DEVELOPMENT OF A NOVEL VNT ASSAY USING QRT-PCR-BASED ENDPOINT ASSESSMENT FOR RAPID DETECTION - PowerPoint Presentation

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DEVELOPMENT OF A NOVEL VNT ASSAY USING QRT-PCR-BASED ENDPOINT ASSESSMENT FOR RAPID DETECTION AND TITRATION OF NEUTRALIZING ANTIBODIES AGAINST FMDV Zhidong ZhangState Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences(CAAS), China

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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ConclusionA qRT-PCR-VNT was developed for rapid detection of neutralizing antibodies against FMDV Asia 1The endpoint assessment was carried out as early as 20 hpi The developed qRT-PCR-VNT with endpoint at 20hpi has good correlation with traditional VNT Compared with traditional VNT, the developed qRT-PCR-VNT was robust with respect to assay duration (20 hours vs.72 hours)

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Outline BackgroundResultsConclusion Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Outline BackgroundResultsConclusion Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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5

Diagnosis

Detection of FMDV neutralizing antibodies

Measure the serological response to FMDV infection and vaccination

Vaccine matching studies (in vitro)

Confirms clinical diagnosis

Supports the need for accurate clinical diagnosis

Full epidemiological information

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

Unknown/

Uncertainty

Known/

Certainty

Process

Virus neutralization test

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6a confirmatory testhighly specific But

Time-consuming

Slow, may take 2-3 days

Easily affected by man-made factors

Reading CPE

Diagnosis

Virus neutralization test

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Real-time qRT-PCR assay High sensitivity and specificity Be quantitative Results within hoursWidely used for detection of FMDV

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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qRT-PCR-based VNT assay

The

qRT

-PCR assay is used to quantify the reduction in FMDV replication resulting from sera neutralizing antibodies

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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qRT-PCR-based VNT assay FMDV + antiserum 37oC， I h

37

o

C

，

20

h

Inoculation of IBRS 2 cells

Trizol

added at 20

hpi

The mean % of reduction in FMDV RNA copy number

Quantification of viral RNA

RNA extraction

The RT-qPCR assay is used to quantify the reduction in FMDV replication resulting from sera neutralizing antibodies

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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qRT-PCR-based VNT assay

The mean percentage of reduction in FMDV RNA copy number

= 1- (FMDV RNA copy number in serum sample /FMDV RNA copy number in non-neutralized virus control*) %

*non-neutralized virus control: cells infected with FMDV in absence of sera (virus control wells)

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Outline BackgroundResultsConclusion Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Performance analysis of qRT-PCR assay targeting the 3D geneStandard 3D RNA curveRNA extracted from FMDV Asia-1/JSL/06) Dilutions ranging from 1:10 to 1:107

Virus stock :10

5.6

TCID

50

/ 100

μ

l

Range spans 6 logs ranging from 2 to 8 log FMDV copies per reaction

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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The replication kinetic of FMDV was determined by quantifying the RNA extracted from FMDV Asia 1-infected IBRS-2 cells grown in 96-well plates

100 TCID

50

/well of the virus

each point represents the mean value ± SD (n=5) of

lg

(copy number) against the

hpi

.

The mean of RNA copy number initially be of 1.3×10

3

copieat 1 hour and increased to 10

7

copies at 20 h with absence of CPE.

Assessment of FMDV replication kinetics by qRT-PCR Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Assessment of virus neutralization by qRT-PCR Distribution of viral RNA copy for negative sera

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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The negative cut-off value was set by adding 3 standard deviations (SD) to the mean value of reduction in the FMDV copy number in comparison with a non-neutralized virus control at 1:32 dilutionThe negative： cut-off value =equal to or less than 23 % in reduction The mean percentages of reduction in FMDV RNA copy number in a manner dependent on the dilution of sera.

Dilution

Mean of RNA copy number

SD

Mean percentages of reduction

SD

1:4

3.19

0.96

63%

0.07

1:8

4.03

2.06

42%

0.19

1:16

6.46 2.02 24%0. 121:32

7.69 0.36 8%0.051:647.65 0.33

9%0.05

1:1287.77 0.33 8%0.05

control

8.36

0.16

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

23% cut off

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The mean percentages of the reduction at 1:32 is greater than 23 % in reduction Reduction of detectable FMDV copy number observed in sera collected from pigs infected with FMDV Asia 1 at 35 dpiLanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

23% cut off

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Analytical sensitivity sera collected from pigs infected with FMDV Asia 123% cut offLanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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The mean percentages of the reduction at 1:32 is greater than 23 % in reduction Reduction of detectable FMDV copy number observed in VNT-positive seraDilutionVNT

Mean of RNA copy number

SD

Mean percentages of reduction

Sample 1

P

2.86

0.17

65.13%

Sample 2

P

2.93

0.32

64.27%

Sample 3

P

2.93

0.22 64.21%Sample 4P2.79 0.10 65.95%

Sample 5P 2.79 0.10 65.95%Sample 6P

3.06

0.25 62.65%Sample 7P3.03 0.26

63.07%

Sample 8

P

3.17

0.10

61.35%

Sample 9

P

2.82

0.30

65.61%

Control

N/A

8.20

0.17

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences23% cut off

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no cross-protection between different serotype of FMDV.Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Correlation between Log2(VNT titre) and Log2(qRT-PCR-VNT titre). 20 of FMDV negative sera were performed with VNT and qRT-PCR-VNTLanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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CONCLUSIONA qRT-PCR-VNT was developed for rapid detection of neutralizing antibodies against FMDV Asia 1The endpoint assessment was carried out as early as 20 hpi The developed qRT-PCR-VNT with endpoint at 20hpi has good correlation with traditional VNT Compared with traditional VNT, the developed qRT-PCR-VNT was robust with respect to assay duration (20 hours vs.72 hours)

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Yiming SongYang YangXiangle ZhangYanmin Li Guozhen Cong

National Research

Programe

(2016YFD0500907)

CAAS Innovation fund

CAAS master degree studentship

Acknowledgements

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences

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Thanks