Development of a real-time RT-qPCR assay for the detection of porcine respirovirus 1.

Porcine respirovirus 1 (PRV1) was first reported in the pig nasopharyngeal samples in Hong Kong in 2013. It has been widespread in US swine herds. Recently, PRV1 was also detected in South America and European countries. Currently, there is no validated diagnostic assay available for the detection of this virus. In this study, we developed a real-time reverse transcriptase quantitative PCR (RT-qPCR) assay targeting the hemagglutinin-neuraminidase (HN) gene for molecular diagnosis. The analytical sensitivity of this RT-qPCR assay was evaluated using in vitro transcribed RNA standard, and the limit of detection was 10 copies of viral RNA in a 20 μl reaction. No cross-reactivity was observed with nucleic acid prepared from common swine respiratory pathogens. The diagnostic performance of this assay was determined with 114 pig nasal swabs and 19 oral fluid samples with known PRV1 infection status. The RT-qPCR results were consistent with conventional RT-PCR and DNA sequencing of the HN gene, demonstrating a 100 % sensitivity and 100 % specificity. This assay was further applied to field samples. Among 310 nasal swab samples that were tested, 201 samples from 8 swine farms were PRV1 positive. No viremia was detected in PRV1 infected pigs using the available field samples. Nasal swab and oral fluid samples appear to be reliable for PRV1 detection with the RT-qPCR assay. Taken together, we developed and validated an RT-qPCR assay for accurate detection of PRV1 in nasal swab and oral fluid samples. It will be a useful tool for the rapid diagnosis of PRV1 infection and in aid of PRV1 epidemiological surveillance.

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