Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip.

Abstract

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50 min in a completely enclosed environment. The detection limit of this assay was found to be 1 pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.