禽呼肠孤病毒与禽白血病病毒双重RT-PCR检测方法的建立及应用

Establishment and Application of a Double RT-PCR for ARV and ALV

根椐GenBank中禽呼肠孤病毒(ARV)、禽白血病病毒(ALV)基因序列,设计2对引物,在建立鉴别各病毒单项RT-PCR技术的基础上,优化双重RT-PCR反应条件,建立2种病毒的双重RT-PCR。对同一样品中的ARV、ALV核酸模板进行双重RT-PCR扩增,结果可同时扩增ARV 485bp、ALV 673bp的特异性片段,而对其他5种禽病病原的PCR扩增结果均为阴性。敏感性试验结果表明,该双重RT-PCR技术能检出10pg的ALV和10pg的ARV模板。用31份临床病料对本研究双重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示,两者的总符合率为100%。结果表明建立的双重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这2种病毒的同时检测和鉴别诊断。

To identify and differentiate rapidly the cause of clinical diseases, a double RT-PCR was developed. Two sets of specific primers were designed according to the sequences of avian reovirus （ARV） and avian leukosis virus （ALV） in the Gen Bank. A double polymerase chain reaction was optimized to simultaneously detect two pathogens of ARV and ALV in this article. The double RT-PCR system would amplify a 485 bp fragment for ARV and a 673 bp for ALV simultaneously or separately in the samples,depending on its infection status. But not specific band amplified from other six avian pathogenic viruses. As lit- tle as 10 pg of ARV and 10 pg of ALV RNA were detected using gel electrophoresis after double RT-PCR. To evaluate the double RT-PCR, 31 clinical samples were comparatively detected. The data showed that the double RT-PCR method was 100 coincidence with the single RT-PCR, and it could be used for detection and differential diagnosis of these two viruses.