应用反转录多聚酶链反应（RT－PCR）对鸡传染性法氏囊病毒的基因检测研究

Detection of chicken Infectious Bursal Disease Virus by Reverse Transcription and Polymerase Chain Reaction

参考Ｃｕｌ株核酸序列自行设计一对２０ｂｐ序列为引物，经反转录和ＰＣＲ扩增传染性法氏囊病毒（ＩＢＤＶ）核酸高度保守的Ｖｐ４编码区中１５０ｂｐ的片段，用于检测ＩＢＤＶ。对德国Ｃｕｌ株、美国标准强毒ＳＴＣ和变异株１０８４Ｅ、中国ｑ８０１以及７个国内野外分离株分别进行扩增，同时对新城疫病毒（ＮＤＶ）、马立克氏病毒（ＭＤＶ）、禽呼肠孤病毒（ＲＥＯＶ）、禽腺病毒（ＨＡＡ）、Ｖｅｒｏ细胞、ＳＰＦ鸡法氏囊组织进行扩增后，２％琼脂糖凝胶电泳分析，１１株ＩＢＤＶ都出现分子量大小与设计相符合的ＤＮＡ扩增带，４种其他病毒、囊组织和Ｖｅｒｏ细胞都未出现任何扩增带。建立了直接采用细胞毒或组织毒进行ＰＣＲ的快速简便的核酸提取方法，应用二级ＰＣＲ扩增出了与一级ＰＣＲ相符的更加清晰的扩增带。

With reference to the published IBDV-Cul strain’s nucleotide sequence a pair of 20-baseoligonucleotide primers were designed and synthesized. Reverse transcription and polymerase chain reactionof IBDVs resulted in amplification of a 150 bp fragment located in the region encoding VP4 that detected theIBDVs.The USST-C andE/Del and Chinese strains CJ801(bursal tissue),BJ836 and 7 other field isolatesfrom 6 provinces were chosen for the experiment. Meanwhile,Newcastle disease virus(NDV).Marek’sdisease virus(MDV),avian reovirus(Reov),hemogglutinating avian adenovirus(HAAV), mock-infectedVero cell culture and bursae from SPF chickens were also amplified by RT-PCR, electrophoresized on 4%polyacrylamide gel or 2%agar gel and screenedby silver stainning or UV transilluminator as the virus-con-taining samples.The above 11 strains of IBDV universally elicited a band of molecular weight equivalent tothe size in between the 2 primers. In contrast,heterologous viruses and mock-infected Vero cell and unin-fected SPF bursae did not produce similar bands. Our experiment established a method to directly amplify thenucleic acid fragment from cell-culture adapted and bursa contained IBDVs; Secondary PCR showed moredistinct bands than primary PCR.