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Validation of a Taqman based real time RT-PCR assay suitable for surveillance anddiagnosis of Viral Haemorrhagic Septicaemia Virus worldwide

Jonstrup, Søren Peter; Kahns, Søren; Skall, Helle Frank; Boutrup, Torsten Snogdal; Olesen, NielsJørgen

Publication date:2011

Document VersionPublisher's PDF, also known as Version of record

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Citation (APA):Jonstrup, S. P., Kahns, S., Skall, H. F., Boutrup, T. S., & Olesen, N. J. (2011). Validation of a Taqman basedreal time RT-PCR assay suitable for surveillance and diagnosis of Viral Haemorrhagic Septicaemia Virusworldwide. Poster session presented at 15th International Conference on Diseases of Fish and Shellfish, Split,Croatia.

Validation of a Taqman based real time RT PCR assay suitable for surveillance and Validation of a Taqman based real time RT-PCR assay suitable for surveillance and q ydiagnosis of Viral Haemorrhagic Septicaemia Virus worldwidediagnosis of Viral Haemorrhagic Septicaemia Virus worldwide

S P Jonstrup S Kahns H F Skall T S Boutrup and N J OlesenS.P. Jonstrup, S. Kahns, H.F. Skall, T.S. Boutrup, and N.J. OlesenDTU Vet Section for Fish Diseases Aarhus DenmarkDTU Vet, Section for Fish Diseases, Aarhus, Denmark

Genotype Number of isolates Jonstrup GarverGenotype Number of isolates Jonstrup Garver

I 2 21,9 23,2, ,

I 18 20 3 21 3Ia 18 20,3 21,3

Ib 14 19 7 21 0Ib 14 19,7 21,0

Ic 3 19 4 20 3Ic 3 19,4 20,3

Id 3 20 8 21 7Introduction: Viral haemorrhagic septicaemia (VHS) is a serious disease Id 3 20,8 21,7g p ( )in several fish species VHS is caused by the rhabdovirus Viral Ie 2 21,3 23,1in several fish species. VHS is caused by the rhabdovirus Viral Haemorrhagic Septicaemia Virus (VHSV) To prevent outbreaks it is

Ie 2 21,3 23,1Haemorrhagic Septicaemia Virus (VHSV). To prevent outbreaks it is i h f i i d ifi di i l id if

II 4 21,6 25,1important to have a fast, sensitive, and specific diagnostic tool to identify

, ,

III 11 18 8 22 7g

infected fish. Traditional diagnosis based on isolation in cell culture III 11 18,8 22,7infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using e g ELISA is sensitive and specific but IVa 16 20 1 19 2followed by identification using e.g. ELISA is sensitive and specific but

th l S ill f d l i f d f VHS i fi h f i IVa 16 20,1 19,2

rather slow. Surveillance for declaring freedom of VHS in fish farms is IVb 6 19 1 19 1hitherto based on cell cultivation since no serological- or molecular test

IVb 6 19,1 19,1hitherto based on cell cultivation since no serological or molecular test had previously been fully validated and documented to have a competitive Controls 15 No Ct No testhad previously been fully validated and documented to have a competitive

iti it d ifi it t th ll lt th d B it hi t RTControls 15 No Ct No test

sensitivity and specificity to the cell culture methods. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before an Figure 3: Analytical specificity test. The average Ct values obtained by ganswer is available will be considerably shortened and the need for

Figure 3: Analytical specificity test. The average Ct values obtained by using the Jonstrup et al PCR or the Garver et al PCR are shown Each Ct answer is available will be considerably shortened and the need for

maintaining expensive cell culture facilities reduced Here we present the using the Jonstrup et al. PCR or the Garver et al. PCR are shown. Each Ct

l i b d f b f diff t VHSV i l t d maintaining expensive cell culture facilities reduced. Here we present the value is based on an average of a number of different VHSV isolates and validation of a sensitive and specific Taqman based real time RT-PCR that each of these were performed in triplicates. Also a panel of 15 other fish p qdetects all VHSV isolates in a panel of 79 VHSV isolates covering all known

p p pviruses was used Unspecific signals were not observed for the Jonstrup et detects all VHSV isolates in a panel of 79 VHSV isolates covering all known

genotypes and subtypes The sensitivity of the real time RT PCR is viruses was used. Unspecific signals were not observed for the Jonstrup et al PCR indicating an analytical specificity close to 1 It was also genotypes and subtypes. The sensitivity of the real time RT-PCR is

bl di i l ll b d h d d i i ibl b i al. PCR indicating an analytical specificity close to 1. It was also

comparable to traditional cell based methods and it is possible to obtain a concluded that our PCR competes with the Garver et al. PCR and especially diagnosis within one work day.

p p yfor genotype II and III it performs significantly betterdiagnosis within one work day. for genotype II and III it performs significantly better.

Diagnostic specificityg p yKnown positive diagnostic samples (16) from recent Danish VHS outbreaks Known positive diagnostic samples (16) from recent Danish VHS outbreaks

d 3 k i ill l f i h S fand 43 known negative surveillance samples from Danish VHSV free g prainbow trout farms were examined. All positive isolates were detected rainbow trout farms were examined. All positive isolates were detected with Ct values of15 to 25 and none of the known negative samples gave with Ct values of15 to 25 and none of the known negative samples gave i t f l iti ti A i t l ELF1 t l d th t rise to any false positive reactions. An internal ELF1a control ensured that

the lacking signal was not due to degradation of sample. Taken together the lacking signal was not due to degradation of sample. Taken together with the results from the investigations of analytical specificity it was with the results from the investigations of analytical specificity it was

l d d th t th di ti ifi it f thi i l t 1concluded that the diagnostic specificity of this assay is very close to 1.

Di i i i iDiagnostic sensitivityg yRainbow trout (84) from a VHSV infection study were examined using both Rainbow trout (84) from a VHSV infection study were examined using both

ll lt d th J t t l l ti RT PCR I 35 fi h th ll cell culture and the Jonstrup et al. real time RT-PCR. In 35 fish the cell culture titer was below the detection limit (<1.9x102 TCID50 ml-1). The ( 50 )remaining 49 fish had titer values evenly distributed from low to high In

Figure 1: Design of Taqman probe and primers. Alignment of nt 532-remaining 49 fish had titer values evenly distributed from low to high. In 25 of the 35 fish all with titers below detection limit no virus were detected Figure 1: Design of Taqman probe and primers. Alignment of nt 532

608 (according to GenBank accession number Z93412) of the N gene of 25 of the 35 fish all with titers below detection limit no virus were detected

608 (according to GenBank accession number Z93412) of the N-gene of diff t VHSV i l t i t t VHSV

in either test. In 45 fish virus was detected by both methods. 8 fish were different VHSV isolates spanning most genotypes. VHSV sequences were

ypositive in PCR but not in cell culture and 6 vice verse All fish with a titer

retrieved from www.FishPathogens.eu. The probe was designed to target positive in PCR but not in cell culture and 6 vice verse. All fish with a titer ≥ 2 7x103 TCID ml-1 were detected using both methods Assuming no retrieved from www.FishPathogens.eu. The probe was designed to target

an area where no variation between isolates where seen while primers ≥ 2.7x103 TCID50 ml 1 were detected using both methods. Assuming no f l f l h d f h lan area where no variation between isolates where seen while primers

h d i d th t i i t h i th FW i d t false negatives or false positives the diagnostic sensitivity of the real time

where designed so that maximum one mismatch in the FW primer and two g p g y

RT-PCR was determined to 53/59 (0.90), while the diagnostic sensitivity of in the BW primer was observed.

RT PCR was determined to 53/59 (0.90), while the diagnostic sensitivity of the cell culture experiment was 51/59 (0 86)p the cell culture experiment was 51/59 (0.86).

Conclusions:100 101 102 103 104 10510 10 10 10 10 10

BF-2 + + + + + + + + + + + Analytical and diagnostic specificity determined to be very close to 1BF 2 + + + + + + + + + + +EPC + + + + + + +

Analytical and diagnostic specificity determined to be very close to 1N f l iti b dEPC + + + + + + + - No false positives observed

Jonstrup et al + + + + + + + + + + + + + + +Jonstrup et al. + + + + + + + + + + + + + + +Diagnostic sensitivity determined to be around 0 90Diagnostic sensitivity determined to be around 0.90

- When RT-PCR is performed in triplicatesFigure 2: Analytical sensitivity. 10x dilutions of VHSV isolates were

When RT PCR is performed in triplicatesC ll lt h d iti it f d 0 86g y y

made in triplicate for all 4 genotypes (Ia shown as example here) Each - Cell culture had a sensitivity of around 0.86

made in triplicate for all 4 genotypes (Ia shown as example here). Each dilution was tested for VHSV by either traditional diagnosis (cultivation on dilution was tested for VHSV by either traditional diagnosis (cultivation on

Assay also passed tests on (data not shown here):BF-2 or EPC cell lines followed by ELISA) or by the Jonstrup et al. RT-PCR. Assay also passed tests on (data not shown here):y ) y pThe number of plusses indicates the number of positive wells found within - Robustness, rugedness, repeatability, and reproducibilityThe number of plusses indicates the number of positive wells found within a specific triplicate diagnosed by a specific method Sensitivity of the cell

, g , p y, p ya specific triplicate diagnosed by a specific method. Sensitivity of the cell

l f d b h h f ll P f t th l l b tt th th bli h d l ti RTculture versus PCR was found to be within the same range for all Performs at the same level or better than other published real-time RT-ggenotypes (+/- 102). PCR assays for detection of VHSVgenotypes (+/ 10 ). PCR assays for detection of VHSV

G KA H l LM M Cl CA S h d T Ald S D i F S M Garver KA, Hawley LM, McClure CA, Schroeder T, Aldous S, Doig F, Snow M,

National Veterinary InstituteEdes S, Baynes C, Richard J (2011) Development and validation of a reverse National Veterinary Institute, y , ( ) ptranscription quantitative PCR for universal detection of viral hemorrhagic transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus Dis Aquat Org 95:97-112

For further information please contact:septicemia virus. Dis Aquat Org 95:97 112

For further information please contact:Sø P t J t ( j @ t dt dk)Søren Peter Jonstrup (spjo@vet.dtu.dk)