Krzysztoń-Russjan Jolanta, Chudziak Jakub, Bednarek Małgorzata, Anuszewska Elżbieta Lidia
Department of Biochemistry and Biopharmaceuticals, National Medicines Institute (NMI), 00-725 Warsaw, Poland.
Internship at the Department of Biochemistry and Biopharmaceuticals, NMI Chelmska 30/34 Str., 00-725 Warsaw, Poland.
Diagnostics (Basel). 2021 May 14;11(5):876. doi: 10.3390/diagnostics11050876.
, , and sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the , , and sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of , , , , , , , , , , and . Limit of detection values varied between 125-300 and 50-100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from , , and in tested cell cultures.
支原体属、脲原体属和螺原体属是导致体外细胞培养污染的非典型细菌，会使结果出现偏差。这些细菌还会引发人和动物感染，并可能导致慢性病。在本研究中开发聚合酶链反应（PCR）时，应用了带有SYBR Green I荧光染料的定量PCR来促进支原体属、脲原体属和螺原体属的DNA检测与鉴定。筛选试验-1 v.1（三重定量PCR）可检测11种菌。试验-1 v.2（三个单重定量PCR）预先鉴定出三个亚组，从而减少了在试验-2中使用单重定量PCR进行菌种鉴定的次数。两种试验的范围均符合哺乳动物细胞微生物质量控制的药典要求，包括对支原体属、脲原体属和螺原体属中多个菌种（如口腔支原体、精氨酸支原体、发酵支原体、唾液支原体、猪鼻支原体、莱氏无胆甾原体、肺炎支原体、生殖支原体、解脲脲原体、人型脲原体和穿透支原体）的检测。试验-1和试验-2的检测限分别为每毫升125 - 300个拷贝和50 - 100个拷贝。试验-1和试验-2结果完全一致，能够在节省时间的情况下检测和/或鉴定受试细胞培养物中支原体属、脲原体属和螺原体属的选定菌种。
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