Li Yanhua, Sthal Chase, Bai Jianfa, Liu Xuming, Anderson Gary, Fang Ying
Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, United States.
Fairmont Veterinary Clinic, Fairmont, MN 56031, United States.
J Virol Methods. 2021 Mar;289:114040. doi: 10.1016/j.jviromet.2020.114040. Epub 2020 Dec 10.
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.
猪呼吸病毒1型（PRV1）于2013年首次在香港的猪鼻咽样本中被报道。它已在美国猪群中广泛传播。最近，在南美洲和欧洲国家也检测到了PRV1。目前，尚无经过验证的诊断方法可用于检测这种病毒。在本研究中，我们开发了一种针对血凝素神经氨酸酶（HN）基因的实时逆转录定量PCR（RT-qPCR）检测方法用于分子诊断。使用体外转录的RNA标准品评估了该RT-qPCR检测方法的分析灵敏度，在20 μl反应中的检测限为10个病毒RNA拷贝。未观察到与常见猪呼吸道病原体制备的核酸有交叉反应。用114份已知PRV1感染状态的猪鼻拭子和19份口腔液样本确定了该检测方法的诊断性能。RT-qPCR结果与常规RT-PCR以及HN基因的DNA测序结果一致，显示出100%的灵敏度和100%的特异性。该检测方法进一步应用于现场样本。在检测的310份鼻拭子样本中，来自8个猪场的201份样本PRV1呈阳性。使用现有的现场样本在PRV1感染猪中未检测到病毒血症。鼻拭子和口腔液样本似乎对RT-qPCR检测PRV1是可靠的。综上所述，我们开发并验证了一种用于准确检测鼻拭子和口腔液样本中PRV1的RT-qPCR检测方法。它将成为快速诊断PRV1感染和辅助PRV1流行病学监测的有用工具。
