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Detection of white spot syndromevirus

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VIRMET-11751;No.of Pages 4

Journal of Virological Methods xxx (2012) xxx–xxx

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Journal of Virological

Methods

j 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 e

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Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions

Yun-Long Tsai a ,Yu-Chan Lin a ,Pin-Hsing Chou a ,Ping-Hua Teng a ,Pei-Yu Lee b ,?

a Department of Research and Development,GeneReach Biotechnology Corporation,Taichung,Taiwan

Institute of Medical Biotechnology,Central Taiwan University of Science and Technology,Taichung,Taiwan

Article history:

Received 6October 2011

Received in revised form 16January 2012Accepted 18January 2012Available online xxx

Keywords:

Convective PCR

Nucleic acid ampli?cation

Insulated isothermal conditions iiPCR WSSV

a b s t r a c t

Aiming to develop a rapid,low-cost,and user-friendly system for the diagnosis of white spot syndrome virus (WSSV),a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay)was established on the basis of Rayleigh–Benard convection.WSSV amplicons were generated reproducibly within 30min from a target sequence-containing plasmid in an iiPCR device,in which a special polycarbonate capillary tube (R-tube TM )was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baf?e around its upper half.Furthermore,WSSV-speci?c ampli-cons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay,with sensitivity comparable to that of an OIE-certi?ed commercial nested PCR kit (IQ2000TM WSSV Detection and Prevention System).Speci?city of the WSSV iiPCR assay was demonstrated as no ampli-cons were generated from shrimp genomic DNA,and IHHNV,MBV,and HPV DNA.iiPCR has a potential as a low-cost method for sensitive,speci?c and rapid detection of pathogens.

White spot syndrome virus (WSSV),a large double-stranded DNA virus,infects a broad range of freshwater and marine crus-taceans including shrimp,crabs,cray?sh and lobsters (Hameed et al.,2003;Lo et al.,1996a;Syed Musthaq et al.,2006).WSSV infection causes high mortality rates and severe economic losses in the shrimp aquaculture industry worldwide.Pre-screening of WSSV-free broodstock or larvae and regular surveillance of WSSV infection are important strategies to reduce the economic impacts of the disease on shrimp aquaculture.Some nucleic acid-based methods,such as conventional polymerase chain reaction (PCR)(Kasornchandra et al.,1998;Lo et al.,1996b;Vaseeharan et al.,2003),in situ PCR (Jian et al.,2005),real-time PCR (Durand and Lightner,2002),nested PCR (Lo et al.,1996b;Nunan and Lightner,2011),and loop-mediated isothermal ampli?cation (LAMP)(Chou et al.,2011a;Kono et al.,2004)have been developed for WSSV detection.

Although PCR offers high degrees of detection sensitivity and speci?city,the requirements,such as trained technicians and an expensive thermocycler,have limited the application of PCR-based assays in the aquaculture industry.In Rayleigh–Benard convection,PCR cycles can be completed without the need of a thermocy-cler.Simple heating sources are able to generate ?uid density

?Corresponding author at:Institute of Medical Biotechnology,Central Taiwan University of Science and Technology,Rm1311,No.11,Buzih Lane,Taichung 406,Taiwan.Tel.:+886422391647x6936;fax:+886422395474.

E-mail address:pylee@https://www.wendangku.net/doc/c517119515.html,.tw (P.-Y.Lee).

gradients to drive laminar convection of the solution in a closed space,carrying the reaction components through sequen-tial temperature zones to facilitate the completion of PCR cycles (Chandrasekhar,1961;Krishnan et al.,2002).Convection PCR could shorten reaction time from hours to about 30min in comparison to conventional PCR,providing a method for the development of a fast,low-cost,and user-friendly system for nucleic acid ampli?ca-tion.On the basis of Rayleigh–Benard convection,a recent report demonstrated that amplicons could be generated successfully in glass capillary tubes that were heated at one end (bottom)by a sim-ple heating device (Chou et al.,2011b ).However,convection ?ows in the reaction cell could be affected easily by temperature changes in the surrounding environment (Chou et al.,2011b ),leading to erratic termination of the PCR cycles.

In this report,successful convection PCR was carried out in spe-cially designed capillary tubes in an insulated isothermal device (iiPCR)(Chang et al.,2012).A thermal baf?e made of aluminum was installed to shield the upper half of reaction tubes and to reduce impacts of environmental temperature ?uctuations on the convection in reaction tubes.At the same time,the heating block was covered with insulated material to help reduce the in?u-ence of heat radiation on air convection of the surrounding space (Supplementary Fig.1).As a substitute,heating forks connected to the heating block were positioned to make contact with a 2.5mm thick copper ring attached at 2mm above the bottom of each capil-lary tube (Supplementary Fig.1).Polycarbonate (PC)capillary tubes (R-tube TM ,GeneReach)were utilized to substitute for glass tubes or Plexiglas cubes used previously (Chou et al.,2011b;Krishnan et al.,

Y.-L.Tsai et al./Journal of Virological Methods xxx (2012) xxx–

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Fig.1.Ampli?cation of pWSSV1by iiPCR and conventional PCR Different copies (103to 10?1)of the pWSSV plasmid were subjected to WSSV iiPCR or conventional PCR assay.Both systems employed the same set of primers.The amplicons were analyzed on a 12%polyacrylamide gel in 1×TAE buffer.M,DNA size markers;N,non-template control.

2002).Considerable heat conductivity of the copper rings ensured consistent heating at the bottom of the R-tubes.Stable tempera-ture gradients ranged from 94?C (bottom)to 50?C (top)could be generated and maintained in the R-tubes in this simple setup when it is placed at room temperature (Chang et al.,2012).

With the aim of developing a rapid WSSV diagnosis system,a WSSV iiPCR was established using this device.First,plasmid pWSSV1(Chou et al.,2011a )that contains a 1254bp DNA fragment of the WSSV genome was used as the template to establish the opti-mal iiPCR conditions.The forward (5 -AATGGTCCCGTCCTCATCTCA-3 )and reverse (5 -GCTGCCTTGCCGGAAATT-3 )primers speci?c for WSSV were designed by following the criteria described pre-viously for convective PCR (Chou et al.,2011b ).The 50?l iiPCR mixtures,which contained 103copies of pWSSV1,0.5?M for-ward primer,0.5?M reverse primer,0.5mM dNTP,25units of Taq DNA polymerase (BioMi,Taichung,Taiwan),50mM Tris–HCl (pH 8.3),75mM KCl,3mM MgCl 2,and 1mM DTT,were assem-bled in R-tubes and incubated in the device described above for 30min.Amplicons were analyzed subsequently on a 12%poly-acrylamide gel in TAE buffer (40mM Tris,20mM acetic acid and 1mM EDTA)and visualized by ethidium bromide staining.For com-parison,the same mixtures were subjected to convectional PCR ampli?cation performed in a PCR thermocycler (ABI 2720,Life Technologies,Carlsbad,CA).The program included an incubation period at 94?C for 2min,35cycles of 94?C for 20s,50?C for 20s and 72?C for 30s,and one cycle of 72?C for 30s and 20?C for 20s.The results demonstrate that a 71bp WSSV-speci?c product,which was con?rmed by DNA sequencing analysis (data not show),was ampli?ed in both iiPCR and conventional PCR.The sensitivity of WSSV iiPCR,at 101copies per reaction,was similar to that of the conventional PCR (Fig.1),suggesting that iiPCR can reach ampli?ca-tion ef?ciency comparable to that of conventional PCR.Detectable amounts of amplicons were generated consistently from positive samples in 30min,which was comparable to the 10–30min range reported in the various convection PCR systems reported so far (Braun,2004;Chung et al.,2010;Hennig and Braun,2005;Muddu et al.,2011;Wheeler et al.,2004).

PCR ampli?cation may be interfered by inhibitors present in tissue extracts (Wilson,1997).In addition,host nucleic acids could also compete with the primers.To test whether iiPCR can amplify WSSV DNA in shrimp samples ef?ciently,crude nucleic acid extracts of WSSV-infected P.vannamei samples were pre-pared using the Lysis buffer method (BioMi,Taichung,Taiwan)as described previously (Chou et al.,2011a ).Ten-fold serial dilutions of the extracts were subjected simultaneously to the WSSV iiPCR assay and the nested PCR-based IQ2000TM WSSV Detection and Prevention System (DPS),which was assembled and carried out

Fig.2.Evaluation of WSSV detection in shrimp samples by WSSV iiPCR.Serial 10-fold dilutions (10?2,10?3,10?4and 10?5)of genomic DNAs extracted from a WSSV-infected shrimp were analyzed by WSSV iiPCR assay (A)or IQ2000TM WSSV DPS (B).The IQ2000TM WSSV DPS included also serial dilutions (103,102,101,100copies)of a WSSV standard DNA.The kit was designed to generate three amplicons (arrows)from WSSV genome,and the number of the product bands correlates pos-itively with the starting concentrations of target DNA.Each sample was tested in duplicate.Amplicons from iiPCR and IQ2000TM WSSV DPS were analyzed on a 12%polyacrylamide gel in 1×TAE buffer and agarose gel electrophoresis,respectively.M,DNA size markers;N,non-template control.

described by the manufacturer.The endpoints for the detection of WSSV (1:10?4dilution)were comparable between the two assays (Fig.2A and B),suggesting that the primers reacted speci?cally with WSSV DNA with a sensitivity similar to that of IQ2000TM WSSV DPS and extracts of shrimp tissues prepared this way were compatible with the iiPCR reaction.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV),monodon baculovirus (MBV),and hepatopancreatic par-vovirus (HPV)are other common DNA virus pathogens found in shrimp (Lightner,2011).To ensure that the WSSV iiPCR does not cross react with genomes of these viruses,WSSV-,IHHNV,MBV,or HPV-positive P.vannamei identi?ed by IQ2000TM WSSV DPS (Fig.3A,lane 1),IQ2000TM IHHNV DPS (Fig.3B,lane 2),IQ2000TM MBV DPS (Fig.3C,lanes 3and 4),or IQ2000TM HPV DPS (Fig.3D,lanes 3and 4),respectively,were analyzed by the WSSV iiPCR assay (Fig.3E).Results of the iiPCR assay demonstrate that ampli-cons were generated only from WSSV-(Fig.3E,lane 1),but not from IHHNV-,MBV-,and HPV-positive samples (Fig.3E,lanes 2–4),indicating the established iiPCR assay could detect WSSV speci?cally.

Although the established WSSV iiPCR assay demonstrated sen-sitivity similar to that of a conventional WSSV PCR,several primer sets that were able to generate amplicons in conventional PCR could not produce amplicons ef?ciently in iiPCR (data not show).Circulation of the reaction in R-tubes allowed relatively short peri-ods of time (only a few seconds)for each iiPCR cycle (Chou et al.,2011b;Krishnan et al.,2004),implying that the optimal param-eters (such as primer criteria and buffer compositions)for iiPCR may be different from those for conventional PCR and need to be further investigated.In order to include an internal control

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Fig.3.Speci?city evaluation of WSSV iiPCR assay.Genomic DNA extracts of WSSV-(lane1),IHHNV-(lane2),MBV-(lane3),and HPV-infected(lane4),and uninfected(lane 5)shrimp were subjected to ampli?cation by(A)IQ2000TM WSSV DPS,(B)IQ2000TM IHHNV DPS,(C)IQ2000TM MBV DPS,(D)IQ2000TM HPV DPS and(E)WSSV iiPCR.Positive standard DNAs provided by the IQ2000TM kits were diluted(103,102,101copies)and included in the assays.The IQ2000TM kits were designed to generate three amplicons (closed arrow heads)from the target sequences((A)lane1;(B)lane2;(C)lanes3and4;(D)lanes3and4).Samples3and4appeared to be co-infected by both MBV and HPV.The number of the product bands correlates positively with the starting concentrations of target DNA.In addition,the presence of the internal-control signal(open arrow heads)and the absence of target signals implicate that the samples are target-pathogen free(A–D,lane5).Arrows,WSSV iiPCR amplicons;M,DNA size markers;P, 103copies of a WSSV standard DNA;N,water only.

to ensure that the DNA extraction and the PCR procedures have been implemented properly,development of multiplex iiPCR is in progress.Furthermore,iiPCR amplicons were analyzed by gel elec-trophoresis,which is labor-intensive,time-consuming and prone to cross-contamination.Different ways of amplicon detection will have to be integrated into the iiPCR device to make the method more user-friendly.

In order to generate a circulatory?ow that can transport the reaction components through the critical temperature zones involved in the PCR process,heating mechanisms and geome-try of the reaction vessel are major topics in the development of convection PCR.Successful convection PCR has been demon-strated in reactors of cylindrical or looped geometries(Agrawal et al.,2007;Agrawal and Ugaz,2007;Chen et al.,2004;Chou et al.,2011b;Krishnan et al.,2004;Wheeler et al.,2004).Reac-tors of looped geometries,which work in closed design,would require manual elimination of air pockets(Chung et al.,2010). Alternatively,successful convective PCR was reported by dip-ping a constantly heated wire directly into an open vessel (Hennig and Braun,2005).However,repeated use of the wire might lead to higher risks of cross-contamination from sample to sample.These problems could be largely avoided in the iiPCR method,because all components of the open-designed R-tube TM are inexpensive and disposable.Furthermore,less precaution is required to handle polycarbonate tubes than the glass capillary tubes,which were used by the capillary convection PCR method (Chou et al.,2011b).

The WSSV iiPCR assay could amplify its target DNA speci?-cally,sensitively and reproducibly if carried out in the specially designed R-tubeTM in the small iiPCR device designed par-ticularly to stabilize convection PCR.The iiPCR system has a considerable potential to be developed into a fast,user-friendly and portable method for pathogen detection in the aquaculture industry.Appendix A.Supplementary data

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