The most prominent of growth-retarded in black tiger shrimp (Penaeus monodon) is associated with an infection of Laem-Singh virus (LSNV) that has been involved the reduction of annual production volume. To more accurately, rapidly and easily determine its effect on shrimp industries for further control disease, a portable Real-Amp Turbidimeter with real-time detection method was evaluated for LSNV detection.The device exploited the turbidity that produced by-product of magnesium pyrophosphate and displayed turbidity values in real-time on the provided software screen. The results of quantitative can be accomplished by reaction time measuring. The sensitivity of a turbidity detection was comparable to that of the nested RT-PCR, while it was 1000-time more sensitive than RT-PCR. With the use of a portable Real-Amp Turbidimeter. The application of RT-LAMP using a portable Real-Amp Turbidimeter offers a simple and rapid detection platform for LSNV detection in the field assessment. So, This 's awesome technic not only for detection of LSNV but also for many pathogen.

1. Rapid and real-time diagnosis of
Laem-Singh virus
using a portable Real-Amp Turbidimeter
Real Amp
Narong Arunrut
E-mail: narong.aru@biotec.or.th
E
il
@bi t
th
National Center of Genetic Engineering and Biotechnology (BIOTEC)
& CENTEX Shrimp, Thailand

2. Introduction
Penaeus monodon with monodon slow growth syndrome
Source: 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
MSGS(Chayaburakul et al. 2004)
 Later investigations revealed the presence of a virus called
Laem-Singh virus (LSNV) (Sritunyalucksana et al., 2006)

3. Laem-Sing Virus (LSNV)
L
Si Vi
 It’ a positive-sense single stranded RNA virus
It’s
iti
i l t
d d
i
(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
Indo
 Population from different sites (Thailand, India and IndoPacific)
 Listed for exclusion from domesticated stock of the black
tiger shrimp

4. How to prevent outbreak of disease?
• Good culture system
• Efficient and early detection (i.e. field
diagnosis)
g
)
Rapid Diagnosis f L
R id Di
i for Laem-Singh virus
Si h i
@ RT-PCR (Sritunyalucksana et al., 2006)
RT PCR
@ nested RT-PCR (Prakasha et al., 2007)

5. Problems
High cost instrument
PCR machine
Electrophoresis set
Long time usage
usage.
RT-PCR 2 h
Nested RT-PCR 3-4 h (Two steps)
Gel electrophoresis 1 h
Carcinogen
Ethidium bromide (EtBr)

6. LoopLoop-mediated isothermal amplification (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)
(60-65
Small test menu
Point of care
Point-of-care or extralaboratory detection
Originally described by Notomi et al. (2000)
g
y
y
(http://loopamp.eiken.co.jp/e/)

7. 3’
LF
5’
LF
Primer
Target DNA
cDNA
5’
3’
LB
LB
Primer
Primer
Inner Primer
FIP
BIP
:
:
Outer Primer
5’-F1c - F2
B3
5’-B1c - B2
F3
:
:
Loop Primer
LF
LB
:
:
 4-6 primer recognize 6-8 specific gene sequences
 Bst DNA Polymerase strand-displacing activity and autocycling

8. Target DNA
1
3’
F3c F2c F1c
B1
B2
B3
2
5’
5’
FIP
F3 Primer
LB
3’
F1c
F2c
5
5’
3’
5’
F2
F1
B1
3’
B1c B2
FIP F1c
F1c F2
F1
B1c B2c
B3c
3’
3’
F1
B1c
LF
5’
BIP
5’
B3 Primer
F1c
F2c
F1
B1
B1c
B2
(http://loopamp.eiken.co.jp/e/)
B2c
F2 F1c
3’
B2
B1c BIP

9. The
Th product of LAMP reactions (Mori et al., 2001)
d t f
ti
2001)
(DNA)n‐1 + dNTP (DNA)n + P2O7
P2O7
4‐
+ 2Mg2+ Mg2P2O7
4‐
[1]
[2]

10. LAMP PRODUCT DETECTION
+
Naked-eye observation
k d
b
Bias.
+
Electrophoresis
Electrophoresis Instrument
EtBr.
EtBr.

11. RealReal-Time LAMP
Measured the change in turbidity
of LAMP product
No post LAMP processing
postBy using a commercial portable
Real-Amp Turbidimeter
(Mobilis Automata, Thailand)
 Based on the detection and quantitation of Mg2P2O7
 The turbidity signal increases in direct proportion to the amount
of LAMP product generated.

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

13. EXPERIMENT for real-time RT LAMP
f
l ti
RT-LAMP
Optimal
conditions
Sensitivity
test
Specificity
test

14. Optimal conditions
RNA
standard
60oC, 1 H
RNA
standard
63oC, 1 H
RNA
standard
65oC, 1 H
Read the result by
Turbidimeter

15. The primer used for real-time RT-LAMP for
real time RT LAMP
detection of LSNV
Primers: Designed according the RdRp gene of
Leam-singh virus (Gen-Bank accession number: DQ127905;
Arunrut et al., 2011) using Primer Explorer version 3

16. PROTOCOL of LAMP for LSNV
(Arunrut et al., 2011)
65oC for 60 min
 2 uM each of inner primer FIP and BIP
M
h fi
i
d
 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 MgSO4
g
 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 ul

17. Optimization of temperature

18. Sensitivity test
y
RNA Extraction by TRIzol® (Invitrogen)
7
RNA Dilution 100(100 ng) t 10-7(10 fg)
Dil ti
to
Real-time
RT-LAMP
Conventional
RT-PCR system
(RT-PCR and nested RT-PCR)
Compared sensitivity both
a turbidity and gel electrophoresis

19. Sensitivity test
Real-time RT-LAMP
RT-PCR
Nested RT-PCR
N
d RT PCR
RT-LAMP by AGE

20. Specificity test
p
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)
Read th
R d the result by real-time
lt b
l ti
RT-LAMP assay

21. Specificity test
p
y

22. Conclusion
C
l i

23. Comparison of a real-time RT-LAMP and
RT PCR
RT-PCR system
real-time
RT-LAMP
RT LAMP
•High sensitivity
High
(100 fg of total RNA)
•Rapid
Rapid
(1 h)
RT-PCR
Nested
RT-PCR
•Lower sensitivity •High sensitivity
Lower
High
(100 pg of total RNA)
•Long time
L
ti
(3 h)
(100 fg of total RNA)
•Very long time
V
l
ti
(4-5 h)
•Cheap instrument •PCR machine
•PCR machine
PCR
hi
•User friendly
•Carcinogen
Turbidimeter
Read the result by
real-time system
l ti
t
•Carcinogen
 Gel Electro.(EtBr)
Gel Electro.(EtBr)

24. Discussions
Moreover, The real-time RT-LAMP assay is suitable
M
Th
l ti
RT LAMP
i
it bl
not only for detection of LSNV but also for many
pathogen of shrimp

25. ACKNOWLEDGMENT
• National Center for Genetic Engineering and
Biotechnology
gy
• Prof. Timothy W. Flegel, Centex Shrimp, Thailand
• Prof Boonsirm Withyachumnarnkul Shrimp Genetic
Prof.
Withyachumnarnkul,
Improvement Center (SGIC),Thailand
• Mrs Wansika Kiatpathomchai
Mrs.Wansika

26. Thanks attention
you ti
For
F your tt