TY - JOUR
T1 - Development of a real-time RT-qPCR assay for the detection of porcine respirovirus 1
AU - Li, Yanhua
AU - Sthal, Chase
AU - Bai, Jianfa
AU - Liu, Xuming
AU - Anderson, Gary
AU - Fang, Ying
N1 - Publisher Copyright:
© 2020 Elsevier B.V.
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
KW - Hemagglutinin-neuraminidase gene
KW - Nasal swab, oral fluid
KW - Porcine respirovirus 1
KW - RT-qPCR
UR - http://www.scopus.com/inward/record.url?scp=85097891377&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85097891377&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2020.114040
DO - 10.1016/j.jviromet.2020.114040
M3 - Article
C2 - 33309757
AN - SCOPUS:85097891377
SN - 0166-0934
VL - 289
JO - Journal of Virological Methods
JF - Journal of Virological Methods
M1 - 114040
ER -