结核杆菌实验室甲醛终末消毒效果辅助评价方法建立

Establishment of a supplementary evaluation method for mycobacteria laboratory's terminal disinfection

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 explore a new method for supplementary evaluation on the efficiency of mycobacteria la- boratory＇s terminal disinfection. Methods First,Mycobacterium complex specific insert sequence IS6110 was used as a target sequence to design mycobacteria specific primer IS6110P independently, and the culture of mycobacteria standard strain H37Rv was used to test the primer＇s specificity and sensitivity. Second,H37Rv cultivation material pumping filtra- tion membranes were used to evaluate the common nucleotide extraction and genome extraction after formaldehyde termi- nai disinfection for primer chain amplification. Third, both swab and air suction filter samples were collected to extract nucleotide for primer chain amplification by traditional method and genome extraction method separately. At the same time,mycobacteria were cultured for all of the samples to evaluate the efficiency of different methods. Results Myco- bacteria nucleotide could be amplified sensitively and specifically with IS6110P and the limit of the detection was as low as 3.5 colony forming unit （ cfu ）/R. After terminal disinfection, the PCR results of mycobacteria laboratory＇ s smear samples were still positive with traditional method but negative with genome extraction method;meanwhile the mycobac- teria culture tests were ali negative. Conclusion Using IS6110P as primers to amplify nucleotide extracted from samples of mycobactria laboratory with genome extraction is a sensitive, convenient and reliable evaluation method for the evalua- tion on the efficiency of mycobacteria laboratory＇s terminal disinfection.