【作者】 潘盛；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 本实验对异硫氰酸胍一步法、Trizol试剂法和RNA抽提试剂盒法三种不同的病毒RNA提取方法做了对比试验，用紫外光检测RNA纯度和电泳检测RNA的完整性的方法对三种提取方法作为评价指标，以找出适合禽传染性支气管炎病毒RNA提取方法。结果表明：三种RNA提取方法的对比实验表明三种方法中，异硫氰酸胍一步法提取的RNA样品纯度不高，且不完整；Trizol试剂法和RNA抽提试剂盒法提取的RNA样品纯度和完整性都较理想，两者提取效果差异不显著，考虑到成本的因素，本研究选用Trizol试剂法做为今后的RNA提取方法； 通过在IBV非结构基因3abc的保守区域设计一对引物，对引物的特异性进行检验；建立和优化了一步法RT-PCR，在此基础上组装成一步法RT-PCR检测试剂盒。引物的特异性实验表明，用该引物对IBV分离株和IBV标准株检测成阳性，而对IB DNA疫苗和鸡相关病原体检测呈阴性，阳性产物回收后测序表明，扩增出的阳性片段为IBV的3abc基因。 通过对8株IBV国内分离株和3株国内标准株以及IB DNA疫苗（S1、M和N三基因混合）和4株鸡常见病原体的检测来检验试剂盒的特异性。结果表明，试剂盒对IBV分离株和IBV标准株检测成阳性，而对IB DNA疫苗和鸡相关病原体检测呈阴性；用试剂盒对不同浓度的病毒RNA模板进行检测来检验试剂盒的灵敏度结果表明：试剂盒最低可以检测出浓度为10^-3ng／μl的IB病毒RNA模板；通过在试剂盒组装后的6个月内定期用试剂盒对IBV毒株、IB DNA疫苗和鸡常见病原体的检测，来检验试剂盒的稳定性和重复性，结果表明：6个月后，试剂盒的稳定性未下降，具有较好的灵敏度和特异性。 用一步法RT-PCR试剂盒、两步法RT-PCR和ELISA方法对人工感染鸡进行对比检测。结果表明：试剂盒和两步法RT-PCR均能特异地检测出IBV，试剂盒比两步法RT-PCR节省了1.5小时的检测时间，并节约了成本，减少了试剂污染的可能。试剂盒和ELISA方法均能特异地检测出IBV，试剂盒比ELISA方法提前36小时检测出IBV。 试剂盒对IB DNA疫苗免疫鸡、人工IBV感染鸡和自然感染病例进行检测。结果表明，试剂盒对IB DNA疫苗免疫的检测全部呈阴性；试剂盒可以对人工IBV感染鸡更多还原

【Abstract】 In order to find appropriate RNA extraction method for IBV, three different RNA extraction methods （iso-sulfuric cyanic guanidine, Trizol reagent and RNA extraction kit） were compared and evaluated. UV and electrophoresis were employed to test the purity and integrity of extracted RNA. The result shows: The RNA which extracted by so-sulfuric cyanic guanidine has got the lowest purity and integrity. And the RNA which extracted by Trizol reagent and RNA extraction kit has got good purity and integrity. For the difference of extraction efficiency between RNA extraction kit and Trizol reagent is not significant, considering the cost, Trizol reagent is chosen for next steps of experiment.A pair of primers located at the conservative district of IBV nonstructural gene 3abc was designed, and the specificity of the primers was also tested; One-step RT-PCR was established and optimized, and then one-step RT-PCR kit was constructed according to the optimized condition and concentration. The result shows: when the primers were employed to detected IBV isolates and IBV standard strains, positive results were got; when it came to IBV DNA vaccine and other pathogens of chicken, negative results were got. The positive products were collected and sequenced, and the result shows: the positive product was 3abc gene of IBV.In order to test the specificity of the kit, 8 domestic IBV isolates, 3 domestic standard strains, IBV DNA vaccine （S1, M and N gene mixed） and 4 other pathogens of chicken were detected by the kit. The result shows: positive results were got when IBV isolates and IBV standard strains were detected, negative results were got when IBV DNA vaccine and other pathogens of chicken were detected. Different concentration of IBV RNA were dectected by the detection kit, the result shows: the concentration of 10^-3 ng/μl RNA template can be detected at least. In order to test the stability and reproducibility of the detection kit, IBV strains, IB DNA vaccine and other pathogens of chicken were detected regularly for 6 monthes since the construction of the kit. The result shows: the kit kept good stability and specificity.更多还原