa aggcacagaa gaattcatag ctgtgtatat tgatgacatc 120
ttggtcttca gcgagactat ggcggaacac accaagcata ttggaatcat gctaacaatc 180
tgccaagaaa atgggctggt cctaagccca aacaaaatat gtcttgctca acgagagatt 240
gaatttttgg gcacaatcat ccctcaaggt caaatgaagc ttcagcctca tgtcataaag 300
aagataglca acaaggcaga aatggagctc gaaacaacta agggcctaag atcatttttg 360
ggcctcctga actatgcccg aatciacata cctaatttgg ggaagaagct aagtccacta 420
tatgccaaaa ccagtcccac cggagaaaag aagtttaatc gacaggattg gcatctgata 480
aaggagatta aaaatatggt ccaaaagctc ccaaacctcg ctatcccacc agcaagatgc 540
tgcattatca tagaaagtga tggttgtatg gaaggctggg gagccgtttg caagtgg 597

Figure 11: Sequences of PCR products generated using the new primers badna 

1+4.

A: sequence from PCR DNA (badna 1 +4) obtained for CSSV 1A (Kofi Pare).

B: sequence from PCR DNA (badna 1 +4) obtained for CSSV Enchi.

69



CHAPTER FIVE

5.0 DISCUSSION AND CONCLUSION

This project set out first, to develop a sequence database from as many viral DNA 

clones as possible and second, to identify or design new oligonucleotide primers 

based on a conserved sequence of the viruses for the rapid detection of bacilliform 

DNA virus infection in cocoa.

To achieve these objectives, 36 virus isolates were collected from the CRIG 

museum, based on their serological and biological characteristics (Sagemann et al., 

1985; Hughes and Ollennu, 1993).

Isolates in groups A2, A3 and A4 had similar leaf symptoms that resembled those 

induced by the 1A strain. They also had similar ELISA and ISEM reaction results. 

All the members in groups Ai, C and A2 had decoration at antiserum dilution 1:1600 

results less than 0.150. Isolates in groups B and D did not react whilst groups A3, A* 

and E were not tested. The similarities could be due to serological resemblance of 

virus isolates (Table 1). The five main isolate groups covered all the cocoa growing 

regions in Ghana (Brong Ahafo, Ashanti, Volta, Western, Eastern and Central 

regions), (Figure 2). Since the primers to be designed had to be able to