用半套式PCR检测犬瘟热病毒的研究

Studies On Detection of Canine Distemper Virus by Semi-nested PCR

根据GenBanK中Barrett报道的犬瘟热病毒Onderstepoort弱毒株的附着或血凝蛋白基因序列，设计合成了能扩增760bp基因片段的半套式引物。用异硫氰酸胍—酚—仿一步抽取法提取细胞总RNA进行反转录，再以此产物进行半套式PCR扩增，并筛选出最佳PCR扩增。结果经半套式PCR扩增能得到与设计片段大小相同的产物，并且不扩增犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒的核酸。敏感性试验表明，此法能扩增出CDV强毒细胞培养物（毒价为10－5.8TCID50／0．1mL）10－6稀释的反转录产物，远高于常规PCR。经初步应用表明，此法可用于天瘟热的临床诊断和实验研究。

Three semi-nested primers which amplify 760 bp fragment had been designed and synthesized based on the H protein gene of the Onderstempoort strain of canine distemper virus (CDV)according GenBank. The total RNA were extracted from CDV infected DK cells or the tissues of infected animals by single step method of acid guanidinium thiocyanate- phenol - chloroform extraction and was used for reverse transcription. Anut 760 bp cDNA bont had been amplified from theproduct of RT - PCR.The conditions for semi - nested PCR had been optimized .The length of cCNA frgrnt was found same as that of the CDV Onderstepoort stram. No gene fragment had been amplified from the cells infected by canine parvovirus, canine adenovirus - I, canine coronavirus, rabies virus. The sensitiVy of experiment signifies the methed can amplify the specific bont fm the RT-PCR pnduct diluted by 10-6, which was more sensitive than normal PCR. It was shown tha this method may be used in clinical diagnosis and ladoratoy research.