实验室快速检测细胞株支原体污染的方法研究

目的    建立快速可靠的实验室检测细胞株培养中支原体污染的方法。方法    采用DNA荧光染色试剂对本实验室11株细胞进行荧光染色观察与分析；同时从常见污染细胞的支原体16S rRNA选择两段高度保守的核酸序列，作为支原体引物，采用聚合酶链反应（PCR）法对同一批细胞株进行检测，并对两种检测结果进行比较。 结果    采用DNA荧光染色法检出11株细胞中，有2株细胞出现阳性，阳性率为18.2%；采用PCR方法检测11株细胞，有4株细胞出现阳性，阳性率为36.4%。通过两种方法比较可以发现：DNA荧光染色法检测结果阳性的细胞株用PCR方法检测结果也是阳性。 结论    上述两种方法均能有效地检出细胞株中的支原体污染，DNA荧光染色法虽较PCR法灵敏度低，但操作简便易观察；而PCR法成本较高，将两种方法结合应用，对DNA荧光染色法可疑阳性的标本再进行PCR检测，既提高阳性检出率，又节约了成本。

Investigation of fast methods for detection of mycoplasma contamination in cell lines in laboratory

Objective To establish fast and reliable methods for detection of mycoplasma contamination in cell culture in laboratory. Methods Eleven cell lines were stained by DNA fluorescein stain, and the results of staining were analysed. Two pieces of highly conserved oligonucleotide sequences from mycoplasmal 16S rRNA of contaminating cells were selected and used as primers of mycoplasma. The mycoplasma contamination in cell culture was determined by PCR. A comparison of the results of determination was made between the two methods. Results Out of 11 cell lines, 2 ( 18.2% ) gave a positive result with DNA fluorescein staining method, and 4 (36.4%) gave a positive result with polymerase chain reaction (PCR) method. A comparison between the two methods indicated that those cell lines positive by DNA fluorescein staining were also positive by PCR. Conclusions The above-mentioned two detection methods can effectively detect mycoplasma contamination in cell lines. The sensitivity of DNA fluorescein staining is lower than that of PCR, but the cost of PCR is higher. Combined use of the two detection methods, for example, the cell lines being suspectedly positive by DNA fluorescein staining are detected by PCR, can raise positive detection rate, and lower cost.