Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction

Jinho Shin, Elida M. Bautista, Yung Bai Kang, Thomas W. Molitor

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is in boar semen for extended periods of time as determined by reverse transcription- nested polymerase chain reaction (RT-nPCR) assay. The concentration of PRRSV RNA in semen and the biological significance of the detection level, however, remain to be resolved. In order to determine the concentration of PRRSV VR- 2332 (a prototypic strain of North American isolates) in semen following infection, we established a 'standard curve'-quantitative competitive (SC- QC)-RT-nPCR assay as well as an equimolar QC-RT-nPCR assay. A deletion-type competitor RNA derived from the Lelystad virus, a European strain of PRRSV, ORF-7 gene standard which shares the nested sets of primer recognition sequences with the VR-2332 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPCR assay results revealed that the number of copies of PRRSV RNA in 1 TCID50/ml of virus derived from CL-2621 cell culture supernatants varied depending upon the type of samples in which virus was added; 143 ± 24.0 and 266.5 ± 48.5 copies in serum and semen samples spiked with PRRSV VR-2332, respectively. For the establishment of SC-QC-RT-nPCR assay, a standard curve was generated from band intensity ratios versus a series of known initial numbers of wild-type RNA copies which were quantified by the equimolar QC-RT-nPCR assay. Various initial numbers of copies of wild- type PRRSV RNA and each band intensity ratio with 1000 copies of competitor RNA were well correlated within the range of 100 to 200000 copies (R2 = 0.947). A 'standard curve' quantitation assay using competitive single-tube RT-nPCR will offer a rapid and reliable way to quantify low concentrations of PRRSV RNA in semen.

Original languageEnglish (US)
Pages (from-to)67-79
Number of pages13
JournalJournal of Virological Methods
Volume72
Issue number1
DOIs
StatePublished - May 1 1998

Keywords

  • Competitive RT-PCR
  • Porcine reproductive and respiratory syndrome virus
  • Quantitation
  • Semen

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