结核杆菌实验室甲醛终末消毒效果辅助评价方法建立

发布时间：2018-02-24 11:00

本文关键词： 结核分枝杆菌实验室终末消毒效果评价方法建立　出处：《中国公共卫生》2016年11期 　论文类型：期刊论文

【摘要】：目的建立一种便捷可靠的辅助评价结核杆菌实验室终末消毒效果的新方法。方法首先以结核分枝杆菌复合群特异性插入序列IS6110为靶标自主设计引物IS6110P,并经结核分枝杆菌标准株H37Rv的培养物验证其特异性和灵敏度;其次,采用H37Rv培养物抽滤膜经甲醛终末消毒后分别采用普通核酸提取法和完整基因组提取核酸法提取核酸,再经IS6110P引物进行特异性PCR扩增验证其辅助评价甲醛终末消毒效果的情况,同时对抽滤膜进行分离培养以验证其评价结果;最后,采集经甲醛终末消毒结核杆菌实验室内的拭子和大气样本采用上述方法检测,并与分离培养结果相验证,以检测该方法的评价效果。结果 IS6110P可灵敏特异地对结核分枝杆菌进行扩增,检测限可达3.5 cfu/R;H37Rv培养物抽滤膜甲醛终末消毒后经普通核酸提取扩增仍有特异性核酸片段检出,而经完整基因组提取核酸扩增无特异性核酸片段检出;终末消毒后的结核分枝杆菌实验室采用普通核酸提取仍可扩增出结核分枝杆菌特异性核酸片段,而完整基因组提取核酸无特异性核酸扩增片段检出;平行的结核分枝杆菌分离培养实验结果均为阴性。结论采用IS6110P扩增结核分枝杆菌完整基因组的核酸提取物为辅助评价结核分枝杆菌实验室终末消毒效果的灵敏、便捷和可靠的新方法。
[Abstract]:Objective to establish a convenient and reliable method to evaluate the efficacy of terminal disinfection of Mycobacterium tuberculosis in laboratory. Methods the primer IS6110Pwas designed with the specific insertion sequence IS6110 of Mycobacterium tuberculosis as the target. The specificity and sensitivity of the culture of the standard strain H37Rv were verified. Secondly, H37Rv culture was used to extract the nucleic acid from the filter membrane by formaldehyde terminal disinfection, and then the nucleic acid was extracted by ordinary nucleic acid extraction method and complete genome extraction method, respectively. Then the specific PCR amplification with IS6110P primer was used to verify the effect of formaldehyde terminal disinfection. At the same time, the filter membrane was separated and cultured to verify the evaluation results. The swabs and atmospheric samples collected from the laboratory of formalin terminal disinfection of Mycobacterium tuberculosis were detected by the above method and verified with the results of isolation and culture. Results IS6110P could sensitively and specifically amplify Mycobacterium tuberculosis, and the detection limit could be up to 3.5 cfur / r ~ (37) Rv culture after the final disinfection of formaldehyde in the filter membrane, and the specific nucleic acid fragments could still be detected by ordinary nucleic acid extraction and amplification. However, the nucleic acid fragments of Mycobacterium tuberculosis were amplified by complete genome extraction, and the isolated fragments of mycobacterium tuberculosis could be amplified by ordinary nucleic acid extraction in the laboratory of Mycobacterium tuberculosis after final disinfection. However, the complete genome extracted nucleic acid with no specific nucleic acid amplification fragment was detected. Conclusion the nucleic acid extract from the complete genome of Mycobacterium tuberculosis amplified by IS6110P is a sensitive method to evaluate the efficacy of terminal disinfection of Mycobacterium tuberculosis in laboratory. Convenient and reliable new method.
【作者单位】：辽宁省疾病预防控制中心结核病防制所;
【基金】：辽宁省科技厅计划项目(2013225079);辽宁省科技厅卫生安全重点实验室项目(2007403016)
【分类号】：R378.911;R440

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