Journal of Virological Methods 92 (2001) 105–111

Optimized protocols for the detection of porcine circovirus2 DNA from formalin-fixed paraffin-embedded tissues usingnested polymerase chain reaction and comparison of nested

PCR with in situ hybridization

Junghyun Kim, Chanhee Chae *Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology,

Seoul National Uni6ersity, Suwon 441-744, Kyounggi-Do, South Korea

Received 12 June 2000; received in revised form 31 August 2000; accepted 1 September 2000

Abstract

Optimized DNA extraction method and nested polymerase chain reaction (PCR) were developed for the detectionof porcine circovirus 2 (PCV2) from formalin-fixed, paraffin-embedded tissues. Conventional PCR, nested PCR, andin situ hybridization methods were also compared for the detection of PCV2 in archival tissues. A method based onxylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for PCR analysesreliably and consistently. Twenty-six (70%) of the 37 tissues examined gave positive results with conventional PCR,whereas all the 37 tissues gave positive results using the nested PCR. A distinct positive signal for PCV2 was detectedin spleen and lymph node from all the 37 pigs by in situ hybridization. The nested PCR and in situ hybridizationcould be applied successfully to archival tissues for the detection of porcine circovirus 2 DNA. © 2001 ElsevierScience B.V. All rights reserved.

Keywords: DNA extraction; Formalin-fixed tissue; In situ hybridization; Paraffin section; Polymerase chain reaction; Porcinecircovirus

www.elsevier.com/locate/jviromet

1. Introduction

Postweaning multisystemic wasting syndrome(PMWS) is a new porcine disease that affectsmainly nursery and early growing pigs, and clini-cally is characterized by poor body condition,

dyspnea, pallor of the skin, and sometimes byicterus (Ellis et al., 1998; Choi et al., 2000).PMWS was first seen in western Canada (Ellis etal., 1998). Since then, the PMWS has been de-scribed in the USA (Allan et al., 1998; Kiupel etal., 1998; Morozov et al., 1998), Ireland (Kennedyet al., 1998), and Korea (Choi et al., 2000).Porcine circovirus 2 (PCV2) antigens and nucleicacids have been detected in tissues of pigs withPMWS (Allan et al., 1998; Choi and Chae, 1999;

* Corresponding author. Tel.: +82-31-2902736; fax: +82-31-2944588.

E-mail address: swine@plaza.snu.ac.kr (C. Chae).

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0166 -0934 (00 )00255 -X

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111106

Ellis et al., 1998; Choi et al., 2000). The demon-stration of PCV2 antigen and nucleic acid, closelyassociated with lesions in a wide range of tissuesfrom diseased pigs, has led to speculation thatPCV2 may be an etiologic agent of PMWS, andlesions of PMWS have now been reproduced ingnotobiotic pigs (Ellis et al., 1999).

Molecular analyses of nucleic acids have re-quired traditionally genomic DNA derived fromfresh or frozen tissues. The recovery of DNAfrom formalin-fixed, paraffin-embedded tissuesand the development of the polymerase chainreaction (PCR) method have vastly expanded theopportunity for molecular analysis of paraffin-em-bedded, as well as fresh and frozen tissues. Al-though formalin-fixed, paraffin-embedded tissuesare commonly regarded as poor sources of DNA,due to supposed damages occurring during thefixation and embedding processes (Greer et al.,1991; Jackson et al., 1991), optimized extractionprotocols can be performed with only minor mod-ifications to obtain DNA of sufficient quality toundertake most of the PCR analyses. In situhybridization has been reported for the detectionof PCV2 in formalin-fixed, paraffin-embedded tis-sues (Morozov et al., 1998; Choi and Chae, 1999).

One objective of this study was to develop theoptimized protocol for the detection of PCV2DNA from formalin-fixed, paraffin-embedded tis-sues through a nested PCR assay derived fromconventional PCR (Ellis et al., 1999). The secondobjective was to compare nested PCR with con-ventional PCR and in situ hybridization for thedetection of PCV2 DNA from archival tissues.

2. Materials and methods

2.1. Samples

Formalin-fixed, paraffin-embedded tissues from37 pigs with PMWS were used in the presentinvestigation. Cases were selected on the basis ofclinical signs and histopathological lesions as de-scribed previously (Choi et al., 2000). Paraffin-em-bedded tissue blocks from lymph node and spleenwere used to detect PCV2 DNA using a PCRtechnique.

2.2. Virus isolation

Specimens from a total of ten pigs with PMWSwere processed to attempt virus isolation fromfrozen tissues after diagnosing of PWMS as de-scribed previously (Ouardani et al., 1999). Lymphnode and spleen from each pig were pooled forvirus isolation. PCV-free PK-15 cells, kindly pro-vided by Dr Keith West of Prairie DiagnosticServices in Saskatchewan, Canada, were used toisolate PCV2 from tissue suspensions.

2.3. DNA extraction

For each pig, a 10-mm wide section was pre-pared from tissue blocks, and excess paraffin wastrimmed. Sections were placed in 1.5-ml sterilemicrofuge tubes. The microtome blade, tweezers,and other equipments that could come into con-tact with the samples were carefully sterilizedbefore processing each tissue block.

To extract the DNA from the formalin-fixed,paraffin-embedded tissue, the following three pro-cedures were used. In method A, tissue sectionswere deparaffinized with toluene for 10 min andwashed twice with ethanol to remove the solvent.Ethanol was allowed to evaporate under vacuumfor 10 min. To isolate genomic DNA, 500 ml ofdigestion buffer (10 mM Tris [pH 8.5], 1 mMethylenediaminetetraacetic acid, plus 0.5% Tween20) containing 200 mg/ml of proteinase K wasadded to the extracted dried samples. The resus-pended tissues were incubated overnight at 56°Cand then at 100°C for 8 min to inactivate theproteinase K (Lambert et al., 1996). Method Bwas a slightly modified version of method A.Xylene was used as the solvent instead of toluene,and digestion buffer did not contain 0.5% Tween20. In method C, tissue sections were de-paraffinized with xylene for 10 min and washedtwice with ethanol to remove the solvent. Ethanolwas allowed to evaporate under vacuum for 10min. To isolate genomic DNA, 500 ml of digestionbuffer (10 mM Tris [pH 8.5], 1 mM ethylenedi-aminetetraacetic acid, plus 0.5% SDS containing200 mg/ml of proteinase K was added to theextracted dried samples (Imai et al., 1998).

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111 107

The resuspended tissues were incubatedovernight at 55°C followed by further incubationat 65°C for 1–2 h with constant agitation. Thedigested samples were extracted three times usingthe standard phenol–chloroform–isoamyl alcoholprocedure and precipitated in ethanol to collectDNA.

2.4. Primers

The conventional PCR was performed usingprimers described previously (Ellis et al., 1999),which amplified a 481-base pair (bp) region fromopen reading frame (ORF) 2. The forward prim-er was 5%-CGGATATTGTAGTCCTGGTCG-3%(nucleotide positions 1095-1115), and the reverseprimer was 5%-ACTGTCAAGGCTACCACA-GTCA-3% (nucleotide positions 1570–1549). Theprimers for the nested PCR were designed usingcomputer software (Oligo 4.0 program, NationalBiosciences, Plymouth, MN, USA). A species-spe-cific region was chosen. The primers amplified a225-bp fragment that was in the 481-bp regionamplified in the first reaction. The forward primerwas 5%-GATTGTATGGCGGGAGGAGT-3% (nu-cleotide positions 1286-1305), and the reverseprimer was 5%-ATTGACGACTTTGTTCCCCC-3% (nucleotide positions 1510-1491).

2.5. Polymerase chain reaction

Ten microliters of the supernatant containingextracted DNA was used as PCR templates in thefirst reaction, and 10 ml of the product was usedfor the second reaction. The amplification wasperformed in a 50 ml reaction mixture containing1.25 mM MgCl2, 1×PCR buffer, 0.2 mM of eachdNTP, 1.00 m M of each primer, and 2.5 U of TaqDNA polymerase. Both the reactions were run ina thermocycler and required the same conditions,35 cycles of denaturation at 95°C for 1 min;primer annealing at 65°C for 1 min; and extensionat 72°C for 1 min. The PCR was ended with afinal extension step at 72°C for 10 min.

The amplified product was visualized by stan-dard gel electrophoresis of 10 ml of the finalreaction mixture on a 2% agarose gel. AmplifiedDNA fragments of specific sizes were located by

ultraviolet fluorescence after staining with ethid-ium bromide. Their lengths were verified by adigested lambda DNA standard run simulta-neously. The PCR reactions were repeated threetimes. Control DNA from reference strain wasincluded in each reaction.

2.6. Sensiti6ity and specificity assays

Sensitivity of PCV2 detection was estimatedthrough serial dilutions of DNA extracts preparedfrom formalin-fixed, paraffin-embedded tissues. Inthe specificity studies, porcine reproductive andrespiratory syndrome virus (PRRSV), porcineparvovirus (PPV), and porcine circovirus 1(PCV1) were tested independently with both setsof primers.

2.7. In situ hybridization

A 481-bp region DNA fragment from ORF 2was used as a probe. The PCR was carried out asdescribed previously (Ellis et al., 1999). The PCRproducts were purified with Wizard PCR Preps(Promega Biotech, Madison, WI, USA) and la-beled by random priming with digoxigenin-dUTPwith a commercial kit (Boehringer Mannheim,Indianapolis, IN, USA) according to the manu-facturer’s instructions. In situ hybridization wascarried out as described previously (Choi andChae, 1999).

3. Results

3.1. Sensiti6ity and specificity assay

The conventional PCR detected PCV2 DNA toa dilution of 10−3. Conversely, the nested PCRdetected DNA to a dilution of 10−5 (Fig. 1).Neither the outer nor the inner primers cross-re-acted with any of the viruses tested in this study.

3.2. Comparison of DNA extraction methods byPCR and nested PCR

Table 1 summarizes the results of 37 samples.The samples that were found positive by PCR

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111108

Fig. 1. Conventional and nested PCR sensitivity. Ten-folddilutions of the extracted PCV2 DNA from 10−1 to 10−4

were used. Lane M=1-kb DNA ladder; lane 1=conventionalPCR for 10−1 dilution; lane 2=conventional PCR for 10−2

dilution; lane 3=conventional PCR for 10−3 dilution; lane4=conventional PCR for 10−4 dilution; lane 5=nested PCRfor 10−1 dilution; lane 6=nested PCR for 10−2 dilution; lane7=nested PCR for 10−3 dilution; lane 8=nested PCR for10−4 dilution.

Fig. 2. Comparison of three DNA extracted methods byconventional PCR and nested PCR. Lane M=1-kb DNAladder; lane 1=positive conventional PCR; lane 2=positivenested PCR; lane 3=conventional PCR using method A; lane4=nested PCR using method A; lane 5=conventional PCRusing method B; lane 6=nested PCR using method B; lane7=conventional PCR using method C; lane 8=nested PCRusing method C.

3.3. In situ hybridization

A distinct positive signal was detected in all 37weaned pigs by in situ hybridization. The signalintensity varied within and between histologicalstructures in every one section, and between pigs.Positive cells typically exhibited a dark brown toblack reaction product mainly in the cytoplasmbut occasionally in the nucleus, without back-ground staining. PCV2-infected cells were de-tected in lymph node and spleen. In the spleen,PCV2-positive cells, which were concentratedaround periarteriolar lymphoid sheaths, generallyappeared to be macrophages, with large oval nu-clei and moderate cytoplasm. In addition, scat-tered macrophages exhibited a tense, dispersedhybridization signal in the cytoplasm of the whitepulp (Fig. 3). The positive cells were clustered inthe germinal centers of lymphoid tissues. A stronghybridization signal was detected in the cytoplasmof macrophages and multinucleated giant cells inlymph node (data not shown).

3.4. Virus isolation

Attempts were made to isolate PCV2 from cellcultures of ten clinical samples. After two succes-sive passages and an incubation period of 5 days,no cytopathic effect was observed in PK-15 cell

were always found positive by nested PCR re-gardless of DNA extraction methods. In methodA, 17 and 34 of the 37 samples were foundpositive by PCR and nested PCR, respectively.All the 17 samples that were found positive byPCR were also found positive by nested PCR. Inmethod B, 26 of the 37 samples were foundpositive by PCR but all the 37 samples examinedwere found positive by nested PCR. In method C,4 and 7 of the 37 samples were positive by PCRand nested PCR, respectively (Fig. 2). NestedPCR products from each method were sequenced,and their identity was confirmed as PCV2 (datanot shown).

Table 1Comparison of three DNA extracted methods through con-ventional PCR and nested PCR using formalin fixed, paraffin-embedded tissue samples of 37 pigs naturally infected withporcine circovirus 2

DNA extraction methods (%)

CBA

26 (70.3)17 (45.9)PCR 4 (10.8)34 (91.9)Nested PCR 37 (100) 7 (18.9)

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111 109

cultures inoculated with pooled tissue ho-mogenates from the PCR-positive animals. How-ever, in eight of these samples (80%), viral nucleicacid in the cytoplasm of infected cells could bedetected by in situ hybridization. For these sam-ples, the presence of PCV2 in culture supernatantswas detected by PCR after the second passage.

4. Discussion

Both nested PCR and in situ hybridization areable to detect PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with PMWS. Thesemethods also offer the advantage of using routineformalin-fixed, paraffin-embedded histologic spec-imens in retrospective study, obviating the needfor frozen or fresh materials. The development ofsuch techniques is most pertinent since formalinfixation of tissues allows veterinary practitionersto ship tissue samples for PCV identification in awell-preserved, noninfectious state.

The ability of the PCR procedure to amplifytarget sequences is particularly useful for analyz-ing DNA of formalin-fixed, paraffin-embeddedspecimens. The increased sensitivity of the nestedPCR assay in this report may be related to the

method used for DNA extraction. The quality ofthe DNA extracted from archival specimens de-pends on such factors as the length of time be-tween surgical removal of the tissues and formalinfixation, variable levels of nucleases in differenttissues, and length of storage in paraffin blockform (Goelz et al., 1985). The presence of in-hibitors of PCR in paraffin-embedded tissue spec-imens is a well-known diagnostic problem thatfrequently leads to false-negative results (An andFleming, 1991; Bascunana and Belak, 1996). Inorder to remove the inhibitors of the PCR, someinvestigators have used complex purification pro-tocols (An and Fleming, 1991). However, exten-sive purification procedures must be weighedagainst the increased risk of sample contamina-tion with each step of manipulation. Our datademonstrated that xylene and digestion bufferwithout detergent produce better results than anyother combination of solvent and digestion buffer.

One persistent problem that these highly sensi-tive techniques have is contamination during ma-nipulation of the samples such as samples’collection, DNA extraction, PCR setting, and gelanalysis. Care must be taken during each of thesesteps, and negative controls must be included foreach reaction. The consistently negative resultsobtained with the numerous negative controls in-dicate that false positive reactions were not aproblem in the present study. Three separatedrooms were also used to perform major phases,the extraction, amplification, and analysis of thePCR steps. Another problem with PCR assays isthe occurrence of false negative results. To detectthose cases where the negative results are due to alack of amplification in that particular sample, aninternal standard could be added in each reaction.

In contrast to PCR, in situ hybridization pro-vides cellular detail and histological architecturemaking it possible to study small numbers ofPCV2-infected cells and lesions in the same sec-tion with more confidence. Occasionally, falsepositives may occur if the slides are allowed to dryduring the procedure or if improper concentra-tions of saline sodium citrate solution are used.Conversely, false negatives may occur if stringen-cies are too high (Cheon and Chae, 1999; Choiand Chae, 1999; Kim and Chae, 2000). Multiple

Fig. 3. Spleen of pig naturally infected with porcine circovirus2. In situ hybridization with a PCV2-probe. Positive hybridiza-tion signals for PCV2 nucleic acid occur in the macrophages inthe red pulp. Hybridization signals are seen as black grains.Methyl green counterstain.

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111110

testing of known infected and uninfected tissuesare necessary to determine the proper stringencyof washes for probes used for in situ hybridiza-tion. A comparison of in situ hybridization andimmunohistochemistry techniques for the detec-tion of PCV2 in formalin-fixed paraffin-embeddedtissues has shown immunohistochemistry to bemore sensitive than in situ hybridization (Mc-Neilly et al., 1999). However, in this study, thehybridization probes used were based on thePCV1 whole genomic sequence. In view of thesignificant genomic differences now known to ex-ist between PCV1 and PCV2 isolates (Hamel etal., 1998; Meehan et al., 1998; Morozov et al.,1998), probes for the detection of PCV2 should bemodeled on the PCV2 genomic sequence.

In conclusion, a sensitive and reproduciblemethod was established for the detection of PCV2DNA in formalin-fixed, paraffin-embedded tis-sues. The detection of the PCV2 DNA using PCRhad been done on fresh, frozen, or fixed tissues(Ellis et al., 1999; Ouardani et al., 1999; Ellis etal., 2000). The optimized protocols allow us toextend molecular analyses to even smalleramounts of archival tissue, such as microdissectedsamples. This study has extended the possibility ofusing archival tissues for use in large-scale molec-ular epidemiologic studies of viral DNA.

Acknowledgements

The research reported here was supported byministry of agriculture, forestry and fisheries-spe-cial grants research program (MAFF-SGRP), andBrain Korea 21 Project, Republic of Korea.

References

Allan, G.M., McNeilly, F., Kennedy, S., Daft, B., Clarke,E.G., Ellis, J.A., Haines, D.M., Meehan, B.M., Adair,B.M., 1998. Isolation of porcine circovirus-like virusesfrom pigs with a wasting disease in the USA and Europe.J. Vet. Diagn. Invest. 10, 3–10.

An, S.F., Fleming, K.A., 1991. Removal of inhibitor(s) of thepolymerase chain reaction from formalin fixed, paraffinwax embedded tissues. J. Clin. Pathol. 44, 924–927.

Bascunana, C.R., Belak, K., 1996. Detection and identificationof mycobacteria in formalin-fixed, paraffin-embedded tis-sues by nested PCR and restriction enzyme analysis. J.Clin. Microbiol. 34, 2351–2355.

Cheon, D.-S., Chae, C., 1999. Distribution of a Korean strainof porcine reproductive and respiratory syndrome virus inexperimentally infected pigs, as demonstrated immunohis-tochemically and by in-situ hybridization. J. Comp. Pathol.120, 79–88.

Choi, C., Chae, C., 1999. In-situ hybridization for the detec-tion of porcine circovirus in pigs with postweaning multi-systemic wasting syndrome. J. Comp. Pathol. 121,265–270.

Choi, C., Chae, C., Clark, E.G., 2000. Porcine postweaningmultisystemic wasting syndrome in Korean pig: detectionof porcine circovirus 2 infection by immunohistochemistryand polymerase chain reaction. J. Vet. Diagn. Invest. 12,151–153.

Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Will-son, P., Strokappe, J., Martin, K., McNeilly, F., Meehan,B., Todd, D., Haines, D., 1998. Isolation of circovirusfrom lesions of pigs with postweaning multisystemic wast-ing syndrome. Can. Vet. J. 39, 44–51.

Ellis, J., Krakowka, S., Lairmore, M., Haines, D., Bratanich,A., Clark, E., Allan, G., Konoby, C., Hassard, L., Mee-han, B., Martin, K., Harding, J., Kennedy, S., McNeilly,F., 1999. Reproduction of lesions of postweaning multisys-temic wasting syndrome in gnotobiotic piglets. J. Vet.Diagn. Invest. 11, 3–14.

Ellis, J.A., Bratanich, A., Clark, E.G., Allan, G., Meehan, B.,Haines, D.M., Harding, J., West, K.H., Krakowka, S.,Konoby, C., Hassard, L., Martin, K., McNeilly, F., 2000.Coinfection by porcine circoviruses and porcine parvovirusin pigs with naturally acquired postweaning multisystemicwasting syndrome. J. Vet. Diagn. Invest. 12, 21–27.

Greer, C.E., Peterson, S.L., Kiviat, N.B., Manos, M.M., 1991.PCR amplification from paraffin-embedded tissues. Effectsof fixative and fixation time. Am. J. Clin. Pathol. 95,117–124.

Goelz, S.E., Hamilton, S.R., Vogelstein, B., 1985. Purificationof DNA from formaldehyde fixed and paraffin embeddedhuman tissue. Biochem. Biophys. Res. Commun. 130,118–126.

Hamel, A.L., Lin, L.L., Nayar, G.P., 1998. Nucleotide se-quence of porcine circovirus associated with postweaningmultisystemic wasting syndrome in pigs. J. Virol. 72,5262–5267.

Imai, K., Mase, M., Yamaguchi, S., Yuasa, N., Nakamura,K., 1998. Detection of chicken anaemia virus DNA fromformalin-fixed tissues by polymerase chain reaction. Res.Vet. Sci. 64, 205–208.

Jackson, D.P., Hayden, J.D., Quirke, P., 1991. Extraction ofnucleic acids from fresh and archival material. In: McPher-son, M.J., Quirke, P., Taylor, G.R. (Eds.), PCR: A Practi-cal Approach. Oxford University Press, New York, pp.29–50.

J. Kim, C. Chae / Journal of Virological Methods 92 (2001) 105–111 111

Kennedy, S., Allan, G., McNeilly, F., Adair, B.M., Hughes,A., Spillane, P., 1998. Porcine circovirus infection inNorthern Ireland. Vet. Rec. 142, 495–496.

Kim, O., Chae, C., 2000. In situ hybridization for the detec-tion and localization of porcine epidemic diarrhea virus inthe intestinal tissues from naturally infected piglets. Vet.Pathol. 37, 62–67.

Kiupel, M., Stevenson, G.W., Mittal, S.K., Clark, E.G.,Haines, D.M., 1998. Circovirus-like viral associated diseasein weaned pigs in Indiana. Vet. Pathol. 35, 303–307.

Lambert, A.-J., Houde, A., Drolet, R., D’Allaire, S., 1996.Determination of halothane gene mutation associated withmalignant hyperthermia in sows dead of cardiac failure. J.Vet. Diagn. Invest. 8, 513–515.

McNeilly, F., Kennedy, S., Moffett, D., Meehan, B.M., Fos-ter, J.C., Clarke, E.G., Ellis, J.A., Haines, D.M., Adair,B.M., Allan, G.M., 1999. A comparison of in situ hy-

bridization and immunohistochemistry for the detection ofa new porcine circovirus in formalin-fixed tissues from pigswith post-weaning multisystemic wasting syndrome(PMWS). J. Virol. Methods 80, 123–128.

Meehan, B.M., McNeilly, F., Todd, D., Kennedy, S., Jew-hurst, V.A., Ellis, J.A., Hassard, L.E., Clark, E.G., Haines,D.M., Allan, G.M., 1998. Characterization of novel cir-covirus DNAs associated with wasting syndromes in pigs.J. Gen. Virol. 79, 2171–2179.

Morozov, I., Sirinarumitr, T., Sorden, S.D., Halbur, P.G,Morgan, M.K., Yoon, K.-J., Paul, P.S., 1998. Detection ofa novel strain of porcine circovirus in pigs with postwean-ing multisystemic wasting syndrome. J. Clin. Microbiol. 36,2535–2541.

Ouardani, M., Wilson, L., Jette, R., Montpetit, C., Dea, S.,1999. Multiplex PCR for detection and typing of porcinecircoviruses. J. Clin. Microbiol. 37, 3917–3924.

..