应用半套式RT-PCR技术诊断番鸭呼肠孤病毒病

Diagnosis of Muscovy Duck Reovirus Disease by Semi-Nested RT-PCR

参考GenBank中番鸭呼肠孤病毒(muscovy duck reovirus,MDRV)S1基因序列设计并合成3条引物C1、HP11和HP12,组成半套式RT-PCR,扩增片段为300 bp.应用该半套式RT-PCR对MDRV、禽呼肠孤病毒(ARV)、番鸭细小病毒(MPV)、鹅细小病毒(GPV)、鸡胚成纤维细胞(CEF)和番鸭胚成纤维细胞(MDEF)培养物进行扩增.结果仅能从MDRV中扩增出300 bp特异片段,而不能从ARV、MPV、GPV、CEF和MDEF细胞培养物中扩增出特异片段;该方法检测灵敏度为1 fg核酸,重复性好.对人工感染和临床疑似病例用病毒分离和半套式RT-PCR进行检测,2种方法符合率为100%.因此,该半套式RT-PCR可以用于番鸭呼肠孤病毒病的临床快速检测.

According to the nucleotide sequence of the S1 gene of MDRV registered in GenBank, a set of primers C1 ,HPll and HP12 were designed to amplify a specific 300 bp fragment by semi-nested RTPCR. The specific product could only be amplified from MDRV, but not from ARV,GPV,MPV,CEF and MDEF cell cultures by this method. The detectable dsRNA amount by this method was 1 fg and which had high specificity and repeatability. Pathological tissues of artificial infection and suspicious clinical samples were diagnosed by virus isolation and semi-nested RT-PCR. The coincidence rate of the two methods was 100%. So this semi-nested RT-PCR could be used for rapid detection of the muscovy duck reovirus disease.