检测乙型脑炎减毒活疫苗中分枝杆菌污染的环介导恒温扩增法的建立  

Development of loop-mediated isothermal amplification method for detection of mycobacterium contamination in live attenuated Japanese encephalitis vaccine

作　　者：王静[1,2] 吕冰凌[2] 孟丽[2] 邓雪莲[2] 姚亚夫[2] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,四川成都610041 [2]成都生物制品研究所有限责任公司,四川成都610023

出　　处：《中国生物制品学杂志》2013年第11期1675-1678,共4页Chinese Journal of Biologicals

摘　　要：目的建立检测乙型脑炎减毒活疫苗中分枝杆菌污染的环介导恒温扩增(loop-mediated isothermal amplification,LAMP)法。方法选择皮内注射BCG疫苗、人结核分枝杆菌标准株和临床株、牛分枝杆菌标准株和临床株、草分枝杆菌标准株和临床株、偶然分枝杆菌、瘰疬分枝杆菌、堪萨斯分枝杆菌、龟脓分枝杆菌、戈登分枝杆菌、土地分枝杆菌、鸟分枝杆菌、施氏分枝杆菌、亚洲分枝杆菌共16株,对建立的LAMP法的灵敏度、精密度、适用性及特异性进行验证,分析其在乙型脑炎单一病毒收获液质量控制中的可行性。结果该方法对偶然、堪萨斯和鸟分枝杆菌的检测灵敏度为103CFU/ml,对其他分枝杆菌的检测灵敏度可达102CFU/ml,灵敏度良好;用建立的方法对同一批样品的6次检测结果均一致,2名试验人员分别每日检测每批样品,连续检测3 d,检测结果均一致,表明该方法精密度良好;乙型脑炎单一病毒收获液中的成分对实验结果无干扰作用,表明该方法适用性良好;分别加入百日咳杆菌、金黄色葡萄球菌和大肠埃希菌C3000 3种非分枝杆菌的乙型脑炎单一病毒收获液的检测结果均为阴性,表明该方法可区分分枝杆菌属细菌和非分枝杆菌属细菌,特异性良好。结论建立了检测乙型脑炎减毒活疫苗中分枝杆菌污染的LAMP法,该方法可用于检测乙型脑炎单一病毒收获液是否被分枝杆菌污染。Objective To develop a loop-mediated isothermal amplification method for detection of mycobacterium contamination in live attenuated Japanese encephalitis （JE） vaccine. Methods The developed LAMP method was verified for sensitivity, precision, suitability and specificity by using BCG for intradermal injection, standard strains and clinical isolates of human Mycobacterium tuberculosis, M. bovis, M. phlei, as well as M. fortuitum, M. scrofulaceum, M. kansasii, M. chelonei subsp, abscessus, M. gordonae, M. terrae, M. avium, M. shimoidei Tsukamura and M. asiaticum strains, based on which the feasibility of the method in quality control of single harvest of JEV was analyzed. Results The sensitivities of the developed method for Mo fortuitum, Mo kansas and M. avium were 103 CFU/ml, while those for other mycobacteria were 102 CFU/ml. The same sample was tested by the developed method for 6 times, and the results were in agreement. Each batch of samples were tested by two laboratroy staff once a day for 3 d, and the results were in agreement, indicating high precsion of the method. The component in a single harvest of JEV showed no interference to the test result, indicating high suitability of the method. All the test results of Bordetella pertussis, Staphylococcus aureus and E. coli added to the single harvest of JEV were negative, indicating high specicity of the method. Conclusion The LAMP method for detection of mycobacterium contamination in live attenuated JE vaccine was developed, which was suitable for a single harvest of JEV.

关 键 词：乙型脑炎减毒活疫苗 分枝杆菌 环介导恒温扩增法 质量控制 

分 类 号：R373.31[医药卫生—病原生物学] R392-33[医药卫生—基础医学]