鸽腺病毒通用型实时荧光PCR检测方法的建立与应用

Establishment and Application of a Universal Real-time Fluorescent PCR for Pigeon Adenovirus

鸽腺病毒(pigeon adenovirus,PiAdV)是影响鸽产业健康发展的重要病原之一。为实现PiAdV的高通量快速检测,根据GeneBank登录的PiAdV-A种和PiAdV-B种病毒pⅧ基因序列的共同保守区域设计特异性检测引物和探针,经反应条件优化,建立了一种PiAdV通用型实时荧光PCR检测方法,并对该方法进行了特异性、敏感性、重复性试验。结果显示:该方法的最佳探针浓度为0.2μmol/L,与鸽群其他常见病毒无交叉反应,对PiAdV的最低检测限为56.9 copies/μL,比常规PCR敏感性高100倍,组内和组间重复变异系数均小于1.8%。利用该方法和常规PCR方法对20份疑似PiAdV感染样品进行检测,发现PiAdV通用型实时荧光PCR检测方法的病毒阳性检出率高于常规PCR方法。结果表明,本研究建立的PiAdV通用型实时荧光PCR检测方法特异性强、敏感性高、可靠性好,可用于PiAdV感染的临床检测及流行病学调查。

Pigeon adenovirus(PiAdV)is one of the major pathogens affecting healthy development of pigeon industry.In order to rapidly detect PiAdV at high-throughput,based on the common conserved region of pⅧ gene sequences of PiAdV-A and PiAdV-B registered in GeneBank,specific primers and probes were designed,after optimization of reaction conditions,a universal real-time fluorescent PCR assay for detecting PiAdV was established,followed by tests of its specificity,sensitivity and repeatability.The results showed that the optimal probe concentration of the established method was 0.2μmol/L,without any cross reactivity with other common pigeon viruses,the minimum detection limit was 56.9 copies/μL,100 times more sensitive than conventional PCR,and all the coefficients of variation in repeated inter and intra groups were less than 1.8%.20 suspected samples were detected by the method and the conventional PCR,the detection rate of positive samples by the former was higher than that by the latter.In conclusion,the universal real-time fluorescent PCR assay for PiAdV established in the paper,with its advantages of strong specificity,high sensitivity and good reliability,could be used for clinical detection and epidemiological investigation of the virus.