兽用疫苗中霉形体污染的套式PCR检测技术建立及初步应用

Development and application of nested PCR technique for detection of mycoplasma contamination in live vaccines

根据GenBank公布的鸡毒霉形体、猪肺炎霉形体、猪滑液霉形体和絮状霉形体的16s rRNA基因序列,利用引物设计软件DNAStar和Primer5.0自行设计3对特异性引物,以市场上常见的兽用疫苗为检测对象进行试验,优化反应体系,建立了兽用疫苗霉形体污染检测的套式PCR方法。同时,本试验还对市场上的兽用疫苗进行随机抽查检测,其检出率分别为24.2%(23/95)、21.1%(20/95)和14.7%(14/95)。

According to the published sequence of 16s rRNA gene of M. gallisepticum, M. hyopneurnoniae, M. hyosynoviae and M. flocculate, three specific pairs of primers were designed for detecting Mycoplasma contamination in samples by nested PCR assay with the software DNAStar and Primer 5.0. To establish the optimized conditions of PCR with the DNA of contaminated Mycoplasma in live vaccines, this method was performed to detect different live vaccines from the market randomly. The result showed that this method was rapid with detection dositive rates at 24. 2%（23/95）, 21.1%（20/95） and 14.7%（14/95） respectively using different primers.