【作者】 李立；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2000， 硕士

【摘要】 本研究成功地建立了检测牛病毒性腹泻/粘膜病的反转录-套式聚合酶链式反应（RT-nested PCR）技术。根据BVDV NADL株、Oregon C₂₄V和猪瘟病毒5，端非编码区保守核酸序列的异同，利用计算机设计合成了两套引物，分别对BVDV NADL株、Oregon C₂₄V、BVDV地方分离株和猪瘟兔化弱毒疫苗株等不同毒株的RNA进行了提取、反转录、PCR和套式PCR扩增，经琼脂糖凝胶电泳检查，用外部引物扩增BVDV和猪瘟病毒都获得了预期长度约为280bp的片段。用内部引物扩增第一次PCR的产物，三株BVDV获得191bp片段，猪瘟病毒未被扩出，与预期结果相符。对伪狂犬病毒和未接种病毒进行PCR扩增，结果均为阴性。该技术检测到病毒最低浓度达10^-1TCID₅₀水平。以上结果表明RT-nested PCR技术对BVBV检测不仅具有特异、敏感、快速诊断的优点，而且可以与抗原性相似的猪瘟病毒加以区分。 另外，对RT-nested PCR技术中摸板RNA的制备及保存和BVDV的诊断技术进行了试验和探讨。更多还原

【Abstract】 In this study, reverse transcription nested polymerase chain reaction (RT-nested PCR) technique was successfully developed for detection bovine viral diarrhea-mucosal disease virus (BVD/MDV).Two pair of primer were designed using computer and synthesized respectively, according to 5 ’ untranslated cDNA region of the NADL stain BVDV and classical swine fever virus (CSFV).Viral RNA was extracted, reverse transcripted and amplified by PCR and nested PCR. Using corresponding primer P, and P2 , BVDV and CSFV could be detected by RT-PCR. When analyzed by agarose gel electrophoresis, a single band with the expected size of about 280bp was abstain. When primer P3 and P4 was used, only three stains BVDV were amplified by nested PCR, a single band with size of about 191 bp was detected by agarose gel electrophoresis, CSFV wasn’t amplified.51The result was expected. No amplification was observed when using RNAs extracted from Pseudorabies virus (PRV) and uninfected MDBK cell. 10 TCID50 BVDV could be detected by RT-PCR, and 10-1 TCID50 BVDV could be detected by nested PCR. The result demonstrated that the RT-nested PCR was not only a specific, sensitive, conveninent and rapid method for diagnosis of BVD, but also a way to differentiate BVDV and CSFV.In addition, other aspects of the RT-nested PCR technique for detecting BVDV, such as preparation and store condition of template RNA and the techniques for diagnosis BVD were tested.更多还原