联合使用聚合酶链式反应和培养法可以提高培养细胞支原体的检出率

A PCR-based Assay Combined with Microbial Culture for Improving Mycoplasma Detection

目的分析国家实验细胞资源共享服务平台及其他实验室细胞支原体污染的情况,探讨聚合酶链式反应(PCR)和培养法在实验细胞支原体检测中的一致性。方法通过PCR法对245例细胞培养上清进行了支原体检测,并检测了阳性样品支原体感染的种类;对其中127例新鲜上清同时进行了培养法检测,通过Mc Nemar检验分析了二者的一致性。对二者检测不一致的细胞进行复检,同时使用生物发光法进行验证。结果国内培养细胞支原体感染率在19%~66%;培养法和PCR法检测支原体只有假阴性,没有假阳性;延长取样前细胞在无抗生素培养基中的培养时间至96h,可以提高低效价支原体感染样品的检出率。结论 PCR法和培养法检测支原体具有较好的一致性,应联合使用两种方法对培养细胞进行定期检测,提高支原体的检出率。在细胞培养过程中使用合格制剂,严格无菌操作,预防为主。

Objective To assess mycoplasma contamination levels and species in experimental cells from the China Infrastructure of Cell Line Resource and customers＇ laboratories and to discuss the practical methods for mycoplasma detection by comparing the consistency of PCR-based assay and microbial culture. Methods Totally 245 cell culture supernatant samples were assessed by PCR-based assay for mycoplasma detection,and mycoplasma species of positive samples were further detected. A total of 127 fresh samples were tested simultaneously by combination of the PCR-based assay and microbial culture. Mc Nemar＇ s test was used compare proportions in 2-by-2 contingency tables. For samples with inconsistent test results,cells were re-sampled,and tested by PCR,microbial culture,and bioluminescent assays. Results In China,the rate of mycoplasma contamination in experimental cells was 19%-66%. False negatives but no false positives were found for both methods and prolonged culturing for 96 h before sampling for mycoplasma detection could improve the detection of low titer infections. Conclusion The performances of the methods did not significantly differ,and the results were in good agreement. The combined use of these two assays could improve the detection rate of mycoplasma. More strict quality control and more vigorous aseptic techniques must be employed in cell culture studies to prevent mycoplasma contamination.