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目的建立BCG DNA定性用国家参考品,供BCG菌株、卡介菌多糖核酸注射液等卡介菌衍生制品鉴别实验用。方法以紫外分光法检测260nm和280nm的吸光度值,比较A260/280的比值,考量国家参考品BCG DNA的纯度指标;以多重PCR法检测BCG DNA特异性缺失区RD1区是否存在,验证所制备的BCG DNA可作为定性用国家参考品,组织3家实验室对该参考品进行验证;并在不同时间,观察其稳定性。结果所制备的BCG DNA A260/286=1.96>1.8;BCG DNA国家参考品经多重PCR扩增BCG缺失区RD1的产物为约200bp DNA片段,与卡介菌巴斯德株DNA结果相同,3家实验室的验证结果一致,该制品经37℃4周,4℃3个月、6个月、12个月放置后,PCR结果不变。结论该BCG DNA制品纯度高,稳定,可作为BCG DNA定性用国家参考品,在卡介苗相关制品的质量控制中,做为BCG菌株、卡介菌多糖核酸注射液等卡介菌衍生制品鉴别实验用的国家参考品。
OBJECTIVE: To establish a BCG DNA qualitative use of national reference materials for BCG strains, BCG polysaccharides nucleic acid injection and other derivatives of BCG derivative experiments. Methods The absorbance values of 260nm and 280nm were detected by ultraviolet spectrophotometry. The ratio of A260 / 280 was compared with the purity of BCG DNA of national reference samples. The presence of RD1 region in BCG DNA specific deletion region was detected by multiplex PCR. BCG DNA can be used as a qualitative national reference, the organization of three laboratories to verify the reference; and at different times, observe its stability. Results BCG DNA A260 / 286 = 1.96> 1.8 was prepared. The BCG DNA reference sample was amplified by multiplex PCR. The product of RD1 in BCG deletion region was about 200 bp DNA fragment. Laboratory validation results are consistent, the product after 4 weeks at 37 ℃, 4 ℃ 3 months, 6 months, 12 months after placing the PCR results unchanged. Conclusion The BCG DNA product is of high purity and stability and can be used as a national reference material for the qualitative identification of BCG DNA. In the quality control of BCG-related products, it is used as a differential reagent for identification of BCG strains, BCG polysaccharide nucleic acid injection and other derivative products of BCG. Of national reference materials.