Reverse transcription loop-mediated isothermal amplification for the detection of Porcine reproductive and respiratory syndrome virus

J Vet Diagn Invest. 2009 May;21(3):350-4. doi: 10.1177/104063870902100308.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10(2) and 10(4) 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

MeSH terms

  • Animals
  • DNA, Viral / classification
  • DNA, Viral / isolation & purification
  • Nephelometry and Turbidimetry
  • Nucleic Acid Amplification Techniques / veterinary*
  • Porcine respiratory and reproductive syndrome virus / isolation & purification*
  • RNA, Viral / classification
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / veterinary*
  • Sensitivity and Specificity
  • Swine

Substances

  • DNA, Viral
  • RNA, Viral