牛疱疹病毒gD-PCR鉴别检测方法的建立

Establishment of Differential gD-PCR for Detection of Bovine Herpesvirus Infection

建立一种PCR技术,既能快速检测疱疹病毒1型成员牛传染性鼻气管炎病毒(bovine infectious rhinotracheitisvirus,IBRV),又能区分牛疱疹病毒5型和伪狂犬病毒。根据基因库中牛传染性鼻气管炎病毒的gD基因序列,应用primer 5.0软件设计了gD PCR引物,建立PCR方法,反应条件是:94℃预变性5min,94℃1min,58℃1min,72℃1min,30个循环,72℃7min,4℃5min。该方法能从IBRV阳性样本和参考毒株中扩增出372bp的目的片段,而从同属的牛疱疹病毒5型中扩增出440bp和206bp两条目的片段,从同属的伪狂犬病毒中扩增出303bp的目的片段,但不能从非疱疹病毒属成员猪呼吸与繁殖综合征病毒中扩增出目的条带。该PCR检测IBRV的灵敏度可达1PFU/mL以上。鉴于其灵敏度高、特异性好,可望在牛疱疹病毒感染快速鉴别检测方面发挥重要作用。

A PCR method was established successfully to differentiallly detect bovine herpes viruses including Infectious Bovine Rhinotracheitis Virus（IBRV）,Bovine Herpes Virus 5 and psuedorabies virus.The primers specific to IBRV gD gene were designed and the PCR condition was optimized as follows： 94℃ 5min,94℃ 1min,58℃ 1min,72℃ 1min for 30 cycles,72℃ 7min,4℃ 5min.This PCR can amplify one specific fragment of 372bp for IBRV,two fragments 440bp and 206bp for bovine herpesvirus type 5,and one fragment of 303bp for pseudorabies virus.On the othe hand,it couldnot amplify any fragment for non-related virus Porcine Reproductive and Respiratory Syndrome Virus（PRRSV）.The sensitivity of this gD-PCR is 2PFU/mL or 5×10-5TCID50/mL.Due to its high specificity and sensitivity,it would play an important role in rapid differential diagnosis of bovine herpesvirus infection.