反转录—套式聚合酶链反应检测、鉴别鸡新城疫病毒强、弱毒株的研究
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摘要
为了快速、准确地诊断鸡新城疫(ND)，本研究根据新城疫病毒(NDV)基因组结构特点及强、弱毒株F_0裂解位点的序列差异设计合成了3对引物(P_1、P_2，P_3、P_4和P_3、P_5)，建立了检测、鉴别鸡NDV强、弱毒株的反转录-套式聚合酶链反应(RT-nested PCR)技术。用RT-套式PCR技术对NDV标准强毒株F_(48)E_8、弱毒株La Sota、地方强毒株(HJ)及对照病毒IBV、IBDV感染的SPF鸡胚尿囊液和人工感染NDV鸡、自然发病鸡组织病料进行了检测，结果为：引物P_1、P_2为NDV的通用引物，对2株NDV标准毒和HJ株均可扩增出与预期大小相符的1.7Kb片段，而其它对照病毒IBV、IBDV、正常鸡胚尿囊液、非免疫健康鸡组织的RNA扩增结果为阴性；引物P_3为套式PCR的共用引物，以RT-PCR产物为模板，用引物P_3、P_4只能扩增出NDV强毒株靶序列，而用引物P_3、P_5只能扩增出NDV弱毒株靶序列，得到大小均为254bp的单一片段；该技术最低可以检出10fg的NDV RNA模板。试验表明，该RT-套式PCR技术对NDV的检测具有特异、灵敏、快速、简便的优点，不仅适用于对鸡胚毒的检测，还适用于对病死鸡组织匀浆的检测，而且还可以鉴别NDV强、弱毒株，是新城疫鉴别诊断和流行病学调查的颇具潜力的分子生物学诊断方法。
In order to diagnose Newcastle Disease (ND) rapidly and accurately, three pairs of primers (P1+P2, P3+P4 and P3+P5) were designed and synthesized respectively, according to the genome structure of Newcastle disease virus (NDV) and the sequence differences at F0 cleavage sites between virulent and avirulent strains, and the reverse transcription-nested polymerase chain reaction (RT-nested PCR) technique was developed for detecting NDV and differentiating NDV virulent and avirulent strains. The RNAs extracted from allantoic fluid of SPF chicken
embryos inoculated with standard virulent NDV strain F4gEg, avirulent La Sota strain, local virulent isolate ( HJ strain) and control viruses (i.e. IBV, IBDV) respectively and the samples of tissue from chickens infected with the above-mentioned NDV strains experimentally and naturally were tested by this RT-nested PCR technique. The results were as follows: ?l andP2 are general primers for NDV strains. The RNAs extracted from two standard strains of NDV and HJ
strain were amplified using primer pair Pj+Pj, and a single band was observed with the expected size of about 1. 7kb, in addition, as a control, no specific products were observed when RNAs extracted from IBV, IBDV, normal allanotic fluid of SPF chicken embryos and samples of non-vaccinated healthy chickens' tissue were performed on. P3 is the shared primer for nested PCR. Taking the products of RT-PCR as model, the nested PCR using primer pair P3+P4 resulted in a fragment of 254bp only with RNA from virulent NDV, whereas the nested PCR using primer pair P3+P5 resulted in a 254bp fragment only with RNA of avirulent NDV. The lowest content of lOfg RNA of NDV could
be detected by this technique. These results demonstrated that this RT-nested PCR technique was a specific, sensitive, rapid and convenient method for diagnosis of ND. It could be used not only for detection of NDV strains derived from the inoculated chicken embryo and the samples of tissue from morbid or dead chicken, but also for differentiation of virulent and avirulent NDV strains. It is suggested that this molecular biological method is potential for diagnosis and epidemiological investigation of ND and differentiation of NDV.
In order to diagnose Newcastle Disease (ND) rapidly and accurately, three pairs of primers (P1+P2, P3+P4 and P3+P5) were designed and synthesized respectively, according to the genome structure of Newcastle disease virus (NDV) and the sequence differences at F0 cleavage sites between virulent and avirulent strains, and the reverse transcription-nested polymerase chain reaction (RT-nested PCR) technique was developed for detecting NDV and differentiating NDV virulent and avirulent strains. The RNAs extracted from allantoic fluid of SPF chicken
embryos inoculated with standard virulent NDV strain F4gEg, avirulent La Sota strain, local virulent isolate ( HJ strain) and control viruses (i.e. IBV, IBDV) respectively and the samples of tissue from chickens infected with the above-mentioned NDV strains experimentally and naturally were tested by this RT-nested PCR technique. The results were as follows: ?l andP2 are general primers for NDV strains. The RNAs extracted from two standard strains of NDV and HJ
strain were amplified using primer pair Pj+Pj, and a single band was observed with the expected size of about 1. 7kb, in addition, as a control, no specific products were observed when RNAs extracted from IBV, IBDV, normal allanotic fluid of SPF chicken embryos and samples of non-vaccinated healthy chickens' tissue were performed on. P3 is the shared primer for nested PCR. Taking the products of RT-PCR as model, the nested PCR using primer pair P3+P4 resulted in a fragment of 254bp only with RNA from virulent NDV, whereas the nested PCR using primer pair P3+P5 resulted in a 254bp fragment only with RNA of avirulent NDV. The lowest content of lOfg RNA of NDV could
be detected by this technique. These results demonstrated that this RT-nested PCR technique was a specific, sensitive, rapid and convenient method for diagnosis of ND. It could be used not only for detection of NDV strains derived from the inoculated chicken embryo and the samples of tissue from morbid or dead chicken, but also for differentiation of virulent and avirulent NDV strains. It is suggested that this molecular biological method is potential for diagnosis and epidemiological investigation of ND and differentiation of NDV.
引文
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