检测疫苗中猪肺炎支原体污染的PCR-斑点杂交法和套式PCR法比较研究

为建立一种敏感、特异的检测疫苗中猪肺炎支原体污染的方法,设计了猪源支原体PCR通用外套引物和猪肺炎支原体种特异性内套引物及地高辛标记寡核苷酸探针,建立了PCR-斑点杂交技术,应用于猪瘟活疫苗中猪肺炎支原体污染的检测,并与套式PCR技术进行比较研究.结果显示,探针最低检出DNA量为23.4 pg,比套式PCR(最低检出量为62.5 pg)敏感性更高,且探针与鸡毒支原体和大肠杆菌DNA无交叉反应.对来自6个生产厂家共90批疫苗的检测结果显示,PCR-斑点杂交法比套式PCR法对疫苗猪肺炎支原体污染的检出率高10%.以上结果表明PCR-斑点杂交法具有更高的敏感性和特异性,在疫苗猪肺炎支原体污染的检测中具有推广应用价值.

Detection of Mycoplasma hyopneumoniae contamination in vaccineby PCR-dot blot and nested PCR:A comparative study

To establish a sensitive and specific method for detecting Mycoplasma hyopneumoniae(Mhp) contamination in vaccine,a pair of outer primers was designed according to the 16S rRNA gene sequence of several swine mycoplasma;a pair of inner primers and a digoxin-labeled oligonucleotide probe were designed according to the 16S rRNA gene sequence of M.hyopneumoniae.PCR-dot blot and nested PCR technique were compared in detecting mycoplasma contamination in swine fever live vaccines.The results showed that the detection limit of PCR-dot blot was 23.4 pg and that of nested PCR was 62.5 pg ,indicating the former was more sensitive.MG DNA and E.coli DNA were not hybridized with the non-radioactive digoxin labeled probe.Results of the 90 batches swine fever live vaccines from 6 manufacturers detected by PCR- dot blot and nested PCR showed that the Mhp contamination rate of vaccines by PCR- dot blot was 10% higher than which by nested PCR,indicating PCR-based dot blot hybridization was more sensitive and specific.Thus,PCR-based dot blot hybridization assay appeared to be a useful test to detect M.hyopneumoniae contamination in live vaccines.