Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10 -1 median tissue culture infection dose (TCID 50 ) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID 50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05349-8.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus

“…Serum and tissue samples from different days post infection (dpi) of PRRSV-2 Lineage 1 stain-challenged pigs were used to detect the coincidence rate between the developed ELISA and a previous real-time PCR method. For the real-time PCR, all samples were analyzed as previously described [31].…”

Development of Enzymatic Recombinase Amplification Assays for the Rapid Visual Detection of HPV16/18

“…Agarose gel electrophoresis can be applied to analyze the length and amounts of amplicons, but it takes a relatively long reaction time and requires other equipment [ 26 ]. In contrast, the fluorescent probe can monitor and analyze the results in real-time [ 27 - 29 ], while LFD is extremely portable and easy to operate and can read the results within 5min by the naked eye [ 30 - 32 ]. The probes and enzymes applied for the above methods are quite different.…”

“…The THF can be identified and split by exonuclease III and the separation of fluorophore and quencher will generate fluorescence signals [ 28 , 29 ]. For ERA-LFD assay, endonuclease IV can identify the THF of the nfo-probe and convert the probe to a forward primer carrying a fluorophore at the 5¢ end [ 30 ]. With the function of a biotin-labeled backward primer, the amplicons will contain two antigenic markers at both ends.…”