【摘要】 选择鸡传染性喉气管炎病毒保守TK基因的蛋白编码区域 ,设计并合成了一对外引物和一对内引物 ,建立并优化了检测鸡传染性喉气管炎病毒DNA的套式PCR法。通过检测ILTV感染的鸡胚绒毛尿囊膜、实验室病料和临床病料 ,结果表明 ,套式PCR法能检测出ILTV感染后的非免疫鸡胚和SPF鸡胚绒毛尿囊膜研磨液中的被稀释了10 5倍的病毒 (约 1fg的ILTVDNA) ,攻毒后第 10天还能从非免疫鸡和SPF鸡气管拭子中检出ILTV ,第 10天非免疫鸡气管拭子中ILTV的最大检出率为 7/ 10 ,第 10天SPF鸡气管拭子中ILTV的最大检出率为 8/ 10。对非免疫鸡和SPF鸡的气管拭子中ILTV最佳检出时间均在攻毒后第 5天。对临床样品中的ILTV的最大检出率为 7/ 7。经过核酸杂交验证 ,套式PCR法具有很高的特异性和敏感性 ,为从分子水平探讨ILTV的发病机理、临床早期快速诊断提供了新的研究手段。更多还原

【Abstract】 Two pairs of oligonucleotides flanking Thymidine Kinase(TK)segment of infectious laryngotracheitis virus(ILTV) were chosen as primers for polymerase chain reaction(nested PCR).The method of nested PCR was used to detect of virus DNA in ILTV infected different choriollantoic membrane , specimens from laboratory and 7 clinical specimens from various species,with ILTV gene TK segment plasmid DNA as positive control.The virus DNA was extracted by acid guanidinium thiocyanate-phenol-chloroform single-step method.The results showed that all ILTV infected different choriollantoic membrane were positive.The highest detection rate of ILTV from tracheal swabs of non-immunized chicken was 7/10 on the tenth day p.i.,and that from tracheal swabs of SPF chickens was 8/10 on the tenth day p.i..The best time for ILTV detection in tracheal swabs from both non-immunized chickens and SPF chickens was the fifth day p.i. ILTV detection from clinical samples by nested PCR was at a rate of 7/7.To verify the nucleotide hybridization test with TKc probe, it suggests that nested PCR is sensitive,specific,rapid and simple,and can be applied for study of pathogenesis on molecular level and for early clinical diagnosis.更多还原