Valuation of primers for the detection of hiv-1 viruses from Kelantan by using reverse transcription polymerase chain Reaction (RT-PCR)

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EVALUATION OF PRIMERS FOR THE DETECTION OF HIV-1 VIRUSES FROM KELANTAN BY USING REVERSE TRANSCRIPTION POLYMERASE CHAIN

REACTION (RT-PCR)

by

NOOR HAMIZAH BINT! MINAL

Dissertation submitted in partial fulfillment of the requirements for the degree of Bachelor of Health Sciences (Biomedicine)

May 2013

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CERTIFICATE

This is to certify that the dissertation entitle “EVALUATION OF THE PRIMERS FOR THE DETECTION OF HIV-1 VIRUSES FROM KELANTAN BY USING REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR)” is the bonafide record of research work done by Ms. Noor Hamizah binti Minal during the period from September 2012 to June 2013 under my supervision.

Supervisor,

Dr. Maizan Mohamed, PhD Researcher & Lecturer

Institute for Research in Molecular Medicine (INFORMM) Universiti Sains Malaysia

Health Campus 16150 Kubang Kerian Kelantan, Malaysia Date:

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ACKNOWLEDGEMENT

Praise

to God

for blessing

me

with patience and perseverance

to

conduct this

research well.

I

would

liketo

take

this

wonderful opportunity

toexpressmythanksanddeepest

gratitude for

my supervisor, Dr. Maizan btHaji

Mohamed for

all

her

guidance,

valuable

advice,

constructive criticism and scientific

discussion.

1

also would liketo thank Miss

Che Wan Salma

Che Wan Zalati,

Miss Eliana

Saffie,

Miss Julia Abdullah

and

Miss

Fadhilah Usuki,

who have

taught and

assisted

me in molecular

work

and

also

intellectual

and

interesting

conversation that we had together during

thisperiod. Not

to forget,

Dr.

Wan

Nazirah

Wan Yusof for providing

thesamples.

I

also

want to express my

deepest gratitude

to

members

of

INFORMM Health

Campus

for their great support and cooperation.

I also would like

to

thank

Prof.

Norazmi

MohdNor, Directorof

INFORMM for

acceptingmetocompletemy

final

year project

at

INFORMM.

Also thanks

to

INFORMM

administration and

laboratory

staff

for

their

cooperation

in

allowing

me to

use

theirequipment

and

performing lab work

at INFORMM.

Not to

forget,

I would like to express my

heartful thanks

to my beloved

family

and friend,

especially

my parents

for

their blessings,

wishes,

and encouragements

for

thesuccessful

of

this

final year

project.

Last but not least, thanks

to all of

those

who have supported

me inany

aspects

duringthe

completion of

thisdissertation

especially all

my

classmates.

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TABLE OF CONTENTS

i ii List of Tables iv

v vi vii

1 1.1 1

1.2 4 1.3 6

2.1 7

2.1.1 8 2.1.2 9 2.1.3 12 2.2 15

2.2.1 15 2.2.2 17 2.2.3 20 2.2.4 20 2.3 23

23 27 Acknowledgement

Table of Contents

List of Figures

Symbols and Abbreviations Abstract

Properties and Structure

Long Terminal Repeat (LTR) region Replication Cycle

HIV-Entry

HIV-1 Reverse Transcription HIV-1 DNA Integration

HIV-1 Assembly, Budding and Maturation Diagnosis

2.3.1 Enzyme Immunoassay (EIA) 2.3.2 Molecular Diagnosis

CHAPTER 3: MATERIALS AND METHODS CHAPTER 1: INTRODUCTION

Acquired Immunodeficiency Syndrome (AIDS) Problem & Rationale of The Study

Objectives

CHAPTER 2: LITERATURE REVIEW Etiology - HIV

Classification - HIV

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36 37 37 38

39 4.1

4.2 45

4.3 49

49 49 50 50 4.4

56 4.5

4.6 57

CHAPTER 5: DISCUSSION

5.1 59

5.2 61

5.3 61

5.3.1 62

5.3.2 62

5.3.3 63

5.4 64

5.5 64

5.6 65

66

REFERENCES 67

Primer Design Primers Evaluation

Optimization of PCR Parameters

4.3.1 Optimization of Annealing Temperature 4.3.2 Optimization of MgCh

4.3.3 Optimization of dNTP and Taq DNA polymerase Evaluation of Sensitivity and Specificity of Primers

Detection of HIV-1 viruses in 30 Clinical Samples from Kelantan Evaluation of HIV LTRF2&HIV LTR2 primers for Real-Time PCR

Optimization of PCR Parameters

Optimization of Annealing Temperature Optimization of MgCl2

Optimization of dNTP and Taq DNA Polymerase Evaluation Sensitivity and Specificity of Primers

Detection of HIV-1 Virus in Clinical Samples Evaluation of Primers for Real-Time PCR (rtPCR) CHAPTER 6: CONCLUSION

3.2.5 Complimentary DNA (cDNA) synthesis 3.2.6 Polymerase Chain Reaction (PCR) 3.2.7 Agarose Gel Electrophoresis 3.2.8 Real-Time PCR

CHAPTER 4: RESULT Primer Design Primer Evaluation

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LIST OF TABLES

Table 3.1 29

Table 3.2 30

List of equipments used in this study

Table 3.3 30

List of bioinformatic tools used in this study 31 Table 3.4

List of consumable items used in this study 31 Table 3.5

List of HIV-1 samples used in this study

Table 3.6 34

Primers used for amplification of HIV-1 viruses

Table 3.7 35

Result of Ct values of real-time PCR on 6 HIV-1 viruses 58 Table 4.1

from Kelantan using HIV LTRF2 and HIV LTR2 as primers.

List of chemicals and reagents used in this study List of commercial kit used in this study

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LIST OF FIGURES Figure 2.1 11

14 19 22 24 39 40 41 42 43 44 46

Figure 4.8 47

50 51 52

54 55

Figure 4.14 56

Figure 4.15 A dissociation curve of real-time PCR 57 Figure 4.9

Figure 4.10 Figure 4.11 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7

Figure 4.12 Figure 4.13

Optimization of annealing temperature for PCR reaction Optimization of magnesium concentration for PCR reaction Optimization of dNTP and Tag DNA polymerase

concentration for PCR reaction

Sensitivity evaluation of the primers by a 10-fold serial dilution.

Specificity evaluation of the primers on the three RNA of viruses

Result from PCR reaction in detecting HIV-1 viruses from Kelantan

Illustrated basic structure of human immunodeficiency virus (HIV)

HIV-1 Long terminal repeat (LTR) structure of proviral DNA.

Overview of HIV entry.

Overview of HIV-1 replication cycle.

The generation of immunoassay in HIV diagnostic.

Result from BLAST analysis for HIV LTRF2 Result from BLAST analysis for HIV LTR2 Result from BLAST analysis for HIV ENV IF Result from BLAST analysis for HIV ENVRNew Result from BLAST analysis for HIV DRF Result from BLAST analysis for HIV RTR2N Comparison in primers evaluation between

HIV DRF+HIV RTR2N and HIV LTRF2+HIV LTR2 Primers evaluation in PCR reaction using

HIV ENV1F+HIV ENVRNew

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°C

cDNA Ct DNA DNase dNTP EDTA EtBr

kDa LTR

Nuclease Free Water NFW

RNase Ribonuclease

g HIV

Base pair

Basic Local Alignment Search Tool Complementary deoxyribonucleic acid Cycle threshold

Deoxyribonucleic acid Deoxyribonuclease

deoxynucleoside Triphosphate Ethylene Diamine Tetra Acetic acid Ethidium bromide

Gravity

Human immunodeficiency virus kiloDalton

Long terminal repeat Miligram

Magnesium Chloride miliMolar

Nanogram

Polymerase Chain Reaction

Qualitative Polymerase chain reaction Ribonucleic acid

Revolution per minute Reverse Transcriptase mg

MgCl2 mM

ng PCR qPCR RNA

Rpm RT

SYMBOLS AND ABBREVIATIONS Degree Celcius

Microgram Microlitre Pg

pL bp BLAST

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ABSTRACT

Human

immunodeficiency

virus

type 1

(HIV-1) has been

recognized

as

the

causative agent

of

acquired immunodeficiency syndrome

(AIDS). Approximately 42

million people

carryingthe virus

at

present, but

its

case

fatality

rateis

close

to

100%,

making it an

infection

ofdevastating

ferocity. Since

the evolutionary

change of

the virus is

at

challenge on HIV

diagnostic development, vaccine

development,

antiretroviral

drug sensitivity and drug resistance. Rapid and

sensitive methods for

the

detection

of

HIV-1 viruses would

be valuable in controlling

this

disease.

Currently, nucleic

acid amplification

tests

such

as reverse transcription

polymerase

chain

reaction

(RT-PCR)

and real-time PCR

(rtPCR)

are widely

used in detecting HIV-1

viruses.

These

testsarecommonlyapplied

in

blood

donation screening and early detection

of

HIV-1 in infants.

High sequence

variations of

the HIV-1 viruses

from

Kelantan

have

led

to

the

failure

to

obtain

the amplification product

using published primers. Therefore,

inthis

study,

a

few set of

primerswere

designed based

on the

sequence

alignmentsof

published

HTV-1 viruses from

Malaysia and

Kelantan

which

were

retrieved

from

NCBI

genebank.

The

primers

were

evaluated on 30

confirmed

chosen as

the

best

primerstodetecttheseviruses

since

they

were successfully amplified all

the

30 samples used in

this

study. The primers were

further evaluated

their

sensitivity and specificity

against 3 viruses which are Japanese encephalitis (JE),

Chikungunya and Western

encephalitis

virus

(WEE)

where no

expected band yielded.

These primers were

sensitive

and specific

enough in detecting HIV-1 viruses from

Kelantan.

They

were furtherevaluated

for use

in the real-time

PCR

and can

be used in

thistest. Thus, these

primers

could

be

potentially

use

forthe future diagnosis

of HIV-

1 viruses from

Kelantan either

byconventional

or

realtime

PCR.

vii

HIV-1 positive

samples

from

Kelantan, Malaysia.

HIV LTRF2

and

HIV LTR2

were

rapid rate,

it

imposes

a

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ABSTRAK

“Human Immunodeficiecy Virus” (HIV) jenis 1 telah dikenal pasti sebagai agen penyebab kepada Sindrom Kurang Daya Tahan Penyakit (AIDS). Sehingga kini dianggarkan hampir 42 juta orang pembawa virus ini, dengan kadar kematiannya hampir kepada 100%, menjadikannya sebagai satu jangkitan yang boleh membunuh.

Memandangkan perubahan evolusi virus tersebut pada kadar yang sangat cepat, ini menimbulkan satu cabaran dalam pembangunan diagnostik dan vaksin, sensitiviti ubat antiretroviral dan ketahanan terhadap ubat. Ujian amplifikasi asid nukleik merupakan pendekatan yang digunakan secara meluas dalam mengesan virus HIV-1. Pendekatan ini juga banyak dilaksanakan untuk memeriksa darah yang didermakan dan pengesanan awal HTV-1 dalam bayi. Variasi yang tinggi dalam jujukan virus HTV-1 yang terdapat di Kelantan telah menyebabkan virus tersebut tidak dapat dikesan dalam proses PCR dengan menggunakan primer rujukan yang dihasilkan oleh kajian sebelum ini. Justeru, beberapa set primer telah direka dan dinilai keatas 30 sampel positif HIV-1 dari Kelantan. Dalam kajian ini, HIV LTRF2 dan HIV LTR2 telah dipilih sebagai primer yang terbaik untuk mengesan virus HIV-1 yang terdapat di Kelantan memandangkan primer-primer ini berjaya mengamplifikasikan kesemua 30 sampel yang digunakan dalam kajian ini. Sensitiviti dan spesifisiti primer ini kemudian dinilai keatas tiga jenis virus lain iaitu Japanese encephalitis (JE), Chikungunya dan ‘Western equine encephalitis virus’ (WEE) di mana tiada amplifikasi yang berlaku. Primer ini didapati sensitif dan spesifik dalam pengesanan virus HIV dari Kelantan. Primer ini seterusnya