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Detection of PCV2 Nucleic Acids

AVAILABLE TESTS

Detection of PCV2 Nucleic Acids

Detection of Anti-PCV2 Antibodies

Detection of PCV2 Virus or Viral Antigen

Further Characterization of PCV2 Isolate

Available Tests

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Polymerase Chain Reaction (PCR)

There are several PCR assays for the detection of PCV2-specific nucleic acids described in the literature (Choi et al., 2000; Grierson et al., 2004; Hamel et al., 2000; Morozov et al., 1998; Shibata et al., 2003). Variants of the regular PCR assay include the following:

Multiplex PCR for the detection of
PCV1 and PCV2

Multiplex PCR. More than one target sequence is detected in a single PCR step. The following multiplex PCR assays have been described in the literature: PCV2/PCV1 (Pogranichnyy et al. 2000; Ouardani et al., 1999), PCV2/PPV (Kim et al., 2001a), and PCV2/pseudorabies virus/PPV (Huang et al., 2004).

Nested PCR. To increase the ability to detect very small amounts of the target sequence the PCR assay is done in two steps. The first step generates a product that serves as template for the second step (Kim and Chae, 2001; Kim and Chae, 2004, Larochelle et al., 2000).

Multiplex-nested PCR. This assay combines multiplex and nested PCR and is designed to detect very small amounts of several target sequences. Assays have been described for the simultaneous detection of PCV1/PCV2/PPV (Kim et al., 2003; Kim and Chae, 2003) and PCV1/PCV2 (Kim et al., 2001b).

 
   

Quantitative real time PCR. Quantitative real-time PCR-assays have been developed and these assays allow for determination of the amount of PCV2 genomic copy numbers in the serum or tissues. PCR reaction and detection are combined in one step which decreases the turn-around time (Brunborg et al., 2004; Chung et al., 2005, Ladekjær-Mikkelsen, 2002; Liu et al., 2000; Olvera et al., 2004; Opriessnig et al., 2003; Rovira et al., 2002)

Reverse transcriptase (RT-) PCR. This is used to detect PCV2 RNA which is only present if PCV2 replicates (Yu et al., 2005). Reverse transcription of RNA is required to make a complementary DNA for further amplification.

Most pigs in the field (healthy or diseased pigs) are infected with PCV2 at some point in their lives. Therefore the use of PCR, which theoretically can detect one genomic copy, is considered by many to be too sensitive for most applications. One notable exception may be the detection of PCV2-nucleic acids in semen. PCR assays to detect PCV2 in semen have been described (Larochelle et al., 2000; Kim et al., 2003; Kim et al., 2001b); however, the infection dose required to transmit PCV2 infection via semen is unknown.

The amount of PCV2 nucleic acids in serum and tissues has been demonstrated to be predictive of the clinical outcome which might be an application for quantitative real-time PCR on individual pigs (Brunborg et al., 2004; Olvera et al., 2004). Those pigs with 107 or greater PCV2 genomic copies per milliliter of serum likely have PCV2-associated lymphoid lesions and disease (Brunborg et al., 2004).

In Situ Hybridization (ISH)

 
  Kidney: PCV2 -In situ hybrization (black staining is PCV2 antigen)

ISH for PCV2 uses a labeled DNA probe that corresponds to a specific portion of the PCV2-genome (Kim and Chae, 2003; McNeilly et al., 1999; Rosell et al., 1999; Sirinarumitr et al., 2000). Hybridization is usually done over-night and followed by a color reaction. This method allows localization of PCV2 but the method in general is time consuming (over-night hybridization step) and the reagents are expensive. Several ISH assays that detect multiple viruses within the same tissue section have been described: PCV1/PCV2 (Kim and Chae, 2001; Nawagitgul et al., 2000) PCV2/PRRSV (Choi and Chae, 2001; Sirinarumitr et al., 2001), and PCV2/PPV (Choi and Chae, 2000; Kim and Chae, 2002). 

References:

Brunborg IM, Moldal T, Jonassen CM: Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR. J Virol Methods. 122:171-178, 2004

Choi C, Chae C: Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephrology syndrome by double-labeling technique. Vet Pathol. 38:436-441, 2001

Choi C, Chae C: Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization. J Comp Pathol. 123:302-305, 2000

Chung WB, Chan WH, Chaung HC, Lien Y, Wu CC, Huang YL: Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs. J Virol Methods. 124:11-19, 2005

Grierson SS, King DP, Wellenberg GJ, Banks M: Genome sequence analysis of 10 Dutch porcine circovirus type 2 (PCV-2) isolates from a PMWS case-control study. Res Vet Sci. 77:265-268, 2004

Hamel AL, Lin LL, Sachvie C, Grudeski E, Nayar GP: PCR detection and characterization of type-2 porcine circovirus. Can J Vet Res. 64:44-52, 2000

Huang C, Hung JJ, Wu CY, Chien MS:Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Vet Microbiol. 101:209-214, 2004

Kim J, Chae C: Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffin-embedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization. J Virol Methods. 92:105-111, 2001

Kim J, Chae C: Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome. Can J Vet Res. 67:133-137, 2003a

Kim J, Chae C:A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs. J Vet Diagn Invest. 16:45-50, 2004

Kim J, Choi C, Han DU, Chae C: Simultaneous detection of porcine circovirus type 2 and porcine parvovirus in pigs with PMWS by multiplex PCR. Vet Rec. 149:304-305, 2001a

Kim J, Han DU, Choi C, Chae C: Differentiation of porcine circovirus (PCV)-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction. J Virol Methods. 98:25-31, 2001b

Kim J, Han DU, Choi C, Chae C: Simultaneous detection and differentiation between porcine circovirus and porcine parvovirus in boar semen by multiplex seminested polymerase chain reaction. J Vet Med Sci. 65:741-744, 2003c

Ladekjær-Mikkelsen AS, Nielsen J, Stadejek T, Storgaard T, Krakowka S, Ellis J, McNeilly F, Allan G, Bøtner A: Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2). Vet Microbiol. 89:97-114, 2002

Larochelle R, Bielanski A, Muller P, Magar R: PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen. J Clin Microbiol. 38:4629-4632, 2000

Liu Q, Wang L, Willson P, Babiuk LA: Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol. 38:3474-3477, 2000

McNeilly F, Kennedy S, Moffett D, Meehan BM, Foster JC, Clarke EG, Ellis JA, Haines DM, Adair BM, Allan GM: A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS). J Virol Methods. 80:123-128, 1999

Morozov I, Sirinarumitr T, Sorden SD, Halbur PG, Morgan MK, Yoon KJ, Paul PS: Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol. 36:2535-2541, 1998

Nawagitgul P, Morozov I, Sirinarumitr T, Sorden SD, Paul PS: Development of probes to differentiate porcine circovirus types 1 and 2 in vitro by in situ hybridization. Vet Microbiol. 75:83-89, 2000

Olvera A, Sibila M, Calsamiglia M, Segalés J, Domingo M:Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. J Virol Methods. 117:75-80, 2004

Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares F, Thacker EL, Brockus CW, Ackermann MR, Thomas P, Meng XJ, Halbur PG: Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol. 40:521-529, 2003

Ouardani M, Wilson L, Jetté R, Montpetit C, Dea S: Multiplex PCR for detection and typing of porcine circoviruses. J Clin Microbiol. 37:3917-3924, 1999

Pogranichnyy RM, Yoon KJ, Harms PA, Swenson SL, Zimmerman JJ, Sorden SD: Characterization of immune response of young pigs to porcine circovirus type 2 infection. Viral Immunol. 13:143-153, 2000

Rosell C, Segalés J, Plana-Durán J, Balasch M, Rodríguez-Arrioja GM, Kennedy S, Allan GM, McNeilly F, Latimer KS, Domingo M: Pathological, immunohistochemical, and in-situ-hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. J Comp Pathol. 120:59-78, 1999

Rovira A, Balasch M, Segalés J, García L, Plana-Durán J, Rosell C, Ellerbrok H, Mankertz A, Domingo M: Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J Virol. 76:3232-3239, 2002

Shibata I, Okuda Y, Yazawa S, Ono M, Sasaki T, Itagaki M, Nakajima N, Okabe Y, Hidejima I: PCR detection of Porcine circovirus type 2 DNA in whole blood, serum, oropharyngeal swab, nasal swab, and feces from experimentally infected pigs and field cases. J Vet Med Sci. 65:405-408, 2003

Sirinarumitr T, Morozov I, Nawagitgul P, Sorden SD, Harms PA, Paul PS: Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome. J Vet Diagn Invest. 12:562-565, 2000

Sirinarumitr T, Sorden SD, Morozov I, Paul PS: Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV). J Vet Diagn Invest. 13:68-71, 2001

Yu S, Carpenter S, Opriessnig T, Halbur PG, Thacker E: Development of a reverse transcription-PCR assay to detect porcine circovirus type 2 transcription as a measure of replication. J Virol Methods. 123:109-112, 2005