硫乙醇酸盐流体培养基中微量细菌16S核糖体基因快速提取及检测方法的研究  

Study of Rapid Extraction and Detection Methods about 16S Ribosome Genes from Micro Bacteria in Fluid Thioglycolate Medium

作　　者：易大为 周少彤 李昊 仲瑶 傅蓉 孙苓苓 YI Dawei;ZHOU Shaotong;LI Hao;ZHONG Yao;FU Rong;SUN Lingling(Liaoning Inspection Examination and Certification Center,Liaoning Institute for Drug Control,Shenyang 110023,China)

机构地区：[1]辽宁省检验检测认证中心辽宁省药品检验检测院,辽宁沈阳110023

出　　处：《药物生物技术》2024年第6期588-593,共6页Pharmaceutical Biotechnology

基　　金：辽宁省自然科学基金联合基金(No.2023-BSBA-176);中心青年人才创新创业项目(No.SC202328)。

摘　　要：建立硫乙醇酸盐流体培养基中微量细菌16S核糖体基因的快速提取方法,开发基于荧光定量PCR技术宽泛的16S核糖体基因的快速分析方法用于无菌检验。以无菌检查法中标准菌株定量污染硫乙醇酸盐流体培养基,采用Takara微生物裂解液、溶菌酶、Chelex-100螯合树脂不同裂解方式的组合提取微量细菌核酸,采用常规PCR及电泳比较目的DNA的提取效果。分别采用Tagman杂交探针和荧光染料-qPCR法,分析各试验菌株提取的系列浓度的核酸模板;应用荧光染料-qPCR法检测重组乙型肝炎疫苗(汉逊酵母)的硫乙醇酸盐流体培养物中16S核糖体基因水平以评价其无菌性。采用溶菌酶结合Takara微生物裂解液及Chelex-100螯合树脂的核酸提取方法,常规PCR、杂交探针-qPCR法和荧光染料-qPCR法的灵敏度分别可检测到硫乙醇酸盐流体培养基中10~2、10~3和10~1 CFU/mL的污染细菌;荧光染料-qPCR法的各试验菌浓度的对数值与扩增曲线的Ct值呈一定的线性关系(0.969 8≤r≤0.996 8);三批次重组乙型肝炎疫苗(汉逊酵母)的7 d培养物的Ct值均大于微生物污染判读限,均判定为无菌生长,与现行药典检测结果一致。建立的硫乙醇酸盐流体培养基中微量细菌核酸快速提取及荧光定量PCR检测方法在无菌产品快速检查中具有应用潜力。To establish the method for rapid extraction of 16S ribosomal nucleic acid gene from micro bacteria in fluid thioglycolate medium(FTM),and to develop a rapid broad-range 16S rDNA assay for Sterile test,based on fluorescence quantitative PCR technology(qPCR),FTM was quantitatively inoculated by the standard strains in Sterility tests of Chinese Pharmacopoeia,and the extraction results of micro bacterial nucleic acid with serial combination of Takara Lysis Buffer for Microorganism,Chelex-100 Resin and lysozyme were compared by conventional PCR and electrophoresis assay.Both Tagman hybridization probe and fluorescent dyeqPCR were used to analysis the nucleic acid templates extracted from different strains in FTM culture,as well.Fluorescent dye-qPCR was used to test the 16S ribosome gene level in FTM culture of recombinant Hepatitis B vaccine(Hansenula Polymorpha),to assay the sterility of the product.Using the combined nucleic acid extraction method of lysozyme,Chelex-100 Resin and Takara Lysis Buffer for microorganism,the sensitivities of conventional PCR,Tagman hybridization probe-qPCR and fluorescent dye-qPCR were detected 10°,103 and 10'CFU/mL contaminated bacteria in FTM,respectively.There was a linear relationship between the logarithm of each bacterial concentration and the Ct value of the amplification curve by fluorescent dye-qPCR(0.9698≤r≤0.9968).All three batches of FTM culture(7 days)of recombinant Hepatitis B vaccine(Hansenula Polymorpha)were judged to be bacteria-free,because their Ct values were all above the microbial contamination limit,and the results were consistent with the current Chinese Pharmacopoeia.The methods of rapid extraction and fluorescence quantitative PCR technology for microbiological control of microbacterial nucleic acid in FTM culture will permit potential application in rapid detection of aseptic products.

关 键 词：硫乙醇酸盐流体培养基 微量细菌 16S核糖体基因 提取 无菌检验 杂交探针 荧光染料-qPCR 重组乙型肝炎疫苗 

分 类 号：R927.1[医药卫生—药学]