Study on the RT-PCR Method for Detecting Sendai Virus in Vaccine Producing Medium and Live Vaccine
  Revised:November 13, 2000
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YAO Zhihui,ZHU Xiaochun,HE Zhengming,DONG Guanmu,WEI Li,GONG Wei,WU Huiying,XING Ruichang
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Abstract:
      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.
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