牛传染性鼻气管炎病毒LUX^(TM)新型荧光PCR检测方法的建立

Development of a LUX^(TM) real time polymerase chain reaction assay for rapid detection of infectious bovine rhinotracheitis virus

本研究针对牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)高度保守的gC基因设计单标记并具有自身荧光淬灭功能的LUX^(TM)引物,建立LUX^(TM)新型实时荧光PCR方法用于快速检测IBRV。该方法对四株IBRV细胞培养物的检测均呈典型阳性反应,而对其它动物疱疹病毒以及健康牛组织DNA和细胞对照的检测结果为阴性,检测时间包括核酸提取仅需1h～2h。试验表明,LUX^(TM)荧光PCR法对IBRV细胞增殖病毒液的检测敏感性可达0.04 TCID_(50),比病毒分离敏感性至少提高10倍;对10倍系列稀释的纯化IBRV核酸样品,LUX^(TM)荧光PCR的检测敏感性比常规PCR可提高10~3倍。将病毒液添加到健康牛精液和血液样品中,该荧光PCR可检测到牛冻存精液中40 TCID_(50)、牛抗凝全血、血清和临床精液中0.04 TCID_(50)的病毒,说明对临床样品的检测有效。本研究所建立的LUX^(TM)荧光PCR方法快速敏感,适合应用于活牛及其遗传物质的进出口检疫、养牛业疾病防控等领域对IBRV的快速检测。

A real-time PCR assay for rapid detection of infectious bovine rhinotracheitis virus （1BRV） was developed based on LUX^TM （ Light upon extension） fluorogenic primer and light cycle technology. The assay was performed employing LUXTM primers that are specific to the highly conserved virus glycoprotein gC gene. The results showed that the real-time PCR assay was highly specific in detecting viral DNA from four strains of IBRV with no cross reactions with other herpesvirues. The assay was fast and could be performed within 2 hours including nucleic acid extraction. It is highly sensitive and was able to detect 0.04 TCID50 1BRV in cell culture, which was at least 10-fold higher than that of virus isolation. It was 10^3-fold more sensitive than the traditional gel-based PCR method. The LUX^TM PCR could detect 40 TCID50 viruses on mimic-infected bovine extended semen and 0.04 TCID50 viruses on mimic-infected bovine serum, whole blood and raw semen. This study indicates that the realtime PCR may be a reliable method for rapid detection of IBRV infection in live cattle and genetic materials.