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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。
Objective To establish a convenient and reliable new method to evaluate the effectiveness of terminal sterilization of Mycobacterium tuberculosis. Methods The specificity and sensitivity of Mycobacterium tuberculosis isolates H37Rv were tested by using IS6110P, an IS6110 specific target gene of Mycobacterium tuberculosis complex, as the target, and the specificity and sensitivity of mycobacterium tuberculosis isolates H37Rv were tested. Secondly, Formaldehyde was finally disinfected by nucleic acid extraction method and intact genome DNA extraction method respectively, and then by IS6110P primer specific PCR amplification to verify its secondary evaluation of formaldehyde formaldehyde disinfection effect, while the membrane was isolated and cultured Finally, the swabs and atmospheric samples collected from the laboratory of formaldehyde-terminated Mycobacterium tuberculosis were detected by the above method and verified with the isolation and culture results to test the evaluation effect of the method. Results IS6110P could sensitively and sensitively amplify Mycobacterium tuberculosis with a detection limit of 3.5 cfu / R. After the H37Rv culture membrane was denatured, the specific nucleic acid fragments were detected by normal nucleic acid extraction and amplification. But no specific nucleic acid fragment was detected by complete genome extraction nucleic acid amplification. Mycobacterium tuberculosis bacterin still could amplify mycobacterium tuberculosis specific nucleic acid fragment after the terminal disinfection of Mycobacterium tuberculosis laboratory, but intact genome extracted nucleic acid No specific amplification of nucleic acid fragments detected; parallel Mycobacterium tuberculosis isolation culture experiments were negative. Conclusion It is a sensitive, convenient and reliable new method to amplify the complete genome of Mycobacterium tuberculosis using IS6110P as an assistant to evaluate the terminal sterilization of Mycobacterium tuberculosis.