基于16S核糖体序列分析对无菌药品检出菌溯源的分析

Traceability analysis of aseptic drug detection bacteria based on 16S ribosome nucleic acid/protein sequence

目的:采用基于16S核糖体的序列分析,对无菌检查药品检出菌进行溯源,确保无菌检查结果的准确可靠。方法:采用ABI 3730测序仪和基质辅助激光解离飞行时间质谱(MALDI-TOF MS),对本实验室无菌检查区收集的42株环境菌及无菌检查不合格样品中分离纯化的1株检出菌进行鉴定分型,建立系统进化树;在实验环境符合要求的条件下对不合格样品严格按照标准操作规程(SOP)进行再分析,并对再分析阳性培养物中阳性菌分离鉴定。结果:本实验室无菌检查区内共分离环境菌42株,以革兰阳性球菌(29株,占69.4%)和革兰阴性杆菌(8株,占19.0%)为主,其中表皮葡萄球菌8株(19.0%),沃氏葡萄球菌7株(16.7%),藤黄微球菌5株(11.9%),奥斯陆莫拉菌、解脲葡萄球菌及蜡状芽胞杆菌各3株(均占7.1%)。无菌检查样品中检出菌鉴定为1株纺锤形赖氨酸芽孢杆菌(Lysinibacillus fusiformis),在无菌保障条件都符合要求的情况下,不合格样品再分析检出该菌而本实验室洁净环境菌中未发现该菌。结论:无菌样品检出的纺锤形赖氨酸芽孢杆菌来自样品本身而非环境带入。

Objective:To ensure the accuracy and reliability test using sequence analysis based on 16 S ribosome to trace the source of bacteria detected in sterile drug. Methods:ABI 3730 sequencer and matrix assisted laser dissociation time-of-flight mass spectrometry were adopted to identify and classify 42 strains of environmental bacteria isolated in the sterile examination area in our lab.1 strain of bacteria isolated and purified from the unqualified samples in the sterility test was also assayed,and a phylogenetic tree was established Under the condition that the experimental environment meets the requirements,the unqualified sample was re-analyzed in strict accordance with SOP,and the positive bacteria was isolated and identified. Results:42 strains of clean environmental bacteria were isolated in the sterile examination area in our lab. The main type of the isolated bacteria were Gram-positive cocci(29 strains,69.4%) and Gram-negative bacilli(8 strains,19.0%),including 8 strains of Staphylococcus epidermidis(19.0%),7 strains of Staphylococcus wallichii(16.7%),5 strains of Micrococcus luteus(11.9%). At the meaning time,Moraxella Oslo,Staphylococcus urealyticus and Bacillus cereus were all 3 strains,accounting for 7.1% respectively. The bacteria detected in the samples were Lysinibacillus fusiformis,which was not isolated in the clean environmental area in our lab. Conclusion:The bacteria detected in the sterile drug comes from the sample itself rather than environmental pollution or personnel.