RAPID DETECTION AND QUANTIFICATION OF Vibrio

RAPID DETECTION AND QUANTIFICATION OF Vibrio parahaemolyticus IN COCKLES

(Anadara granosa) BY USING REAL-TIME PCR ~

Patricia Rowena Anak Mark Baran Saging

QR 81 S6 P314 1012

Bachelor of Science with Honours (Resource Biotechnology)

2012

ampR gl

~ ~

nlt 0 2

Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

Rapid detection and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR

PKHIDMAT MAKLUMAT AKADEMIK

PATRICIA ROWENA ANAKMARK BARAN SAGING (24823)

A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARAW AK

2012

ACKNOWLEDGEMENTS

First and foremost I would like to thank God the Almighty for listening to my

prayers upon the completion of this Final Year Project A greatest appreciation and heartfelt

gratitude also goes to my respected supervisor Dr Lesley Maurice Bilung and to my coshy

supervisor Dr Micky Vincent for their endless guidance support and advice throughout

conducting this project

Furthermore I dedicate many thanks to the post-graduate students from

Microbiology Laboratory Velnetti Linang Christy and Zena for their help concern and

suggestion on laboratory work until final accomplishment of this project Besides that I

would like to thank Mr Azis who has assisted in term of technical and material matters Lest

to forget this acknowledgment is also expressed especially to my laboratory mates Sherry

Sim Nur Quraitu Aini Aimi Syazana Dellroy Marlina and Miza for their guidance as well

as for making this project filled with positive atmosphere

Last but not least special thanks to my family and friends for their endless moral

supports and for their word full of compliments throughout -thiS Final Year Project and thesis

preparation

DECLARATION

I hereby declare that the study entitled Rapid detection and quantification of V

parahaemolyticus in cockles (Anadara granosa) by using Real-Time peR is my original

work and that all the sources that I have quoted and referred to have been acknowledged by

means of complete references It has been submitted and shall not be submitted in any form

to any institution or other university

Students signature Date

II

- -~ ~ -------~

PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK

T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENT I

DECLARATION II

LIST OF TABLES

III

VI

VIII

LIST OF FIGURES VII

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

XABSTRACT

XABSTRAK

CHAPTER 1 INTRODUCTION

CHAPTER 2 LITERA TURE REVIEWS 3

21 Vibrio Genus 3

22 Vibrio parahaemolyticus 3

23 Cockles 4

24 Route of Transmission of V par~haemolyticus 5

525 Clinical Manifestation of V parahaemolyticus

626 Virulence Properties of V parahaemolyticus

727 Isolation and Identification

728 Real-Time Polymerase Chain Reaction (RT-PCR)

9CHAPTER 3 MA TERIALS AND METHODS

31 Sample Collection 9

32 Sample Preparation and Enrichment of Bacterial 9 Sample

1033 Genomic DNA Preparation

III

J

34 Selective Isolation and Detection of 10 V parahaemolyticus

35 Polymerase Chain Reaction (PCR) 11

36 Agarose Gel Electrophoresis 12

37 Sensitivity Study with Pure Culture for Standard 12 Curve

371 Dilution and Bacterial cell count 12

372 Bacterial cell count by Hemocytometer l3

14373 Spiking of Pure Culture into Raw Fish

1438 Specificity Study using Specific PCR

1539 Real-Time PCR Assay

40 RESULTS

1741 Bacterial Identification

1942 Sensitivity Studies of Pure Culture of V parahaemolyticus

421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking

Purpose - ~

20422 Vibrio parahaemolyticus Cell Count by Hemocytometer

423 Spiking and Dilution Samples for Standard 21 Curve

43 Specificity Study using Specific PCR 23

44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples

2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES

Table 1 PCR parameters

Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)

Table 3 Bacterial strains for specific PCR

Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)

Table 5 RT-PCR reagent component

Table 6 Raw data for spiking samples with standard deviation value for standard curve

Table 7 Raw data for initial concentration for spiking samples

Table 8 Raw data for serial dilution samples with standard deviation value for standard curve

Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page

Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate

Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio

Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control

Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification

Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments

Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments

Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)

Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature

Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct

VII


APW Alkaline Peptone Water

ATCC American Type Culture Collection

em centimetre

Ct Threshold cycle

DNA Deoxyribonucleic acid

dNTPs deoxynucleoside triphosphates

EDTA Ethylenediamine tetra-acetic acid

epsM extracellular secretion protein

EtBr Ethidium bromide

g gram

kbp kilo base pair

LB Luria Bertani

m meter

MgCh magnesium chloride

min minute(s)

mL mililiter

mM milimolar

NaCI sodium chloride

run nanometer

PCR Polymerase Chain Reaction

pH potential hydrogen

(shy correlation coefficient

rpm revoluhon per minute

VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC

Real-Time Polymerase Chain Reaction

second(s)

Tris-Borate-EDTA

Thiosulphate-Citrate-Bile Salts-Sucrose

thermostable direct hemolysin

thermolabile hemolysin

thermostable direct hemolysin related hemolysin

ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan

Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR

Patricia Rowena aDak Mark Baran Saging

Resource Biotechnology Program Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers

Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR

ABSTRAK

Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai

E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna

E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR

x

CHAPTER 1

Introduction

Seafood products of high protein content are a great significant contribution for

human nutritions globally However with the continual wave outbreaks of food-borne

disease related to seafoods consumption associated to pathogenic bacteria this raises

awareness towards the public health concern (Gubala and Proll 2006) The disease may lead

to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio

parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the

world and is reported to be the causative organism of acute food-borne gastroenteritis as well

as septicemia in human by the consumption of raw or undercooked seafood and hence arise

public concerns (Ward and Bej 2006)

In this paper the aim of study is to determine the occurrence of etiological V

parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood

preferred in local cooking ingredients by the human populations in Malaysia Samples were

collected randomly from different locations of wet markets in ~uching- i h Mile district Due

to increase in demand of seafood intake and the increase of marine surface temperatures that

may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)

thus rapid detection and quantification of V parahaemolyticus can be determined by using

real-time peR

A major foodbome-disease caused by V parahaemolyticus is most common in Asia

and United States although there were only few cases stated in Europe Statistically food-

borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations

and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia

Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand

1

there were about one-third of the people indicated with diarrhea after cockles intake

(FAOIWHO2011)

The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction

(PCR) which include the molecular and biochemical testing for isolation and identification of

organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some

extend of study and hence it gives a less precise result (Huang et al 2009) In addition

DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel

electrophoresis is able to give end point detection but it is not very quantitative to the PCR

product and hence due to the currently developed fluorogenic chemistries a new method

acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and

quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides

a rapid and sensitive detection of reaction products through the fluorescence emission

(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based

quantitative peR strategies which monitor the amplification reaction are the most accurate

(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid

detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both

seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus

the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria

such as in this case for detecting V parahaemolyticus present in environment and food

samples

11Objectives

1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by

1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method

To quantify V parahaemolyticus by using Real-Time (RT-PCR)

2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus

Vibrio can be characterized as a significant group in the family of Vibrionaceae This

genus is mainly found in the marine environment whereby some Vibrios are pathogenic to

human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and

possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average

length (Kelly et ai 1991 Ambrose 2005)

Generally Vibrios can be divided into two groups by characterisation non-halophilic

and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)

Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe

intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They

show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC

with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~

There are so far 12 species have been reported to be pathogenic to humans in which

among these species V parahaemolyticus and V cholerae and V vulnificus are those

associated with foodbome infections of the gastrointestinal tract (FAO 2011)

Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium

iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy

_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)

3

lIDt~re

This organism possesses a single polar flagellum and is halophilic that grows best in I - 8

salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)

There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains

which is pathogenic and causes food poisoning and Kanagawa negative strains which is

known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that

there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis

which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a

number of outbreaks in many different states of the world (Nair et al 2007) V

parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on

solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI

2006)

V parahaemolyticus is most profoundly be found in both estuarine and temperate

coastal marine environment globally The organism is a free living or may act as commensals

to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation

to the latter statement fact also is discussed according to Sakazaki et al (2006) that V

parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on

surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer

temperature and is often related to seafoods from estuarine and coastal marine environment

8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus

Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a

aquatic product in Malaysia It can be classified into the genus Anadara from

family hence Anadara granosa (A granosa) is the scientific name and this species

4

Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK

are mainly widespread in the warm temperate areas In addition A granosa is able to adapt

living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum

temperature in 20 m water depth This intertidal species usually live shallowly buried in

sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6

em which in adult stage when harvested and they normally feed on organic detritus

phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)

14 Route of Transmission of V parahaemolyticus

The wide distribution of V parahaemolyticus in nature and in manne coastal

environment especially precludes the possibility from reservoir to infection According to

Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria

from environment through filter-feeding process leads to human infections Another

important issue of the transmission route is understood that this bacterium is generally

transmitted through the consumption of raw improperly cooked or cooked recontaminated

fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection

in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)

COnical Manifestation of V parahaemolyticus

V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et

2000) This type of gastroenteritis infection in human may be observed as mild to

iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia

In some cases of an affected human the onset of gastroenteritis symptoms that follows

48 hours upon the ingestion of V parahaemoiyticus include hyper secretion

5

gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps

(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the

bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy

96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the

small intestine of the host and excrete toxin However in more severe cases there is a need

for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery

period (park et al 2004)

26 Virulence properties of V parahaemolyticus

The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and

thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V

parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported

that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus

aeleC110n since it is presence in all V parahaemolyticus strains although the gene is

(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-

Apart from that there were cases involving V parahaemolyticus in the outbreaks of

I118troentleritis related to seafood consumptions and this occur in various states include Spain

__n Japan Russia India North America and Southeast Asia (Chiou et al 2000

iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001

1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat

of Thailand there were about one-third of the people indicated with diarrhea after

6

IecIlmlclue that


Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance

the isolation of bacteria as the enrichment media works to increase recovery of the bacteria

from environmental stress and other samples (Wong 2002) Besides that alkaline peptone

water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)

Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is

recommended that optimum 3 salt concentration (Curtis 2006) is added into the

enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two

regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will

normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al

2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will

indicate the presence of V parahaemolyticus The detection of colonies morphologies grown

on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)


Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive

somewhat evaluate the bacterial presence by quantitative means The

IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their

detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent

amplification of peR product throughout cycles thus decreasing analytical time and

In addition fluorogenic probes target-gene-specific sequences internal to the primer

thus RT-PCR provide enhanced specificity as compared to conventional PCR-based

(Blackstone et al 2003) In Real-Time PCR the commonly used reaction

include the peR amplification reaction buffer magnesium chloride

7

deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic

probe Basically a complete amplification cycle involves denaturation annealing and

extension steps for the template DNA to be replicated (Dorak 2006) The first step involve

denaturation step which is crucial in separating the double stranded of DNA double helix in a

temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60

middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences

in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds

in which the polymerase extends from the primer in 5 to 3 direction The accumulated

fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles

(Blackstone et al 2003)

Moreover there were researches which have been conducted by many researchers by

applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus

One of the research conducted by Ward and Bej (2006) they had detecting V

ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed

with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)

analysis for -deecting epsM gene from V

JlJllrJIRJMP in seafood

8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

MATERIALS AND METHODS

a total of 30 samples of cockles (Anadara granosa) were collected

randomly from different wet market in Kuching-i h Mile area Locations of sampling sites

include Stutong wet market and 7th Mile wet market All of these samples were collected

from the month of December 2011 until February 2012 During

JI1Dpling all samples taken were kept in polysterene box containing ice which act as a

temporary storage for cockles to maintain its freshness until analysed immediately in the

SlIIIple preparation and enrichment of bacterial sample

Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -

lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to

ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples

liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth

laboratories) in order to enhance their growth Five hundred microliter of each

samples tested was inoculated into a universal bottle containing 10 mL LB broth

_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker

BrumlWi(~ Innova model 4000) for overnight at 37 DC

9

33 Genomic DNA preparation

The protocol for isolation of total genomic DNA by boiling method is as described by

Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL

microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute

in order to separate the meat particle Next the supernatant was obtained and was subjected

to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then

removed and 500 ~ sterile distilled water was added into the microcentrifuge tube

containing pellet The content was mixed by vortexing the tube gently The microcentrifuge

tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was

immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again

at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new

microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use

bull Real-Time PCR assay The number of DNA template obtained for each cockles sample

- tive isolation and detection of Vibrio parahaemolyticus

The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth

LB broth CONDA laboratories) was then streaked onto TCBS (MERCK

1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated

__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of

i__riIl culture were observed and recorded Further confirmation of the selected single

the bacterial culture in which V parahaemolyticus produced green colonies on

IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized

10

inoculating loop and incubated overnight at 37degC Again the result was observed and

teCOrded

oS Polymerase Chain Reaction (PCR)

peR was performed to detect the specificity of the primer known as MV2B-tl primer

bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this

study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR

~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of

extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx

buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix

BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10

of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR

IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which

lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which

IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC

1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes

Table 1 peR parameters

Volume per reaction 40

iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30

ftriI_ (Forward) 30 1I1miml~ (Reverse) 30

10 30 110 250

11

Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)

Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)

6 Agarose Gel Electrophoresis

Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel

io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer

(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each

with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into

first well Reference strain of V parahaemolyticus obtained in laboratory was used as

illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour

an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained

eel water for 10 minutes The amplified DNA fragments were then viewed under

1IIrIlyeni()1et (UV) transilluminator and result was analysed

fIIiM1ty study with pure culture for standard curve

1 middot6Ia aad bacterial cell count

bull ptUahaemolyticus known to be presented by growing culture on both TeBS and

Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers

_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step

12

ampR gl

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nlt 0 2

Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

Rapid detection and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR

PKHIDMAT MAKLUMAT AKADEMIK

PATRICIA ROWENA ANAKMARK BARAN SAGING (24823)

A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARAW AK

2012

ACKNOWLEDGEMENTS















preparation

DECLARATION







II

- -~ ~ -------~


T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENT I

DECLARATION II

LIST OF TABLES

III

VI

VIII

LIST OF FIGURES VII

TABLE OF CONTENTS


XABSTRACT

XABSTRAK



21 Vibrio Genus 3


23 Cockles 4










III

J










40 RESULTS




Purpose - ~





2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

ACKNOWLEDGEMENTS















preparation

DECLARATION







II

- -~ ~ -------~


T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENT I

DECLARATION II

LIST OF TABLES

III

VI

VIII

LIST OF FIGURES VII

TABLE OF CONTENTS


XABSTRACT

XABSTRAK



21 Vibrio Genus 3


23 Cockles 4










III

J










40 RESULTS




Purpose - ~





2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

DECLARATION







II

- -~ ~ -------~


T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENT I

DECLARATION II

LIST OF TABLES

III

VI

VIII

LIST OF FIGURES VII

TABLE OF CONTENTS


XABSTRACT

XABSTRAK



21 Vibrio Genus 3


23 Cockles 4










III

J










40 RESULTS




Purpose - ~





2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

- -~ ~ -------~


T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENT I

DECLARATION II

LIST OF TABLES

III

VI

VIII

LIST OF FIGURES VII

TABLE OF CONTENTS


XABSTRACT

XABSTRAK



21 Vibrio Genus 3


23 Cockles 4










III

J










40 RESULTS




Purpose - ~





2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

J










40 RESULTS




Purpose - ~





2650 DISCUSSION

60 CONCLUSION 30

REFERENCES 31

IV

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

35 APPENDICES

-

v

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

LIST OF TABLES









Page

11

12

15

15

16

35

35

36

VI

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

--------

LIST OF FIGURES

Page










VII




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12




em centimetre

Ct Threshold cycle






g gram

kbp kilo base pair

LB Luria Bertani

m meter


min minute(s)

mL mililiter

mM milimolar


run nanometer





VIII

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

RT-PCR

sec

TBE

TCBS

tdh

tlh

trh

UV

IJIll

~

middotC


second(s)

Tris-Borate-EDTA





ultraviolet

micrometer

microliter

degree celsius

percentage

bull

IX

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

Dlat pengiraan





ABSTRACT



ABSTRAK




x

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

CHAPTER 1

Introduction

















real-time peR






1


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12


(FAOIWHO2011)


















samples

11Objectives




2

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

CHAPTER 2

LITERATURE REVIEW

21 Vibrio Genus


















3

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

lIDt~re











2006)












4























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12























5





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12





















6

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

IecIlmlclue that
























7


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12


















8

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12

31 Sample coUection

In this study

monthly (3 months)

CHAPTER 3

















9






















10


teCOrded


















10 30 110 250

11


















12






















10


teCOrded


















10 30 110 250

11


















12


teCOrded


















10 30 110 250

11


















12


















12

Documents

RAPID DETECTION AND QUANTIFICATION OF Vibrio