活疫苗及其生产基质中Sendai病毒RT-PCR检测方法的研究

目的建立检测Sendai病毒的RT-PCR方法并应用于活疫苗及其生产基质中Sendai病毒的检测.方法将Sendai病毒E17株接种9日龄鸡胚尿囊腔,72h后收集尿囊液,用于提取病毒RNA,并逆转录成cDNA,用两对针对Sendai病毒NP基因设计的外引物和内引物分别进行扩增.扩增产物克隆于T-载体,并测序.尿囊液按10倍倍比稀释,进行敏感性实验.将该方法用于检测乙脑减毒活疫苗和用于生产疫苗用的普通级乳地鼠肾中的Sendai病毒.结果外引物和内引物的PCR分别扩增出684bp和248bp的片段,外引物PCR产物的测序结果与Genbank报告的序列完全一致.敏感性实验结果表明,第一次PCR可检测到10-4病毒滴度,巢式PCR可检测到10-7病毒滴度.乙脑减毒活疫苗和乳地鼠肾的检测结果为阴性.结论建立检测Sendai病毒的RT-PCR方法具有很高的特异性和敏感性.

Study on the RT-PCR Method for Detecting Sendai Virus in Vaccine Producing Medium and Live Vaccine

Objective To establish RT PCR method for detecting Sendai virus in live vaccine and vaccine producing medium. Methods The virus RNA was extracted from allantoic fluid of 9 days old chicken embryo inoculated with Sendai virus E17 strain after 72 hours, and was reverse transcribed into cDNA. Then cDNA was amplified by outer primer sets and inner primer sets respectively, designed according to the NP gene sequence of Sendai virus.The PCR products were cloned into T vector and sequenced. The sensitivity experiment was performed by serially diluting allantoic fluid, extracting RNA and then amplifying by RT PCR. This method was used to detect Sendai virus in Japanese encephalitis attenuated live vaccine and the kidney of nurturing hamster, which was used for producing vaccine in China for years. Results Two fragments, 684bp and 248bp, were amplified by using outer primer sets and inner primer sets respectively. The sequencing result of PCR products of outer primer sets was completely homology with the nucleotide sequence reported in Genbank. The sensitivity experiment indicated that 10 -4 virus titer was detected by the first PCR with the outer primer sets and 10 -7 virus titer by nested PCR with the inner primer sets. The results of detecting Japanese encephalitis attenuated live vaccine and the kidney of nurturing hamster were negative. Conclusions The RT PCR method for detecting Sendai virus was highly sensitive and special.