Reverse transcription loop-mediated isothermal amplification for the detection of Porcine reproductive and respiratory syndrome virus.
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.
Rovira
Albert
A
University of Minnesota, Veterinary Diagnostic Laboratory, 1333 Gortner Avenue, St. Paul, MN 55108, USA. rove0010@umn.edu
Abrahante
Juan
J
Murtaugh
Michael
M
Muñoz-Zanzi
Claudia
C
D000818:Animals; D004279:DNA, Viral / Q000145:classification / Q000302:isolation & purification; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D012367:RNA, Viral / Q000145:classification / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013552:Swine
D004279:DNA, Viral; D012367:RNA, Viral