Rapid and real-time diagnosis of Laem-Singh virus

using a portable Real-Amp Turbidimeterusing a portable Real Amp Turbidimeter

Narong ArunrutE il @bi t th

National Center of Genetic Engineering and Biotechnology (BIOTEC)& CENTEX Shrimp, Thailand

E-mail: narong.aru@biotec.or.th

Introduction

Penaeus monodon with monodon slow growth syndromeSource: DV Lightner

Monodon slow growth syndrome (MSGS) has caused serious problems in Thailand since 2002.

Average growth rate in MSGS pond is half that normally expected

No correlation of known pathogens were associated with No correlation of known pathogens were associated with MSGS(Chayaburakul et al. 2004)

Later investigations revealed the presence of a virus called Later investigations revealed the presence of a virus called Laem-Singh virus (LSNV) (Sritunyalucksana et al., 2006)

L Si Vi (LSNV)Laem-Sing Virus (LSNV)

It’ iti i l t d d RNA i It’s a positive-sense single stranded RNA virus (ssRNA, 25-30 nm)

phylogenetic analysis shown to be most closely related the family Leuteoviridae

LSNV considered to be a necessary but insufficient cause of MSGS

Population from different sites (Thailand, India and Indo-Population from different sites (Thailand, India and IndoPacific)

Listed for exclusion from domesticated stock of the black Listed for exclusion from domesticated stock of the black tiger shrimp

How to prevent outbreak of disease?• Good culture system

How to prevent outbreak of disease?

• Efficient and early detection (i.e. field diagnosis)g )

R id Di i f L Si h iRapid Diagnosis for Laem-Singh virus@ RT-PCR (Sritunyalucksana et al., 2006)@ RT PCR (Sritunyalucksana et al., 2006)

@ nested RT-PCR (Prakasha et al., 2007)

ProblemsProblemsProblemsProblems

High cost instrument PCR machinePCR machineElectrophoresis set

Long time usageLong time usage. RT-PCR 2 h

Nested RT-PCR 3-4 h (Two steps)Gel electrophoresis 1 h

Carcinogen

Ethidium bromide (EtBr)

LoopLoop--mediated isothermal amplification mediated isothermal amplification (LAMP) (LAMP)

An approach allows amplified target DNA with High specificity High sensitivity High efficiency (large amounts product # 109 copies) Rapidity under isothermal conditions (60 65oC) Rapidity under isothermal conditions (60-65oC) Small test menu Point-of-care or extralaboratory detection Point of care or extralaboratory detection

Originally described by Notomi et al. (2000)g y y(http://loopamp.eiken.co.jp/e/)

LF5’3’

LFLFPrimerPrimer

5’3’Target DNA

cDNA

LB5’ 3’

LBLBPrimerPrimer

PrimerPrimer

LFLF ::::

BB33 ::55’’--FF11c c -- FF22

55’’ BB11cc BB22

FIPFIP ::::

Inner PrimerInner Primer Outer PrimerOuter Primer Loop PrimerLoop Primer

FF33 :: LBLB ::55’’--BB11c c -- BB22BIP BIP ::

4-6 primer recognize 6-8 specific gene sequences Bst DNA Polymerase strand-displacing activity and autocycling

Target DNA

1 2F3c F2c F1c

5’

3’ 5’B1 B2 B3

FIPF3 Primer

5’

3’ 5’ F1c B1LB

FIP

53’

5’

F2c

F2F1c

F1 3’

B1

B1c B2

F1c F2 F1 B1c B2c B3c3’ 3’

B2cB1cF1

LF

B3 Primer

BIPBIP

5’5’

F1c B1

B2B1c

B2c3’F1cF2

(http://loopamp.eiken.co.jp/e/)F1F2c

B1

B1c B2

Th d t f LAMP tiTh d t f LAMP tiThe product of LAMP reactions The product of LAMP reactions (Mori et al., (Mori et al., 20012001) )

(DNA)n‐1 + dNTP         (DNA)n + P2O74‐ [1]

P2O74‐ +  2Mg2+                 Mg2P2O7 [2] 

LAMP PRODUCT DETECTIONLAMP PRODUCT DETECTIONLAMP PRODUCT DETECTIONLAMP PRODUCT DETECTION

k d b Electrophoresis

+Naked-eye observation

++ +

Electrophoresis InstrumentEtBr.

Bias.EtBr.

RealReal--Time LAMPTime LAMP

Measured the change in turbidityMeasured the change in turbidity of LAMP product

No post LAMP processingNo post- LAMP processing

By using a commercial portableReal-Amp Turbidimeter (Mobilis Automata, Thailand)

Based on the detection and quantitation of Mg2P2O7 The turbidity signal increases in direct proportion to the amount The turbidity signal increases in direct proportion to the amount

of LAMP product generated.

ObjectivesObjectives

To developed a real time RT LAMP assay forTo developed a real-time RT-LAMP assay for detection of Leam-Singh virus (LSNV).

EXPERIMENT f l ti RT LAMPEXPERIMENT for real-time RT-LAMP

Optimal conditions

Sensitivity test

Specificity test

Optimal conditionsOptimal conditions

RNA standard

RNA standard

RNA standard

60oC, 1 H 63oC, 1 H 65oC, 1 H

Read the result by Read the result by Read the result by Turbidimeter

The primer used for real-time RT-LAMP forThe primer used for real time RT LAMP for detection of LSNV

Primers: Designed according the RdRp gene of Leam-singh virus (Gen-Bank accession number: DQ127905;Leam-singh virus (Gen-Bank accession number: DQ127905; Arunrut et al., 2011) using Primer Explorer version 3

PROTOCOL of LAMP for LSNVPROTOCOL of LAMP for LSNV

65oC for 60 min 2 M h f i i FIP d BIP

(Arunrut et al., 2011)

2 uM each of inner primer FIP and BIP 0.2 uM each of outer primer F3 and B3 2 uM each of loop primer LF and LB 2 mM of dNTP mix 0.1 M betaine 6 mM MgSO4g 4 8U of Bst DNA polymerase and 0.25U of AMV reverse transcriptase 1X of the supplied buffer 2 ul of RNA template in a final volume 25 ul2 ul of RNA template in a final volume 25 ul

Optimization of temperatureOptimization of temperature

Sensitivity testy

RNA Extraction by TRIzol® (Invitrogen)RNA Dil ti 100 t 10 7RNA Dilution 100(100 ng) to 10-7(10 fg)

Real-time RT-LAMP

Conventional RT-PCR system (RT-PCR and nested RT-PCR)

Compared sensitivity bothCompared sensitivity both a turbidity and gel electrophoresis

Sensitivity test

RT-PCRReal-time RT-LAMP

N d RT PCRNested RT-PCRRT-LAMP by AGE

Specificity testp y

Both RNA/DNA other shrimp virus

used for the test (IHHNV, WSSV, YHV, IMNV, MrNV and normal shrimp)

Test by turbidimeter(65oC, 1 H)

R d th lt b l tiRead the result by real-time RT-LAMP assay

Specificity testp y

C l iConclusionConclusion

Comparison of a real-time RT-LAMP and RT-PCR system RT PCR system

real-time RT LAMP RT-PCR Nested

RT-PCRRT-LAMP

•High sensitivity •Lower sensitivity

RT-PCR

•High sensitivity

Rapid (1 h) L ti

High sensitivity(100 fg of total RNA)

Lower sensitivity(100 pg of total RNA)

High sensitivity(100 fg of total RNA)

V l ti •Rapid (1 h)

•Cheap instrument

•Long time (3 h)

•PCR machine

•Very long time (4-5 h)

PCR hi•Cheap instrumentTurbidimeter

•PCR machine •PCR machine

•User friendlyRead the result by

l ti t

•Carcinogen Gel Electro.(EtBr)

•CarcinogenGel Electro.(EtBr)

real-time system

DiscussionsDiscussionsM Th l ti RT LAMP i it blMoreover, The real-time RT-LAMP assay is suitable not only for detection of LSNV but also for many pathogen of shrimppathogen of shrimp

ACKNOWLEDGMENT

• National Center for Genetic Engineering and Biotechnologygy

• Prof. Timothy W. Flegel, Centex Shrimp, Thailand• Prof Boonsirm Withyachumnarnkul Shrimp Genetic• Prof. Boonsirm Withyachumnarnkul, Shrimp Genetic

Improvement Center (SGIC),Thailand• Mrs Wansika Kiatpathomchai• Mrs.Wansika Kiatpathomchai

F tt tiThanks you For your attentionThanks you