RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE

CHAIN REACTION (RAPD-PCR) FINGERPRINTING OF

Escherichia coli O157:H7

NOR'AISHAH BINTI HASAN

INSTITUTE OF BIOLOGICAL SCIENCES

FACULTY OF SCIENCE

UNIVERSITI MALAYA

2008

RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE

CHAIN REACTION (RAPD-PCR) FINGERPRINTING OF

Escherichia coli O157:H7

NOR'AISHAH BINTI HASAN

A GRADUATION EXERCISE SUBMITTED TO THE

FACULTY OF SCIENCE

UNIVERSITI OF MALAYA

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE

DEGREE OF MASTER OF BIOTECHNOLOGY

INSTITUTE OF BIOLOGICAL SCIENCES

FACULTY OF SCIENCE

UNIVERSITI MALAYA

2008

ABSTRACT

Twenty (n=20) among beef isolates of Escherichia coli O157:H7 were examined

for the detection of Shiga- toxin 1 and 2 (stx1 and stx2) genes and characterized using

Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR)

fingerprinting. All isolates were obtained from the laboratory of Food Science and

Biotechnology, University Putra Malaysia, Serdang, Selangor.

In the detection of stx1 and stx2 genes, 14 of isolates (14/20) were positive to stx1

and stx2. Whereas, 5 isolates (5/20) were positive to stx1 and 1 isolate (1/20) was

negative by either of stx1 or stx2 genes. Using RAPD-PCR analysis, two

oligonucleotides were chosen because they yielded clearly and reproducible band. There

were OPAR8 (5’-TGGGGCTGTC-3’) and OPAR20 (5’-ACGGCAAGGA-3’).

Subsequently, all 20 isolates of E.coli O157:H7 were subtyped using OPAR8 and

OPAR20. Primer OPAR8 produced 8 RAPD-PCR fingerprinting namely P1 to P11.

Whereas, OPAR20 produced 16 RAPD-PCR fingerprinting of Q1-Q18. Combination of

two primers was analyzed using Unweighted Pair Group Method with Arithmetic mean

(UPGMA). Dendogram performed from cluster analysis showed that all the 20 isolates of

E.coli O157:H7 differentiated into 20 individual isolates which may suggest the high

level of local geographical genetic variation.

ABSTRAK

Dua puluh (n=20) isolat Escherichia coli O157:H7 yang diasingkan daripada

daging lembu telah dikaji untuk mengesan gen toksin Shiga 1 dan 2 (stx1 dan stx2) dan

pencirian dengan menggunakan cap jari Amplifikasi Polimorfik Asid Deoksiribonukleik-

tindakbalas rantaian polimerase (RAPD-PCR). Kesemua isolat telah di perolehi dari

Makmal Sains Makanan dan Bioteknologi, Universiti Putra, Serdang, Selangor.

Dalam pengesanan untuk gen stx1 dan stx2, 14 isolat (14/20) positif terhadap

stx1 dan stx2. Manakala, 5 isolat (5/20) positif terhadap stx1 dan 1 isolat (1/20) negatif

terhadap stx1 atau stx2 gen. Menggunakan analisis RAPD-PCR, 2 oligonukleotida telah

dipilih kerana menghasilkan jalur yang jelas dan keputusan berulang. Mereka adalah

OPAR8 (5’-TGGGGCTGTC-3’) dan OPAR20 (5’-ACGGCAAGGA-3’). Oleh itu,

kesemua 20 isolat E.coli O157:H7 telah disubtaipkan dengan menggunakan OPAR8 dan

OPAR20. Primer OPAR8 menghasilkan 8 cap jari RAPD-PCR dinamakan P1 hingga

P11. Manakala, OPAR20 menghasilkan 16 cap jari RAPD-PCR dinamakan Q1 hingga

Q18. Gabungan dua primer dilakukan dengan menggunakan Kaedah Kumpulan Pasangan

Tanpa penimbang dengan Keertian Arithmatik (UPGMA). Paparan dendrogram dari

analisis kelompok menunjukkan kesemua isolat E.coli O157:H7 boleh dibezakan kepada

20 isolat individu di mana mencadangkan paras tinggi genetik variasi di geografi

tempatan.

ACKNOWLEDGEMENT Assalamualaikum.

Praise to Allah for the strength, wisdom and for the unconditional love that I got

that, I am able to pursue and complete my Degree of Master of Biotechnology. Without

that, I would never have the perseverance to make it until the end.

First of all, I would like to express my utmost appreciation to my supervisor, Dr

Noraida bt Ismail and to my co-supervisor, Dr. Sahilah bt Abdul Mutalib, for their

patience, kindness, guidance and useful advice given throughout this thesis project. Her

wisdom and encouragement has inspired us to work harder, to make this thesis a special,

successful and memorable one.

A million thanks to Prof. Dr. Yaakob Che Man, who is the Director of Halal

Products Research Institute, Universiti Putra Malaysia, for the facilities and helps in

making this, project a successful one

My thanks also go to my coursemates, Kak Suraya, Nadzimah, Faiza, Kak Lina

and Lani who worked together in the Food Biotechnology laboratory for the happy

moment that we shared in the lab. I also would like to express my deepest gratitude to

Amirul Asyraf bin Ismail for his support and guidance. Without your endless love,

support and encouragement, I could never have finished this thesis. Thank you for always

being there for me.

Finally, I would like to dedicate the thesis to my family; to my beloved mom, Ijal,

Aidah, Ajip, Ika, (my loving sisters and brothers) and my dearest relatives, my deepest

appreciation of love and thanks goes to all of you. I can never express my gratefulness

for everything that you have brought into my life. I love you all and this I can do for you.

DEDICACIÓN CON AMOR

-POR MI MADRE Y MI PADRE (1924-2004) POR CONFIANZA EN MI Y MI FAMILIA. ESTE ES MI REGALITOS PARA TODOS.

TABLES OF CONTENTS CHAPTER PAGE

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

LIST OF TABLES viii

LIST OF FIGURES

ix

LIST OF ABBREVIATIONS & SYMBOLS x

CHAPTER ONE INTRODUCTION 1

CHAPTER TWO LITERATURE REVIEW

2.1 Classification of Escherichia coli 4

2.2 Morphology & Colony Characteristics 8

2.3 Biochemical profile 10

2.4 Risk assessment acquiring Escherichia coli 11

2.5 Molecular Epidemiology & Bacterial Typing 13

2.6 Polymerase Chain Reaction (PCR) – Based Methods 14

2.6.1 Random Amplified Polymorphic DNA (RAPD–PCR) 14

2.6.2 Repetitive Element PCR Fingerprinting (REP–PCR)

15

2.6.3 Restricted Fragment Length Polymorphic (RFLP–PCR) 16

2.6.4 Amplified Restriction Fragment Polymorphic 16 (AFLP – PCR)

2.6.5 PCR Ribotyping 17

2.7 Pulsed Field Gel Electrophoresis ( PFGE ) 17

CHAPTER THREE MATERIALS & METHODS

3.1 Bacterial Strains 18

3.2 Preparation of Whole-cell DNA for PCR and RAPD -PCR

19

3.3 Detection of Shiga toxin (stx1 and stx2)genes using multiplex PCR

19

3.4 RAPD-PCR Fingerprinting 20

3.5 Data Analysis 21

CHAPTER FOUR RESULTS

4.1 Detection of Shiga toxin (stx1 and stx2)genes 23

4.2 RAPD-PCR Fingerprinting 24

4.3 Dendogram analysis using Unweighted Pair Group

29

Method with Arithmetic mean (UPGMA)

CHAPTER FIVE DISCUSSION and CONCLUSION 34

CHAPTER SIX REFERENCES 35

APPENDICES

A: GENERAL MEDIA AND SOLUTIONS

B: SOLUTION FOR PCR

LIST OF TABLES

Table Page 2.1 Serotype of Escherichia coli associated with human diarrhea 8 2.2 Comparison between biochemical characteristics of Escherichia coli 10 O157:H7 and Escherichia coli 3.1 Number isolates of E.coli O157:H7 18 3.2 The 2 pairs of primer (Shiga Toxin) with their sequences 20 3.3 The ten oligonucleotides of RAPD-primers with their sequences 21 4.1 Genotypic diversity of Escherichia coli using RAPD-PCR 28

LIST OF FIGURES Figure Page 2.1 The six different pathogenic schemes of diarrheagenic Escherichia .coli. 7

Each of the six categories has unique features in their interaction with eukaryotic cells. 4.1 Representative profile of multiplex detection with 2 pairs of different 24 primers (stx2F, stxR, stx1F and stx1R) of Escherichia coli O157:H7 isolates 4.2 Representative profile of arbitrarily primed-PCR patterns (P1-P4)

26 obtained with primer OPAR 8 of the Escherichia coli O157:H7

isolates. 4.3 Representative profile of arbitrarily primed-PCR patterns (P5-P11) 26 obtained with primer OPAR 8 of the Escherichia coli O157:H7 isolates 4.4 Representative profile of arbitrarily primed-PCR patterns (Q1-Q8)

27 obtained with primer OPAR 20 of the Escherichia coli O157:H7 isolates 4.5 Representative profile of arbitrarily primed-PCR patterns (Q9-Q18) 27 obtained with primer OPAR 20 of the Escherichia coli O157:H7 isolates 4.6 Dendogram generated from the arbitrarily primed-PCR patterns 30

with primer OPAR 8 and OPAR 20 of the Escherichia coli O157:H7 isolates from meat.

ABBREVAITIONS & SYMBOLS Bp Base pair o C Degree celcius/Centigrade DNA Deoxyribonucleic acid dNTPs Dinucleotide(s) triphosphate ds Double stranded et al. Consensus and associates g Gram(s) > Greater than H Hour Kb Kilo base pair µg Microgram(s) µl Microliter(s) µm Micrometer(s) M Molar min Minute mg Milligram(s) mM Millimole(s) ml Milliliter(s) % Per cent Pmol Per mole rmp Rotation per minute UV Ultra violet

CHAPTER ONE

INTRODUCTION

1.1 INTRODUCTION

Enterohaemorrhagic Escherichia coli (EHEC) are collectively, one of the greatest

microbiological challenges to hit the food industry since the scourge of botulism some 80

years ago and are highly infectious in humans (International Life Science Report, 2001).

These organisms can cause illness including very serious illness such as haemorrhagic

colitis and haemolytic uraemic syndrome (HUS). About 5% of cases of haemorrhagic

colitis progress to HUS, in which the case fatality rate is approximately 10%

(Anonymous, 2000).

Outbreaks of infection, generally associated with beef, have been reported in

Australia, Canada, Japan, United States, in various European countries, and in southern

Africa (World Health Organization, 2007). In an outbreak in Japan in 1996, more than

6,300 school children were affected with three deaths (Fukushima et al., 1997). In the

United States in 1999, more than 1000 people (11 children with HUS and two deaths

recorded) were believed to have been infected through contaminated water (Charatan,

1999).

Five types of pathogenic E.coli are categorized according to their differences in

epidemiology, different interaction with intestinal mucosa, clinical syndromes of disease

caused and distinct O: H serotypes. There are Enterohaemorrhagic (EHEC),

Enterotoxigenic (ETEC), Enteropathogenic (EPEC), Enteroinvassive (EIEC), and

Enteroaggregative/Enteroadherent (EnaggEC) (Kauffman, 1947).

Food products associated with E. coli outbreaks include raw ground beef (Institute

of Medicine of the National Academies, 2002),

It has been shown that E.coli O157:H7 had created a major problem in many

developing countries such as United States, Japan and Europe. In Malaysia, no report has

been obtained for E.coli O157:H7 outbreak. Nevertheless, from the data collected in

Malaysia, it shows that the incidence of E.coli O157:H7 isolated from animals and food

sources has increased noticeably in recent years especially from raw ground meat (Son et

al., 1997). The incidence of E.coli O157:H7 in meat samples in local market indicated the

raw seed sprouts or spinach, (Sabin,

2007), raw milk, unpasteurized juice, and foods contaminated by infected food workers

via fecal-oral route (Food Safety, 2006). Transmission of pathogenic E. coli often occurs

via fecal-oral transmission (Sabin, 2007). Dairy and beef cattle are primary reservoirs of

E. coli O157:H7 and they can carry it asymptomatically and shed it in their faeces (Bach

et al., 2002).

An outbreak caused by E.coli O157:H7 requires rapid isolation and identification

in order to identify the reservoir or the vector. Therefore, improved epidemiological

surveillance is essential and bacterial typing is of great value in epidemiologic

investigations. Various molecular typing have been developed; includes plasmid

profiling, PCR-ribotyping, IS 200 profiles, pulsed field gel electrophoresis (PFGE),

random amplified polymorphic DNA(RAPD) and repetitive element PCR fingerprinting

(Gillings and Holley, 1997; Rodrigue et al., 1995; Stubbs et al., 1994; Olsen et al., 1993;

Lagotolla et al., 1996).

exposures of these bacteria to public. It can be serve as a vehicle for the transmission of

the disease to man.

The aim of the project was to study the genetic diversity of E.coli O157:H7

species isolated from meat samples using molecular approaches of RAPD-PCR. In this

study, in view of emerging important of E.coli O157:H7, the prevalence of E.coli

O157:H7 in food samples especially beef will be conducted to determine the presence of

Shiga-toxin 1 (stx1) and Shiga-toxin 2 (stx2) genes in E.coli O157:H7 and to subtype the

Escherichia coli 0157:H7 isolates using randomly RAPD-PCR. This would allow

discrimination below the species level. Besides that the data obtained will provide some

baseline information about the molecular epidemiology of E.coli O157:H7 in Malaysia.

CHAPTER TWO

LITERATURE

REVIEW

2.1 CLASIFICATION OF Escherichia coli

There are five distinct groups of E.coli cause gastrointestinal illness, which are

Enterotoxigenic (ETEC), Enteropathogenic, (EPEC), Enteroinvassive (EIEC),

Enterohaemorrhagic, (EHEC) and Enteroaggregative/Enteroadherent (EnaggEC) (Nataro

and Kaper 1998). However there is one group which is Diffuse Adherent E.coli (DAEC)

cannot be categorized due to the controversial of its pathogenicity (Tamura et al., 1996).

The clinical syndrome, made discovery of a new E.coli phenotype known as

EHEC (Karmali, 1989; Mead and Griffin, 1998). EHEC produce one or both of two

cytotoxin, VT1 and VT2. Gamage et al. (2003) reported that when both toxins are

produced by the same strains, VT1 predominates in cell lysates and VT2 is more active

toxin in supernates (Strocbine et al., 1986). Although this virulence factor (VT) found in

many different serotypes, only a few well characterized bioserotypes and clones (Dorn et

al., 1993) namely 0157:H7 and O26:H11 are the most important host for the verotoxin

phage(s). EHEC strains have become prominent in recent years as causes of hemorrhagic

enteritis and the hemolytic uremic syndrome (Coia, 1998)

Hemolytic Uremic Syndrome (HUS), first described in 1955, is a public human

disease caused by EHEC (International Life Science Report, 2001). E. coli O157:H7 is

responsible for over 90% of the cases of HUS that develop in North America. The

essence of the syndrome is described by its three central features: destruction of red blood

cells (hemolytic anemia), destruction of platelets (those blood cells responsible for

clotting, resulting in low platelet counts, or thrombocytopenia), and acute renal failure

(Karmali et al., 1989). Thrombotic thrombocytopenic purpura (TTP) is a clinical

syndrome defined by the presence of thrombocytopenia and microangiopathic hemolytic

anemia. TTP frequently leads to neurological and renal impairment in patients (World

Health Organization, 2005). Hemorrhagic colitis is an acute disease

EIEC closely resemble Shigella (Kopecko, 1994) and causes diarrhea in humans

(DuPont et al., 1971). EIEC are non-motile and anaerogenic (Peter and Weagent, 2002).

The mechanism is uncertain, but it may attribute to a distinct plasmid encoded

enterotoxin (Robin et al., 1999). PCR assay was reported with primer derived from ial is

and a type of

gastroenteritis which produce a toxin that causes bloody diarrhea (Griffin and Tauxe,

1991).

ETEC strains cause diarrhea in infants and young children in tropical developing

countries, in travelers and contaminated food and water (Public Health, 2005). ETEC

produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT) (Fegan and

Desmarchelier, 2002). LT will influence the metabolism of prostaglandins and stimuli

neurotransmitters of the enteric nervous system (Beuble and Schuligoi, 2000; Mourad

and Nassar, 2000). Sculling mouse, radioimmunoassay and enzyme-linked

immunosorbent assay (ELISA) was the standard test to detect ST (Gianella and Vijg,

1981; Cryan, 1990). Recently, PCR assay has been developed, which are quite sensitive

and specific when used directly on clinical samples or on isolated bacterial colonies

(Lang et al., 2004; James and James 1998; Schultsz et al., 1994; Tornieporth et al.,

1997).

effective in multiplex PCR system to identify EIEC strains simultaneously with other

E.coli categories (James and James, 1998; Frankel et al., 1998).

EPEC causes enteric disease (Larry, 1985). Ability to produce A/E

(attaching/effacing) lesions and intimate adhere of bacteria to the intestinal epithelium is

the main mechanism of EPEC pathogenesis (Roger et al., 2004). There are two

approaches to detection of EPEC in the laboratory; phenotypic and genotypic. The

phenotypic approaches require the use of cell cultures and fluorescence microscopy.

Whereas the genotypic method requires the use of DNA hybridization of PCR, DNA

probes. Makino et al., (1994) suggested that PCR using eae- and bfp-specific primers and

HEp-2 adherence assay are useful to identify EPEC.

Konowalchuk and co-researchers 1977 reported VTEC produced cytotoxic for

vero cells and include representatives of both EPEC and EHEC (Pollard et al., 1990).

Verotoxin (VT) and Shiga-like toxin (SLT) are synonymous (Carter et al., 1987). Toxin

neutralization assay is sensitive and specific technique (immunochromatography and

latex agglutination) (Cermelli et al., 2002) suffer from limited sensitivity and a lack of

specificity (Watanabe et al., 1996). Simple and rapid detection of characteristic (toxin

production and type serotype) is possible in a single dipsticks test device, directly from a

food enrichment culture (Aldus et al., 2003).

Source: Nataro and Kaper (1998)

Figure 2.1: The six different pathogenic schemes of diarrheagenic E. coli. Each of the six

categories has unique features in their interaction with eukaryotic cells

2.2 MORPHOLOGY AND COLONY CHARACTERISTICS

E.coli was discovered in 1885 by Theodor Escherich, a German pediatrician and

bacteriologist (Feng and Weagent, 2002). A specific combination of O and H antigens

defines the serotype of an isolates but a complete O (somatic): H (flagellar): K (capsular)

serotype gives better identification for clonal relationships between strains (Orskov and

Orskov, 1992; Whittam et al., 1993).

Kauffman (1947) described that the O: H system (Table 2.1) was established in

order to differentiate E.coli on the basics of liposaccaharide O, flagellar H and

polysaccharide K antigen.

Table 2.1: Serotypes of Escherichia coli associated with human diarrhea

Enterotoxigenic

(ETEC)

Enteropathogenic

(EPEC)

Enteroinvassive

(EIEC)

Enterohaemorrhagic

(EHEC)

06 026 028ac 026

08 055 029 0111

015 086 0124 0157

020 0111 0136

025 0119 01432

027 0125 0144

063 0126 0152

078 0127 0164

080 0128ab 0167

Source: Levine, 1987

E. coli O157:H7 is a Gram negative bacillus straight rod bacterium. It is

distinguished by their inability to ferment sorbitol rapidly and by their lack of production

of glucuronidase (Hayes et al., 1995, Doyle and Schoeni, 1984). The size of E.coli

O157:H7 is 1.1-1.5 by 2.0-6.0 μm (living) and 0.4-0.7 by 1.0-3.0μm (dried and stained)

(Orskov, 1974). E.coli O157:H7 is motile by peritrichous flagella. However, there are a

few strains, which are non-motile. It is anaerobe and facultative anaerobe bacteria. The

temperature range for E.coli O157:H7 growth is 10-400C (optimum 370

E. coli O157:H7 has its ability to produce one or more Shiga toxins (ST) and it is

indistinguishable from Shiga toxin produced by Shigella dysenteriae (Anantharnarayan

and Jayaram, 1996). Shiga toxin 2 is a more divergent molecule, with only 56% amino

acid homology with Shiga toxin 1. Most E. coli O157:H7 strains produce Shiga toxin 2;

the percentage that also produce Shiga toxin 1 ranges from less than 25% in a series from

Europe to greater than 80% in a series from North America (

C). It can be

grown on ordinary media. Colonies are moist, smooth, large, thick or partially translucent

discs. Some strains may occur in mucoid form. Many pathogenic strains are hemolytic on

blood agar. It ferments lactose, glucose, mannitol, maltose and many other sugars.

Slutsker et al., 1997 , Law

and Kelly, 1995 , Mead and Griffin, 1998) . Other factors thought to contribute to the

virulence of E. coli O157:H7 include a virulence plasmid (pO157) and the locus of

enterocyte effacement (LEE). The LEE contains genes for an adhesion molecule (intimin)

2.3 BIOCHEMICAL PROPERTIES FOR Escherichia coli O157:H7

Table 2.2: Comparison between biochemical characteristics of E.coli O157:H7 and Escherichia coli

Biochemical characteristic Escherichia coli Escherichia coli O157:H7 Ornithine decarboxylase + +

Arginine dihydrolase + - Lysine decarboxylase + +

Urease + + L-Arabitol + -

Galacturonate ND + 5-Ketogluconate ND -

Phenol red + + -Glucosidase ND - Mannitol + + Adonitol + -

Palatinose ND - -Glucuronidase ND -

Indole + + N-Acetyl- -glucosaminidase ND -

-Galactosidase + + Glucose + +

Saccharose + + L-Arabinose + + D-Arabitol + + -Glucosidase - +

-Galactosidase + + Trehalose + + Rhamnose + +

Inositol + + Cellobiose + -

Sorbitol + - -Maltosidase ND +

L-Aspartic acid arylamidase ND - Source: Farmer et al., 2002) + : positive reaction - : negative reaction ND : not determined (different reaction given by different strains of a serotype)

2.4 RISK ASSESSMENT OF ACQURING Escherichia coli O157:H7

Risk assessment studies have been conducted to determine the actual risk of

disease and the parameters contribute to the susceptibility of populations to the disease.

Morgan et al. (1993) suggested that low levels of E. coli O157:H7 can play a role in

acquiring immuno-protection and building disease resistance. More research is needed to

quantify the risk in relation to public health, and to determine the approaches to

government regulation.

The outbreak of E.coli O157:H7 have been reported in many developed countries

such as United States, Japan and European (Beuchat 1996b). In Malaysia, the true burden

of infectious diarrhoea disease is largely unknown. In year 2004, around 6,000 cases of

food poisoning were reported (Ministry of Health, 2000). An attempt was made more

than a decade ago to estimate the burden of diarrhoeal disease in children in Malaysia

using a community-based study (Lim, 2007). In this study, based on the 2000 Malaysian

census, which showed a total population of 23.27 million with 33.3% under the age of 15

years, there would be nearly two million episodes of diarrhoea annually among

Malaysian children. However there have been no outbreaks of E.coli O157:H7 reported

in Malaysia (Health Ministry, 1999) but data collected showed an increasing incidence

among Malaysian (Bach et al., 2002). Son et al. (1998) reported that E. coli O157:H7

strains possessing important virulence traits were shown to be distributed at a

considerable frequency in the beef retailed in Malaysia.

In the last two decades, many new pathogens have been shown to be associated

with diarrhoeal diseases. They include Escherichia coli O157:H7 and other

diarrhoeagenic E. coli. Many of these agents are readily transmitted through food and

water (Walterspeil et al., 2003). Because of the manner processed food is now mass

produced and distributed, a single contaminated food source can cause an outbreak

involving many thousands of cases spread over a wide geographical area (Science Daily,

2007).

Laboratory tests to ascertain the cause of the diarrhoeal episode is not often

performed especially in general practice in Malaysia due to cost factors. The lack of a

specific diagnosis can hinder the institution of appropriate therapeutic and preventive

measures. There is a need for improved surveillance systems including syndromic and

laboratory surveillance to enable us to detect outbreaks in a timely manner in order to

take the necessary preventive measures (Son et al., 1997). Due to improvement in socio

economic status of the population and good access to medical facilities, severe

dehydration resulting from diarrhea has become relatively uncommon in Malaysia.

2.5 MOLECULAR EPIDEMIOLOGY AND BACTERIAL TYPING

Molecular epidemiology is based on characterization of bacterial isolates by

phenotypic assays such as biochemical testing, antimicrobial susceptibility testing and

serotyping (Versalovic et al., 1993). Several approaches had been tried to differentiate

different bacterial strains with biochemical, immunological and molecular methods

(Jordens et al., 1995). Biochemical methods are simple but time consuming and less

sensitive where as immunological method are not reveal full variability of bacterial

(Anand, 2001).

Bacterial typing systems for discriminating bacteria below the species level

subtyping from a single species have been based on phenotype, such as serotype, biotype,

phage typing, or antibiogram (susceptibility to one or more antibiotics)(Eisen et al.,

1995). There are two major categories; phenotypic and genotypic. Phenotyping includes

serotyping, bacteriocins, bacteriophages typing, biotyping, and antibiotic resistance

pattern and protein profiles. Whereas, genotyping have the potential of providing more

consistent, reproducible data and are applicable to other species and genera involve direct

DNA-based analysis of chromosome, plasmid and insertion sequences/ transposon (IS).

The popular approaches for characterizing individual strains are found in the practical

application of molecular biology such as Polymerase Chain Reaction, nucleic acid

fingerprinting and DNA sequence analysis (Relman and Persing, 1996).

2.6. POLYMERASE CHAIN REACTION (PCR) – BASED METHODS

The polymerase chain reaction (PCR) is a biochemistry and molecular biology

technique for isolating and exponentially amplifying a fragment or sequence of interest of

DNA in a short time (Joseph et al., 2001). PCR offer speed and simple protocol (Wernars

and Huevelman, 1993). PCR has an enormous impact and variations of PCR-based

techniques as describe below:

2.6.1 RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD-PCR) or ARBITRARILY PRIMED PCR (AP-PCR)

RAPD-PCR is a technique of utilizing arbitrary oligonucleotides to prime DNA

synthesis at low annealing temperature to divulge genomic diversity. RAPD is great

power and general applicability because does not require any specific knowledge of DNA

sequence of the target organism (Versalovic et al., 2002).

RAPD reaction mixture creates several arbitrary nucleotide sequence, short

primers, and then proceeds with the PCR using to amplify. During the annealing process,

the primer which sequence are not directed to any known genetic locus attaches to the

target DNA at random sites with a complementary sequence to permit initiation of

polymerization. If such sites are located, kilo bases of each other on opposite DNA strand

and in the proper orientation, then amplification of the intervening fragment will occur

(William et al., 1990).

RAPD has been applied to microorganisms such as Camphylobacter jejuni (Owen

and Hernandez, 1993), Listeria monocytogenes (Mazurier and Wernars, 1992) and

Pseudomonas fragi (Tanaka et al., 1993). In Escherichia coli, many reports have been

published regarding RAPD as a typing method such as in E. coli O157:H7 (Son et al.,

1998)

2.6.2 REPETITIVE ELEMENT PCR FINGERPRINTING (REP – PCR)

In rep-PCR technique, primers are designed complementary to bacterial

interspersed repetitive sequences (Versalovic et al., 2002). It is enable a rational approach

to primer design and a limited set of primers can be used with virtually any bacteria. The

primers are longer in length (18 to 22 mers) and, therefore, higher annealing temperatures

that enable greater stringency variation of the PCR can be used. Rep-PCR has multiple

applications in molecular epidemiology; including, Staphylococcus, Xanthomonas and

Pseudomonas (Gillings and Holey, 1997). This technique also widely applied to monitol

enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) (Dalla et al., 1998).

2.6.3 PCR–RESTRICTED FRAGMENT LENGTH POLYMORPHISM (PCR RFLP)

This method involves amplifying a known sequence, cleaving the amplicon with

the suitable restriction endonucleases and comparing the restricted fragments of amplified

DNA from different strains. PCR-RFLP analysis has been applied most widely to genes

encoding toxins or other structural/functional molecules from Camphylobacter (Owen et

al., 1993; Burnens et al., 1995) and Vibrio (Faruque et al., 1994; Suthienkul et al., 1996).

In E.coli, report has been published regarding RFLP as a typing method (Buysse et al.,

1995).

2.6.4 PCR-AMPLIFIED FRAGMENT LENGHT POLYMORPHISM (AFLP – PCR)

AFLP-PCR is a highly sensitive, higher reproducible and resolution method,

described by Vos and Zabeau in 1993. In AFLP analysis, bacterial genomic DNA is

digested with restriction enzymes, ligated to adapters and a subset of DNA fragments are

amplified using primers containing 16 adapter defined sequences with one additional

arbitrary nucleotide (Lin et al., 1996).

AFLP has become widely used for the identification of genetic variation in strains

or closely related species of bacteria such as Vibrio cholerae (Jiang et al., 2000),

Xanthomonas spp.(Rademaker, et al., 2003) and E. coli (Guan et al., 2002, Arnold et al.,

1999)

2.6.5 PCR RIBOTYPING

PCR ribotyping is based on the amplification of the spacer sequences between the

genes coding for 16S and 23S rRNA (Kostman et al., 1992). The rRNA loci are present

in 2 to 11 copies on the chromosomes of the most bacterial species. Whereas, a high

degree of sequence homology exist for rRNA genes, the intergenic spacer regions show

intensive sequence and length variation which can be used to characterize bacteria at the

genus (Jensen et al., 1993), species (Dolzani et al., 2004; Jensen et al., 1993) and

subspecies level (Dolzani et al., 2004; Kostman et al., 1992). This method has been

successfully applied to Salmonella spp. by Jensen et al., (1993) and Lagatolla et al.,

(1996). This technique also had been used by E. coli (Mehwish et al., 2007).

2.7 PULSED FIELD GEL ELECTROPHORESIS ( PFGE )

Pulsed Field Gel Electrophoresis (commonly abbreviated as PFGE) described by

Chu et al. (1986) is a method for separating large DNA molecules. The ability of PFGE

to separate large DNA fragments as large as 1.2 megabases (Zhang et al., 2000), provides

the opportunity to study complete microbial genomes. The use of PFGE has been applied

to other analysis such as taxonomy and bacterial genome mapping (Rompling et al.,

1992). Subtyping has made it easier to discriminate among strains of Listeria

monocytgenes, Salmonella weltevreden (Sahilah et al., 2001) and thus to link

environmental or food isolates with clinical infections. PFGE has been reported in E. coli

O157:H7 by (Son et al., 2001).

CHAPTER THREE MATERIALS

&

METHODS

3.1 Bacterial strains

The Escherichia coli O157:H7 isolates were obtained from the laboratory of Halal

Product Research Institute, Infoport Centre, Universiti Putra Malaysia (UPM), 43400,

Serdang, Selangor Darul Ehsan.

E.coli

O157:H7

Strains no.

1 EC1

2 EC2

3 EC3

4 EC4

5 EC5

6 EC6

7 EC7

8 EC8

9 EC9

10 EC10

11 EC11

12 EC12

13 EC13

14 EC14

15 EC15

16 EC16

17 EC17

18 EC18

19 EC19

20 EC20

Table 3.1: Number of isolates of E. coli O157:H7

3.2 Preparation of whole-cell DNA for PCR and RAPD-PCR fingerprinting

DNA extraction was done by using boiling method. The cells were grown in

1.5 ml of Lauria-Bertani (LB)(Tryptone, 4.0 g/L, Yeast Extract, 5.0g/L, Sodium chloride,

10.0 g/L) at 35 °C for 20 h were harvested and centrifuged at 12,000 rpm for 1 min. The

supernatant was discarded. The pellet was then washed with 1.0 ml sterile distilled water

and vortex. Then, it was boiled at 97 oC for 10 min and immediately was frozen at -20 o

The multiplex detection gene and PCR condition were optimized using

recommendations reported previously by Fode-Vaughan et al. (2003) using 20 strains and

2 pairs of primers were used which stx2F(5’-TTCTTCGGTATCCTATTCCC-3’), stx2R

(5’-ATGCATCTCTGGTCATTGTA-3’) , stx1F(5’-CAGTTAATGTGGTGGCGAAG-

3’), and stx1R (5’-CTGTCACAGTAACAACCGT-3’) designed by Olsvik and

Strockbine (1993). The detection assay was performed in a 25 μl volume containing

5.0 μl of 5 × PCR buffer (100 mmol l

C

for 10 min. The tube was centrifuged at 10,000 rpm for 3 min. The supernatant was used

as a template.

3.3 Multiplex PCR for detection of Shiga-toxin 1 and 2 (stx1and stx2) genes

−1 Tris–HCl, 35 mmol l−1 MgCl2, 750 mmol l−1

KCl, pH 8.8), 1.0 μl of 10 mmol l−1 dNTPs (Promega, Madison, USA) 1.0 μl of

10 pmol μl−1 primer stx2F, stx2R, stx1F and stx1R, 0.2 μl of 1.0 units of Taq DNA

polymerase (Promega, Madison, USA), 12.30 μl of sterile ultrapure deionized water and

2.0 μl of 100 ng DNA template. A negative-DNA control was performed by adding 1 μl

of sterile ultrapure deionized water. Amplification was performed in personal Eppendorf

thermal-cycler (Eppendorf, Germany) with a temperature program consisting of the

initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for

2 min, annealing for 1 min at 35 °C and polymerization at 72 °C for 2 min. Final

elongation was at 72 °C for 10 min.

3.4 Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR) fingerprinting

The discriminatory ability and stability of RAPD-PCR fingerprinting were tested

in a preliminary study against a panel of 4 different bacterial strains of E. coli O157:H7

with 10-mer random primers (Promega, USA). Primer OPAR 8 and OPAR 20 showed

the greatest stability and discriminatory ability among the E. coli O157:H7 isolates, and

was therefore used in this study. The RAPD-PCR fingerprinting assay was performed in

a 25 μl volume containing 2.5 μl of 10× PCR buffer (100 mmol l−1 Tris–HCl,

35 mmol l−1 MgCl2, 750 mmol l−1 KCl, pH 8.8), 0.5 μl of 10 mmol l−1 dNTPs (Promega,

Madison, USA) 1.0 μl of 10 pmol μl−1 primer OPAR 8 & OPAR 20, 0.3 μl of 1.5 units of

Taq DNA polymerase (Promega, Madison, USA), 18.95 μl of sterile ultrapure deionized

water and 1 μl of 100 ng DNA template. A negative-DNA control was performed by

adding 1 μl of sterile ultrapure deionized water. Amplification was performed in personal

Eppendorf thermal-cycler (Eppendorf, Germany) with a temperature program consisting

of the initial denaturation at 94 °C for 5 min followed by 45 cycles of denaturation at

94 °C for 1 min, annealing for 1 min at 35 °C and polymerization at 72 °C for 2 min.

Final elongation was at 72 °C for 7 min. The amplification products were analyzed by

electrophoresis in a 1.0% agarose in 0.5X TBE (0.1 M Tris, 0.1 M Boric acid, 0.1 mM

EDTA) at 90 V for 40 minutes. Gels were stained with ethidium bromide. The amplified

fragments were visualized with UV transilluminator (Syngene, USA). The 100bp DNA

ladder (Promega, USA) was used as a DNA size marker.

3.5 Data analysis

The banding patterns of individuals’ strains were scored based on the presence or

absence of the bands. The banding patterns scored were analyzed using the RAPDistance

Package Software (version 1.04) program. The scoring was made in the form of binary

code with the score ‘1’ indicating presence of band and ‘0’ the absence of band. The data

obtained were recorded and entered in the software CorelDRAW Graphic Suite X3 where

a dendogram was produced for further analysis. Clustering was based on the unweighted

pair of group average method (UPGMA) and was performed with the RAPDistance

software.

CHAPTER FOUR

RESULTS

4.1 Detection of Shiga-toxin 1 and 2 (stx1and stx2) genes

In this study, 2 pairs of primer (stx2F, stx2R stx1F and stx1R) were used for

detection and confirmation of E. coli O157:H7 as described by Fode-Vaughan et al.

(2003). Two pair of primers were used for multiplex PCR analysis of twenty (n=20)

isolates to detect stx gene. Most clinical signs of disease arise as a consequence of the

production of Shiga toxin stx1, stx2 or combinations of these toxins (Griffin et al., 1991).

The average sizes for the two primers are 180 bp and 255 bp, respectively (Paton and

Paton (2002); Genhua et al., 2002).

Among the twenty one samples, 14 isolates (14/20) were positive to stx1 and stx2,

indicated by formation of 2 bands in a range of molecular weight of 180 bp – 255 bp.

Whereas, 5 isolates (5/20); (EC2, EC7, EC12, EC14 and EC17) were positive to stx1,

indicated by formation of only one band in a arrange of molecular weight of 180 bp while

single isolate (1/20); (EC8) was lacked of both stx1 and stx2 which was indicated by no

formation of band (Figure 4.1).

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Figure 4.1: The electrophoresis patterns of multiplex detection of Escherichia coli O157:H7 isolates electrophoresed on 1.0 % agarose gel. M, Molecular weight sizes (base pairs, bp) are indicated by numbers on the left; lane 1-20: EC1, EC2, EC3, EC4, EC5, EC6, EC7, EC8, EC9, EC10, EC11, EC12, EC13, EC14, EC15, EC16, EC17, EC18, EC19, EC20. Lane 21: Positive control (EC 12).

4.2 RAPD-PCR fingerprinting

A total of 10-mer of different oligonucleotide primers was used for RAPD-PCR

analysis of a subset of 4 isolates to detect polymorphism within Escherichia coli

O157:H7. The two primers produced a clear pattern and were used to analyze the whole

set of 20 E.coli O157:H7strains.

Twenty (n=20) strains of Escherichia coli O157:H7 were used for RAPD-PCR

analysis with OPAR8 and OPAR20. RAPD-PCR fingerprinting of E.coli O157:H7

obtained with primer OPAR8 represented by the Figure 4.2 and 4.3. The possible number

of RAPD-PCR fingerprinting was estimated on the basis of changes in one or more clear

bands or band sizes. Eleven (n=11) RAPD-PCR fingerprinting (P1-P11) were apparent

bp

1,517

500

100

from primer OPAR8. The number of RAPD bands produced for a given primer ranged

from 1 to 7, with molecular sizes ranging from 0.1 to more than 2.0 kb with some of the

bands appeared weak. However, no band produced with isolates EC1 and EC8 (Figure

4.3) with primer OPAR8.

The RAPD-PCR fingerprinting of E.coli O157:H7 strains obtained from primer

OPAR20 is shown in Figure 4 and 5. Eighteen (n=18) RAPD-PCR fingerprinting (Q1-

Q18) were obtained from primer OPAR20. The number of RAPD bands produced for a

given primer ranged from 3 to 17, with molecular sizes ranging from 0.1 to more than 2.0

kb. Combination of both primers allowed the all E. coli O157:H7 differentiated into 20

genome types (Table 4.1).

M 1 2 3 4 5 6 7 8 9 10 11

Fingerprintings:

ND P1 P1 P2 P3 P3 P1 ND P4 P2 Figure 4.2: RAPD-PCR fingerprinting (P1-P4) of Escherichia coli O157:H7 isolates obtained with primer OPAR8 electrophoresed on 1.0% agarose gel. Lane M: 100 bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC1, EC2, EC3, EC4, EC5, EC6, EC7, EC8, EC9, EC10; Lane 11: Negative control

M 1 2 3 4 5 6 7 8 9 10 11

Fingerprintings:

P5 P6 P7 P8 P6 P9 P7 P10 P11 P8 Figure 4.3: RAPD-PCR fingerprinting (P5-P11) of Escherichia coli O157:H7 isolates obtained with primer OPAR8 electrophoresed on 1.0% agarose gel. Lane M: 100bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC11, EC12, EC13, EC14, EC15, EC16, EC17, EC18, EC19, EC20; Lane 11: Negative control.

1,517

500

100

100

500

1,517

bp

bp

M 1 2 3 4 5 6 7 8 9 10 11

Fingerprinting: Q1 Q2 Q3 Q4 Q4 Q5 Q6 Q7 Q1 Q8

Figure 4.4: RAPD-PCR fingerprinting (Q1-Q8) of Escherichia coli O157:H7 isolates obtained with primer OPAR20 electrophoresed on 1.0% agarose gel. Lane M: 100 bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC1, EC2, EC3, EC4, EC5, EC6, EC7, EC8, EC9, EC10; Lane 11: Negative control.

M 1 2 3 4 5 6 7 8 9 10 11

Fingerprinting:

Q9 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18

Figure 4.5: RAPD-PCR fingerprinting (Q9-Q18) of Escherichia coli O157:H7 isolates obtained with primer OPAR20 electrophoresed on 1.0% agarose gel. Lane M: 100bp DNA ladder (molecular weight in base pair, bp); lane 1-10: EC11, EC12, EC13, EC14, EC15, EC16, EC17, EC18, EC19, EC20; Lane 11: Negative control

1,517

500

100

1,517

500

100

bp

bp

Table 4.1: Genotypic diversity of Escherichia coli using random amplified polymorphic DNA-PCR (RAPD-PCR).

Strains no. RAPD-PCR profiles for primer Genome types

OPAR8 OPAR20

EC1 ND Q1 1

EC2 P1 Q2 2

EC3 P1 Q3 3

EC4 P2 Q4 4

EC5 P3 Q4 5

EC6 P3 Q5 6

EC7 P1 Q6 7

EC8 ND Q7 8

EC9 P4 Q1 9

EC10 P2 Q8 10

EC11 P5 Q9 11

EC12 P6 Q10 12

EC13 P7 Q11 13

EC14 P8 Q12 14

EC15 P6 Q13 15

EC16 P9 Q14 16

EC17 P7 Q15 17

EC18 P10 Q16 18

EC19 P11 Q117 19

EC20 P8 Q18 20

ND-Not detected

4.3 Combination of two primer using Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis

Combination of two primers, OPAR8 and OPAR20 was analysed using

Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis. Figure

4.6 showed the combination of RAPD-PCR fingerprinting of E.coli O157:H7

obtained from primer OPAR8 and OPAR20, respectively. Dendogram performed

twenty E.coli O157:H7 strains into 2 major clusters. Cluster A contained 2 sub cluster

which are sub cluster I and sub cluster II. Sub cluster I has 6 strains of E. coli

O157:H7 which are EC18, EC14, EC5, EC9, EC11 and EC8. Sub cluster II contained

7 strains which are EC12, EC15, EC16, EC19, EC7, EC4 and EC10. Cluster B

divided into one sub cluster which is sub cluster III. It contained 7 strains which are

EC1, EC2, EC6, EC20, EC17, EC13 and EC3.

Figure 4.6: Dendogram generated from the random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting among 20 beef isolates of the Escherichia coli O157:H7 with primer OPAR 8 and OPAR20.

.1 8 7P 2 7 c

.11 2P 2 2 c (1 )

.1 0 6P 2 4 b (1 )

.0 6 9

.0 8 4

.2 2 9P 2 7 b (2 )

.0 3 3

. 2 4 8P 3 o b (1 )

.3 3P 2 b b (2 ).0 4 2

.0 3 5

.1 9 8P 3 o b (2 )

.1 5 6P 2 4 c (1 )

.0 3 2

. 2 1 8P 2 7 c 2 )

.0 4 3

. 3 2 7P 2 4 c (3 )

.0 9

.2 8 1P 2 4 b (3 )

.0 3 2

.3 4P 2 6 a (1 )

.2 3 7P 2 a b

.1 0 8

.0 3

.3 8P 2 3 a (3 )

.3 6 5P 2 4 a

. 0 6 2

.3 9 6P 2 4 b (2 )

.0 4 1

. 3P 3 o c (3 )

.1 2 3P 2 5 c (2 )

.11 8P 3 o b (3 )

.0 9 3

.3 8 3P 2 4 a (1 )

.0 1 3

.0 3 9

EC18

EC14

EC5

EC9 EC11

EC8

EC12

EC15 EC16

EC19

EC7

EC4

EC1 EC1

EC2

EC6

EC20

EC3

EC17

EC13

CHAPTER FIVE

DISCUSSION

&

CONCLUSION

5.1 DISCUSSION and CONCLUSION

In this study, the detection of stx1 and stx2 genes have been shown among 20 beef

isolates of Escherichia coli O157:H7 were examined. Samples were collected from 3

different wet markets (Location A: EC1-4, Location B: EC5-13 and Location C: EC14-

EC20) in Selangor, Malaysia. Wet markets were chosen due to the unhygienic situation

(Shiklomanov, 2000)

Fourteen (n=14) strains were positive to stx1 and stx2, 5 strains were positive to

stx1 and a single strain was negative by either of stx1 or stx2 genes while a single isolate

was negative either stx1 or stx2. All enterohemorrhagic Escherichia coli (EHEC) strains

cause serious disease in humans and possess at least one Shiga-like toxin (stx1 or stx2)

gene (Jothikumar and Griffiths, 2002). The detection of Shiga-like toxins is very useful

for the identification of EHEC and Non-EHEC strains were negative for both stx1 and

stx2. The stx1 and stx 2 primers gave negative results from other bacteria tested,

including Listeria monocytogenes, Listeria grayii, Listeria ivanovii, Salmonella enterica

serovar Typhimurium var. Copenhagen PT 10 SA, S. enterica serovar Enteritidis,

Shigella sonnei, Yersinia enterocolitica, and Proteus vulgaris (Jothikumar and Griffiths,

2002). In this work, EC8 showed negative result to stx1 and stx2 primers which clearly

indicated the EC8 did not belong to the EHEC E. coli. The primers used are a powerful

primer to amplify stx1 and stx2 sequences in pathogenic EHEC E. coli and able to

distinguish among nonpathogenic E. coli isolates.

All E. coli isolates were examined for random amplified polymorphic DNA-PCR

(RAPD-PCR). Arbitrarily primed-PCR fingerprinting has been shown in recent years to

be useful for classifying a number of bacterial species (William et al., 1990). In addition,

this method can be used as a diagnostic tool in tracing the source of infections associated

with the consumption of beef meat because results can be obtained in less than 24h after

sampling. DNA products produced in RAPD-PCR fingerprinting analysis depend on the

primer used, with different primers producing different banding patterns. Two 10-mer

arbitrary primers (OPAR8 and OPAR20) were used to generate RAPD-PCR fingerprints.

The selections of those primers were based on good yield bands observed on the agarose

gel. Several isolates failed to produce any bands with the two primers used. This can be

interpreted as the loss of specific sites for primers binding in the chromosomal DNAs of

these isolates since these DNAs gave appropriate bands when they examined using the

primers in reciprocal (Table 4.1).

The OPAR20 primer was more powerful in discriminatory of all E. coli O157:H7

tested which generated 18 fingerprints compared to OPAR8 which was only produced 11

fingerprints. The RAPD-PCR analysis using OPAR8 and OPAR20 in combination

allowed all strains of E. coli O157:H7 differentiated into 20 genome types. Cluster

analysis is used to establish the degree of the relatedness among strains, information that

may useful in epidemiological studies (Soto et al., 1991). Consistent with Unweighted

Pair Group Method with Arithmetic mean (UPGMA) analysis, dendogram performed

from cluster analysis showed that all the 20 isolates of E.coli O157:H7 differentiated into

20 individual isolates which may suggest the high level of local geographical genetic

variation.

CHAPTER SIX

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APPENDICES

APPENDIX A: GENERAL MEDIA AND SOLUTIONS

Maintenance of Bacterial Strains All bacterial isolates were maintained on TSA slant in bijou bottle and kept at 4 oC. All

the stock cultures were subculture for every three months.

Luria-Bertani (LB Broth) Tryptone 4.0 g Yeast Extract 2.0 g Sodium chloride 4.0 g Distilled water 400 ml Sterilize by autoclaving at 121o

Arbitrary primer (RAPD-primer)

C for 15 min. Sequences for RAPD-primers.

G+C contents (%) Sequence (5’ to 3’)

OPAR8 70 TGGGGCTGTC

OPAR10 50 CCATTTACGC

OPAR20 60 ACGGCAAGGA

OPAC 01 60 TCCCAGCAGT

OPAC 02 60 GTCGTCGTCT

OPAC 04 70 ACGGGACCTG

OPAC 05 60 GTTAGTGCGG

OPAC 07 70 GTGGCCGATG

OPAC 09 60 AGAGCGTACC

OPAC 11 70 CCTGGGTCAG

OPAC 12 70 GGCGAGTGTG

APPENDIX B: SOLUTION FOR PCR Taq polymerase and other PCR chemicals (store at -20 oC) Gel Electrophoresis 10x Tris-Borate-EDTA (TBE) Buffer Tris-Base 108.0 g Boric acid 55.0 g EDTA 9.3 g Distilled water 1000 ml For routine electrophoresis, 10X TBE were diluted to 0.5 x TBE, with the pH 8.3.Solution can be kept at room temperature. 1.0% Agarose (for RAPD) Agarose 1.0 g 0.5Xtbe Buffer 100ml Heat until the medium boils. Do not overheat Ethidium Bromide solution Ethidium bromide1.0 g Distilled water 100 ml