J Vet Sci.  2010 Sep;11(3):269-271. 10.4142/jvs.2010.11.3.269.

Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system

• Moon HJ
• Park SJ
• Kim HK
• Ann SK
• Rho S
• Keum HO
• Park BK

• KMID: 1093503
• DOI: http://doi.org/10.4142/jvs.2010.11.3.269

Abstract

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

Keyword

DPO; multiplex PCR; PERV; pig

MeSH Terms

Animals
DNA Primers/genetics
DNA, Mitochondrial/*genetics
Gammaretrovirus/*genetics
Polymerase Chain Reaction/*methods
Proviruses/classification/*genetics
Sensitivity and Specificity
Sus scrofa/*genetics/*virology

Figure

• 

  Fig. 1 Specificity (A) and sensitivity (B) of the multiplex PCR assay for detecting porcine endogenous retrovirus pol, envA, B, C, and pig mitochondrial DNA. (A) Lane M: 100 bp DNA ladder, Lane 1: PK 15 cell, Lane 2: Negative control, Lane 3: HRT-18, Lane 4: N2a, Lane 5: MDBK, Lane 6: MDCK, Lane 7: CEF, Lane 8: Vero cell, Lane 9-14: Miniature pig genomic DNA (Lane 9 : pol, Lane 10: envA, Lane 11: envB, Lane 12: envC, Lane 13: pig mitochondrial DNA, Lane 14: pol + envA + envB + envC + pig mitochondrial DNA), (B) Lane M: 100 bp DNA ladder, Lane 1: 10 ng, Lane 2: 1 ng, Lane 3: 100 pg, Lane 4: 10 pg, Lane 5: 1 pg of pig genomic DNA, Lane 6: Negative control.

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