Boutrup, Torsten Snogdal

Jonstrup, Søren Peter

Kahns, Søren

Olesen, Niels Jørgen

Skall, Helle Frank

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 19, 2017Validation of a Taqman based real time RT-PCR assay suitable for surveillance anddiagnosis of Viral Haemorrhagic Septicaemia Virus worldwideJonstrup, Søren Peter; Kahns, Søren; Skall, Helle Frank; Boutrup, Torsten Snogdal; Olesen, NielsJørgenPublication date:2011Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (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 -diagnosis of Viral Haemorrhagic Septicaemia Virus worldwideS P  Jonstrup  S  Kahns  H F  Skall  T S  Boutrup  and N J  Olesen. . , . , . . , . . , . .DTU Vet  Section for Fish Diseases  Aarhus  Denmark, , ,Genotype Number of isolates Jonstrup Garver  I 2 21,9 23,2I 18 20 3 21 3a , ,Ib 14 19 7 21 0, ,Ic 3 19 4 20 3, ,Id 3 20 8 21 7Introduction: Viral haemorrhagic septicaemia (VHS) is a serious disease , ,in several fish species  VHS is caused by the rhabdovirus Viral Ie 2 21,3 23,1.Haemorrhagic Septicaemia Virus (VHSV)  To prevent outbreaks it is .i   h   f  i i  d ifi  di i  l  id if  II 4 21,6 25,1mportant to ave a ast, sens t ve, an spec c agnost c too to ent yIII 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 2. .th  l  S ill  f  d l i  f d  f VHS i  fi h f  i  , ,ra er s ow. urve ance or ec ar ng ree om o n s arms s 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 testiti it  d ifi it  t  th  ll lt  th d  B  it hi  t  RT sens v y an spec c y o e ce cu ure me o s. y sw c ng o -PCR for surveillance and diagnosis of VHS the time needed before an Figure 3: Analytical specificity test. The average Ct values obtained by answer is available will be considerably shortened and the need for using the Jonstrup et al  PCR or the Garver et al  PCR are shown  Each Ct maintaining expensive cell culture facilities reduced  Here we present the . . .l  i  b d    f  b  f diff t VHSV i l t  d . va ue s ase on an average o a num er o eren so a es anvalidation 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 detects all VHSV isolates in a panel of 79 VHSV isolates covering all known viruses was used  Unspecific signals were not observed for the Jonstrup et genotypes and subtypes  The sensitivity of the real time RT PCR is .al  PCR indicating an analytical specificity close to 1  It was also . -bl   di i l ll b d h d  d i  i  ibl   b i   . .compara e to tra t ona ce ase met o s an t s poss e to o ta n a concluded that our PCR competes with the Garver et al. PCR and especially diagnosis within one work day. for genotype II and III it performs significantly better.Diagnostic specificityKnown positive diagnostic samples (16) from recent Danish VHS outbreaks d 3 k i ill l f i h S fan  4  nown negat ve surve ance samp es rom Dan s  VH V ree rainbow trout farms were examined. All positive isolates were detected 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 r se o any a se pos ve reac ons. n n erna a con ro ensure athe lacking signal was not due to degradation of sample. Taken together 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  1conc u e a e agnos c spec c y o s assay s very c ose o .Di i  i i iagnost c sens t v tyRainbow 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 ce cu ure an e ons rup e a . rea me - . n s e ceculture titer was below the detection limit (<1.9x102 TCID50 ml-1). The 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-.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 eren so a es spann ng mos geno ypes. sequences were positive 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 .≥ 2 7x103 TCID ml-1 were detected using both methods  Assuming no an area where no variation between isolates where seen while primers . 50 .f l f l h d f h lh  d i d  th t i   i t h i  th  FW i  d t  a se negatives or a se positives t e iagnostic sensitivity o  t e rea  time w ere es gne so a max mum one m sma c n e pr mer an wo RT-PCR was determined to 53/59 (0.90), while the diagnostic sensitivity of in the BW primer was observed. the cell culture experiment was 51/59 (0 86). .Conclusions:100 101 102 103 104 105BF-2 + + + + + + + + + + + Analytical and diagnostic specificity determined to be very close to 1       EPC + + + + + + + N  f l  iti  b d    - o a se pos ves o serveJonstrup et al + + + + + + + + + + + + + + +  .          Diagnostic sensitivity determined to be around 0 90.- When RT-PCR is performed in triplicatesFigure 2: Analytical sensitivity. 10x dilutions of VHSV isolates were C ll lt  h d  iti it  f d 0 86made in triplicate for all 4 genotypes (Ia shown as example here)  Each - e cu ure a a sens v y o aroun ..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. The number of plusses indicates the number of positive wells found within - Robustness, rugedness, repeatability, and reproducibilitya 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  RTcu ture versus PCR was oun  to e wit in t e same range or a  er orms a e same eve or e er an o er pu s e rea - me -genotypes (+/- 102). PCR assays for detection of VHSVG  KA  H l  LM  M Cl  CA  S h d  T  Ald  S  D i  F  S  M  arver , aw ey , c ure , c roe er , ous , o g , now ,National Veterinary InstituteEdes S, Baynes C, Richard J (2011) Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus  Dis Aquat Org 95:97-112For further information please contact:.Sø  P t  J t  ( j @ t dt dk)ren e er ons rup sp o ve . u.

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

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

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