J Vet Sci.  2017 Jun;18(2):253-256. 10.4142/jvs.2017.18.2.253.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

• Kim HK
• Lyoo KS
• Huynh TM
• Moon HJ
• Nguyen VG
• Park BK

• KMID: 2412579
• DOI: http://doi.org/10.4142/jvs.2017.18.2.253

Abstract

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCIDâ‚…â‚€/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Keyword

duplex nested RT-PCR; porcine circovirus type 2; type 2 porcine reproductive and respiratory syndrome virus

MeSH Terms

Animals
Circoviridae Infections/*diagnosis/virology
Circovirus/*genetics
Coinfection/diagnosis/*veterinary/virology
Limit of Detection
Polymerase Chain Reaction/methods/*veterinary
Porcine Reproductive and Respiratory Syndrome/*diagnosis/virology
Porcine respiratory and reproductive syndrome virus/*genetics
Swine
Swine Diseases/*diagnosis/virology

Figure

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  Fig. 1 Cross-reactivity of the duplex nested reverse transcriptase polymerase chain reaction method. Specific fragments of 347 bp (type 2 porcine reproductive and respiratory syndrome virus [PRRSV]) and 265 bp (porcine circovirus type 2 [PCV2]) were amplified with this method. Shown are a 100 bp ladder (lane M), PCV1-infected PK15 cells of different lots (lanes 1–3), PCV1-free PK15 cells (lane 4), PCV2-infected PK15 cells (lane 5), type 2 PRRSV (lane 6), PRRSV and PCV2 co-infected tissue (lane 7), and MARC-145 cells (lane 8).

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  Fig. 2 Detection limits of the duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method. A mixture of 105 porcine circovirus type 2 (PCV2)-infected PK15 cells/mL and 105.5 TCID50/mL type 2 porcine reproductive and respiratory syndrome virus (PRRSV) was 10-fold diluted. The limited detection of duplex nested RT-PCR was 101.5TCID50/mL for type 2 PRRSV and 102 infected cells/mL for PCV2. A 100 bp DNA ladder (lane M), along with stock of mixture (lane 1) and serial 10-fold dilutions of the stock from 10−1 to 10−5 (lanes 2–6).

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  Fig. 3 Alignment of binding sites for primers N21 (A), N26 (B), N22 (C), and N24 (D). Shaded areas indicate the first 5 nucleotides of the 3' end of each primer. The fractions preceding each primer indicates the number of particular sequences out of the 461 genomic sequences of type 2 porcine reproductive and respiratory syndrome virus (PRRSV) (collected in Europe, Asia, and North American) deposited in Genbank to date. For each primer, the majority of sequences at the primer binding sites (boxes) matched with the sequence of primer.

Reference

1. Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, Walker I, Kennedy S. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication. Arch Virol. 2000; 145:2421–2429.

2. Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J. 1998; 39:44–51.

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

4. Giammarioli M, Pellegrini C, Casciari C, De Mia GM. Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine parvovirus. Vet Res Commun. 2008; 32:255–262.

5. Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, Lum MA, Andrews JJ, Rathje JA. Comparison of the pathogenicity of 2 US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1995; 32:648–660.

6. Harms PA, Sorden SD, Halbur PG, Bolin SR, Lager KM, Morozov I, Paul PS. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet Pathol. 2001; 38:528–539.

7. Kennedy S, Moffett D, McNeilly F, Meehan B, Ellis J, Krakowka S, Allan GM. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J Comp Pathol. 2000; 122:9–24.

8. Kim J, Chung HK, Chae C. Association of porcine circovirus 2 with porcine respiratory disease complex. Vet J. 2003; 166:251–256.

9. Kono Y, Kanno T, Shimizu M, Yamada S, Ohashi S, Nakamine M, Shirai J. Nested PCR for detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs. J Vet Med Sci. 1996; 58:941–946.

10. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis A, Martelli GP, Mayo MA, Summers MD. Virus Taxonomy. New York: Springer;1995.

11. Pogranichniy RM, Yoon KJ, Harms PA, Sorden SD, Daniels M. Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome. J Vet Diagn Invest. 2002; 14:449–456.

12. Rossow KD, Benfield DA, Goyal SM, Nelson EA, Christopher-Hennings J, Collins JE. Chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs. Vet Pathol. 1996; 33:551–556.

13. Yang JS, Song DS, Kim SY, Lyoo KS, Park BK. Detection of porcine circovirus type 2 in feces of pigs with or without enteric disease by polymerase chain reaction. J Vet Diagn Invest. 2003; 15:369–373.

14. 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. 2005; 123:109–112.

15. Yu S, Opriessnig T, Kitikoon P, Nilubol D, Halbur PG, Thacker E. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs. Vet Immunol Immunopathol. 2007; 115:261–272.