皮内注射用卡介苗特异性鉴别试验多重PCR检测方法的建立及验证

Development and verification of a multiplex PCR assay for identity test of BCG vaccine for intracutaneous inoculation

目的建立皮内注射用卡介苗(Bacillus Calmette Guerin vaccine,BCG)特异性鉴别试验的多重PCR法,并进行验证。方法根据GenBank登录的Pasteur 1173P2株序列(AM408590. 1)设计并合成引物,以制备的BCG特异性鉴别试验国家参考品(简称BCG鉴别参考品)DNA为模板,多重PCR法扩增其特异性缺失区RD1,产物经3%琼脂糖凝胶电泳鉴定,验证方法的重复性、中间精密度、特异性、耐用性及灵敏度;采用该方法检测8批皮内注射用BCG供试品。结果 BCG鉴别参考品在重复检测6次、2名检测人员分别重复检测3 d、不同PCR退火温度及不同DNA聚合酶加量时均扩增出约200 bp的核酸片段;最低可检10 pg/mL的目的基因,仅对结核分枝杆菌H37Rv及皮内注射用BCG样品DNA扩增出特异性条带。经该方法检测,8批供试品PCR产物电泳均可见单一的目的条带,无RD1序列存在,大小与BCG鉴别参考品一致。结论多重PCR法的重复性、中间精密度、特异性、耐用性及灵敏度良好,可应用于皮内注射用BCG特异性鉴别试验。

Objective To develop and verify a multiplex PCR assay for identity test of BCG vaccine for intracutaneous inoculation. Methods Primers were designed and synthesized according to the sequence of Pasteur 1173 P2 strain(AM408590)in GenBank,based on which the region 1 of difference(RD1)was amplified by multiplex PCR using the DNA of prepared national reference for identity test of BCG vaccine for intracutaneous inoculation as a template. The PCR product was identified by 3% agarose gel electrophoresis. The developed method was verified for reproducibility,intermediate precision,specificity,durability and sensitivity,and used for test of eight batches of BCG vaccine for intracutaneous inoculation. Results The nucleic acid fragments each at a length of about 200 bp were amplified from the reference for identity test of BCG vaccine in six repeat tests,the tests for 3 d by two persons,at different temperatures for annealing and at different DNA polymerase loadings. The minimum detection limit of the multiplex PCR assay was10 pg/mL. Specific DNA bands were amplified by the PCR assay only from the Mycobacterium tuberculosis H37 Rv and BCG vaccine for intracutaneous inoculation. Each of the eight batches of BCG vaccine showed a single target band on the electrophoretic profile of PCR products,without RD1 sequence,and the size of PCR products were consistent with those of the reference for identity test of BCG vaccine. Conclusion A multiplex PCR assay with high reproducibility,intermediate precision,specificity,durability and sensitivity was developed,which might be used for the identity test of BCG vaccine for intracutaneous inoculation.