Abstract：
Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/μL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/μL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.
摘要：
犬传染性呼吸道疾病综合征(CIRDC)是一种高度传染性疾病。犬呼吸道冠状病毒（CRCoV），犬流感病毒（CIV），犬瘟热病毒（CDV），犬副流感病毒（CPiV）是引起CIRDC的关键病原体。由于这些病毒引起的类似临床症状，仅基于症状的鉴别诊断可能具有挑战性。在这项研究中,开发了一种多重实时PCR检测方法，用于检测CIRDC的四种RNA病毒。设计针对CRCoVM基因的特异性引物和探针,CIV的M基因，CDV的N基因和CPiV的NP基因。CIV或CRCoV的检测限为10拷贝/μL，CDV或CPiV的检测限为100拷贝/μL。组内和组间重复性变异系数(CV)均小于2%。共分析了341个临床犬样本，结果表明，与常规逆转录PCR方法相比，本研究建立的方法具有良好的一致性和更好的特异性。这项研究提供了一种新的方法，能够在单一反应中同时检测所有四种病原体，提高CIRDC中四种病毒流行率的监测效率，这有利于CIRDC的控制。
