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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 establish a loop-mediated isothermal amplification (LAMP) assay for detection of mycobacterial contamination in live attenuated JE vaccine. Methods The intradermal injection of BCG vaccine, the standard and clinical strains of human Mycobacterium tuberculosis, the standard and clinical strains of Mycobacterium bovis, the standard and clinical strains of Mycobacterium phlei, the Mycobacterium fortuitum, Mycobacterium phlei, Kansas Mycobacterium, Mycobacterium turtle Mycobacterium Gordon, Mycobacterium Gordon, Mycobacterium terrengensis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium Asia, a total of 16 strains, the established LAMP sensitivity, precision Degree, applicability and specificity, and analyzed its feasibility in controlling the quality of harvested liquid of Japanese encephalitis virus. Results The sensitivity of mycobacterium Kansas and Mycobacterium avium was 103 CFU / ml and the detection sensitivity of other mycobacteria was 102 CFU / ml. The sensitivity of the method was good. Six test results of the same batch of samples Were consistent, two test workers were tested each batch of samples daily, continuous detection of 3 d, the test results are consistent, indicating that the method is good precision; Japanese encephalitis single virus harvest liquid composition did not interfere with the experimental results, The results showed that the method was of good applicability. The detection results of the single virus harbored by three non-mycobacteria were respectively negative for Bordetella pertussis, Staphylococcus aureus and Escherichia coli C3000, indicating that the method can distinguish Mycobacterium and non-mycobacterium bacteria, good specificity. Conclusion A LAMP method was established to detect mycobacterium contamination in live attenuated Japanese encephalitis vaccine. This method can be used to detect whether the single virus harbored by Japanese encephalitis is contaminated with mycobacteria.