一步血清PT-PCR

Journal of Virological Methods 190 (2013) 1–3Contents lists available at SciVerse ScienceDirectJournal of VirologicalMethodsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j v i r o m etShort communicationDirect RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virusClaudia Bachofen a ,∗,Kim Willoughby a ,Ruth Zadoks a ,Paul Burr b ,Dominic Mellor c ,George C.Russell aaMoredun Research Institute,Pentlands Science Park,Penicuik,Midlothian EH260PZ,UK bBiobest Laboratories Ltd.,The Edinburgh Technopole,Penicuik,Midlothian EH260PY,UK cSchool of Veterinary Medicine,College of Medical,Veterinary and Life Sciences,University of Glasgow,Bearsden Road,Glasgow G611QH,UKArticle history:Received 17December 2012Accepted 13March 2013Available online 26 March 2013Keywords:PestivirusBovine viral diarrhoea Direct RT-PCR Sequencing DatabaseMolecular epidemiologya b s t r a c tStudies of the molecular epidemiology of viral diseases are dependent on the analysis of large numbers of samples from infected individuals,and the assembly of relevant sequence databases are a prerequisite to investigate chains of infection.As part of research in support of the Scottish BVDV eradication campaign,we have established a direct RT-PCR method for the high throughput amplification and analysis of the informative 5 -untranslated region of the BVDV genome.Heat-treatment followed by a one-step RT-PCR,performed in 96-well plates,produced sufficient material for sequence analysis from 0.5␮l of serum or plasma.Of 93samples assayed,only five failed to give full sequence data for the region amplified and these were subsequently successfully analysed in single tube format reactions.This approach improved the speed of analysis,reduced costs,operator time and the potential for contamination,and may allow analysis of samples for which volumes are too low for conventional RNA isolation.It also has the potential for wider application in both human and animal disease research in which high throughput and low cost would increase the size of datasets that can be obtained.© 2013 Elsevier B.V. All rights reserved.Bovine viral diarrhoea virus (BVDV)is a major pathogen of cattle that causes important economic losses for cattle producers around the world.It is a single-strand RNA virus,closely related to classical swine fever virus (CSFV)of pigs and border disease virus (BDV)of sheep,belonging to the genus Pestivirus within the family Flaviviri-dae (Pletnev et al.,2011).BVDV also shares genetic similarity with hepatitis C virus (HCV)of humans,another flavivirus,and has often been used as a model for HCV (Zitzmann et al.,1999;Buckwold et al.,2003).BVDV has the ability to infect the bovine foetus and to cause a persistent infection that is tolerated by the host’s immune system (Peterhans and Schweizer,2010).This complex strategy is only possible if infection occurs between the second and fourth months of gestation (Liess,1985).Persistently infected (PI)animals are virus ‘factories’:BVDV can be isolated from most tissues and secretions and is shed very efficiently.Hence,PI animals enable the virus to spread and persist in cattle herds (Nettleton and Entrican,1995).The clinical signs in affected herds are variable and non-specific:abortions,reduced fertility,pneumonia and diarrhoea are common (Moerman et al.,1994).At the herd level,∗Corresponding author.Tel.:+4401314455111.E-mail addresses:claudia.bachofen@ ,c.bachofen@ (C.Bachofen).BVDV can only be effectively controlled by detecting and removing PI animals (Lindberg,2003).Due to the economic impact of this infection,several European countries,including Scotland,have ongoing national eradica-tion programmes (Heffernan et al.,2009),following the lead of Scandinavian countries.As the example of Sweden has shown,re-infections of previously BVDV-free herds are a major problem towards the end of the eradication programme and can prolong the final phase.In this situation,molecular epidemiology,by means of molecular tracing of chains of infections,has proven to be an important addition to classical epidemiology in order to find sources of infections and routes of virus transmission responsible for herd breakdowns (Ståhl et al.,2005).However,for molecular tracing to be used efficiently,a comprehensive collection of BVDV sequences is required for comparison with ‘new’virus samples.Ideally,BVDV PCR products from all PI animals detected during eradication would be sequenced to form a national database.Since this entails sequencing hundreds or thousands of samples,a fast and cost-effective method is required.In addition,since BVDV is an RNA virus,reverse transcription of purified viral RNA is required before PCR amplification.RNA isolation,reverse transcriptase (RT)-PCR and sequencing in 96-well format constitute a time-consuming and potentially costly workflow.In addition,the multiple steps involved may be susceptible to cross-contamination between samples.Hence,prior to the establishment of a sequence database in support of BVDV0166-0934/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.jviromet.2013.03.0152 C.Bachofen et al./Journal of Virological Methods190 (2013) 1–3eradication in Scotland,we have tested methods for direct RT-PCR of BVDV from serum.RT-PCR of BVDV without prior RNA isolation has been described by Gilbert et al.(1999)and Deregt et al.(2002), who used direct amplification from tissue culture supernatant or whole blood in a nested multiplex RT-PCR.However,in our case,the only samples currently available are frozen sera and plasma sub-mitted for diagnostic testing.In addition,since the use of a nested RT-PCR is time-consuming and susceptible to contamination,we sought a one-step RT-PCR method that was suitable for serum sam-ples.Direct RT-PCR methods have been described for several other viruses,e.g.influenza virus(Song et al.,2009),Chikungunya virus (Pastorino et al.,2005)and Newcastle disease virus(Wambura, 2006).In most cases,however,the template material used was tissue culture propagated virus,whole blood or tissue samples. Abe(2003)described the detection of HCV by direct RT-PCR from serum.The serum was diluted1:5in phosphate buffered saline (PBS),heated at95◦C for4min and cooled on ice for3–5min before using directly in RT-PCR.Because of the relatedness of HCV and BVDV,this method was tested using serum from a BVDV PI animal. Dilutions of serum(heat-treated or controls)were analysed using an established BVDV RT-PCR.Briefly,10␮l of PI serum was diluted 1:5with PBS in a0.2ml PCR tube and heated in a thermal cycler for 3min at95◦C before being cooled on ice for a minimum of5min. We then added2.5␮l samples of heat-treated or control serum to22.5␮l of RT-PCR mix.For RT-PCR,the SuperScript III one-step RT-PCR system with Platinum Taq polymerase(Invitrogen)was supplemented with0.2␮l of recombinant RNasin RNase inhibitor (Promega).The previously described primer pair324/326(Vilˇc ek, 1994)was used to amplify a244–247base pair(bp)fragment of the conserved5 untranslated region(UTR)of BVDV.This fragment is widely used for phylogenetic analyses of pestiviruses(Becher et al.,1997;Harasawa,1996;Vilˇc ek et al.,2004)and for molecular tracing of BVDV(Ståhl et al.,2005).Cycling conditions were as follows:30min at50◦C for reverse transcription;2min at94◦C for Taq activation;40cycles of15s at94◦C for denaturation,30s at55◦C for annealing,1min at68◦C for extension;and afinal extension of5min at68◦C.To visualise the amplified products, 10␮l of the RT-PCR products were used for gel electrophoresis. Amplification of untreated sera gave only very faint bands,but the diluted and heat-treated sera produced bands comparable to those seen using isolated serum RNA as template(data not shown).To estimate the detection limit of the direct RT-PCR compared to RT-PCR after RNA isolation,we diluted serum from the same PI animal1:3,1:10,1:30and1:100with bovine serum(tested free of BVDV RNA or antibodies against BVDV).The undiluted serum and each of the4dilutions were then further diluted1:5in PBS.From the1:5dilutions,140␮l were used for RNA isolation(QIAmp viral RNA mini kit,Qiagen;elution in60␮l)and50␮l were heat-treated for direct amplification.The RT-PCR was performed asdescribed Fig.2.Image of96-well plate containing50␮l aliquots of1:5diluted,heat-treated serum used as RT-PCR templates.The degree of haemolysis can be estimated from the colour of each well.Samples used for additional tests and controls are ringed and numbered as follows:1,Heparin plasma;2,EDTA plasma;3,cell culture medium; 4,PBS control;5,serum storage trial samples;6,water control;7,Qiaxcel size standards.Samples that failed to give complete5 UTR sequences are boxed:solid boxes indicate samples that failed,while dashed boxes indicate samples that gave incomplete or poor quality sequence data.above:2.5␮l of pre-treated serum or eluted RNA were used in a 25␮l reaction and10␮l of each RT-PCR product were visualised after agarose gel electrophoresis.The detection limit of the direct RT-PCR was similar to RT-PCR after RNA isolation(Fig.1).In both cases,the PCR products of the1:100diluted materials could be visu-alised but the bands from the direct RT-PCR were slightly stronger. Thisfinding is quite remarkable when considering that the input volume of serum in the undiluted lanes was only0.5␮l for the direct RT-PCR while the RNA used for the‘normal’RT-PCR was equivalent to1.2␮l of serum(Fig.1,nes).Based on this encouraging result,we tested the suitability of this method for RT-PCR in96-well plates for sequence analysis of serum or plasma samples from PI animals detected during the Scot-tish BVDV eradication programme.This test used86serum samples that had previously been tested positive for BVDV by a commercial antigen capture ELISA(Idexx BVDV Ag/serum plus test)performed by Biobest Laboratories.The sera originated from animals older than one month of age,were tested between January and July2012 and were stored frozen at−20◦C in the interim.In addition to the86serum samples,we also tested one sample each of known BVDV positive EDTA plasma,heparin plasma and cell culture super-natant(Fig.2).Furthermore,serum from one PI animal was used to test the stability of the RNA in heat-treated samples bystoring Fig.1.Agarose gel electrophoresis of RT-PCR products amplified from serum with and without prior RNA isolation.Undiluted positive serum,or positive serum serially diluted up to100-fold in BVDV-negative serum,was further diluted1:5in PBS and used as template for direct RT-PCR(right lanes,B)or RT-PCR after RNA isolation(left lanes, A).The degree of dilution in BVDV-negative serum is given above each gel lane(undil.=undiluted)and sizes(bp)of DNA markers(Hyperladder IV,Bioline)are given on the right of the gel.C.Bachofen et al./Journal of Virological Methods190 (2013) 1–33aliquots for24h at room temperature,4◦C or−20◦C prior to RT-PCR in the96-well plate(Fig.2).After sample pre-treatment and RT-PCR as described above,we screened the plate for the presence of amplicons using the Qiaxcel Advanced capillary gel electrophore-sis system(Qiagen).Except for two samples that showed only a faint band,all positive samples amplified a clear single product of the correct size.The RT-PCR products were diluted1:2in water to reach the DNA concentration required for sequencing and20␮l of each sample were transferred to two fresh96-well plates.The plates were sealed and sent to GATC Biotech(GATC Biotech AG, Konstanz,Germany)for clean-up and Sanger sequencing with the primers used for RT-PCR:one plate with the forward primer(324) and one with the reverse primer(326).Complete BVDV5 UTR PCR product sequences were obtained from all exceptfive of93positive samples analysed(5.4%failure rate).In the complete sequences,the reads from the forward and reverse primers were clear and assembled perfectly,suggesting that inter-well contamination or sample misplacement was not an issue at any stage of the workflow.In addition,the plate contained a number of duplicate samples(storage trial)and several sam-ples that had been analysed previously.In these cases,the samples were identical to each other or to the previously defined sequence, respectively.The successful sequencing of94.6%of the samples was surprising because the quality of the serum was highly variable, including some that were very haemolytic(Fig.2).Indeed,all of the sample types tested,including serum,EDTA plasma,heparin plasma and BVDV cell culture supernatant amplified and sequenced without problems.The wells containing PBS control,RT-PCR nega-tive control(water)and Qiaxcel marker gave no sequencing results. The two samples that showed faint bands in the Qiaxcel analy-sis resulted in low quality sequences that did not cover the entire 5 UTR fragment.Three other sera that gave detectable RT-PCR prod-ucts failed completely.The reason for this failure is unknown but 96-well format sequencing of the same PCR product from purified serum RNA in the Swiss eradication programme has been observed to give a similar failure rate of6.3%(H.P.Stalder,personal com-munication;n=7299).The direct RT-PCR and sequencing reactions were repeated in single tubes for thefive serum samples that failed in the96-well sequencing reaction,and they all yielded complete BVDV5 UTR sequences.The successful amplification and sequencing of heat-treated serum that had been stored for24h at room temperature,4◦C or −20◦C prior to RT-PCR indicates that the viral RNA is relatively stable after heat-treatment.This was unexpected as the heating process should denature the protein capsid of the virus,making the viral RNA accessible for RT-PCR but also susceptible to serum RNases,some of which may be heat resistant.This suggests that the heat-treatment of samples largely inactivates serum RNases.The heat-treated serum samples used for comparison of the detection limit of the direct RT-PCR(Fig.1)were also tested in a BVDV real-time RT-PCR(Willoughby et al.,2006)but,in this format, detection of viral RNA in the heat-treated serum was reduced by nearly10-fold compared to detection of BVDV after RNA isolation (data not shown).As expected,real-time PCR seemed to be more susceptible to inhibition by serum components than conventional RT-PCR.In conclusion,we have shown that the method of Abe(2003), using simple dilution and heat-treatment of serum samples instead of RNA isolation,can be easily adapted for direct RT-PCR of BVDV. The detection limit is comparable to using isolated RNA(Fig.1) but direct RT-PCR can be used to analyse tiny volumes of serum. Furthermore,this approach is a fast,easy and cost-effective way to prepare samples for sequencing of the5 UTR of BVDV for phy-logenetic analysis of large numbers of samples,facilitating the assembly of sequence databases for molecular epidemiology.Since this approach has been shown to work for direct RT-PCR of hep-atitis C virus and direct PCR of hepatitis B virus(Abe,2003)in addition to BVDV,it might be applicable to a wider range of RNA and DNA viruses of animal or human origin,reducing time and costs for conventional(RT-)PCR.AcknowledgmentsThis work was supported by the Scottish government through the Centre of Expertise on Animal Disease Outbreaks(EPIC).The work of C.B.was funded by a fellowship grant from the Swiss National Science Foundation.We are indebted to Jess Gaudy and Richard Irvine from The School of Veterinary Medicine,University of Glasgow,and to col-leagues at Biobest Laboratories for provision of the BVDV-positive serum samples used in this publication.ReferencesAbe,K.,2003.Direct PCR from serum:application to viral genome detection.In: Bartlett,J.M.S.,Stirling,D.(Eds.),PCR Protocols.Tatowa,New Jersey,pp.161–166. 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