摘要
目的：检测细胞培养中支原体的污染，建立支原体通用的ＰＣＲ方法。方法：根据已出版的序列设计并合成一对支原体（柔膜体纲）１６ＳｒＲＮＡ基因组特异引物。对实验室所存的１２种支原体、大肠杆菌、变形杆菌及无支原体污染的组织培养细胞进行了ＰＣＲ扩增。以ＰＣＲ与分离培养比较检测了３３份细胞培养标本。结果：１２种支原体均出现了约６２０ｂｐ左右的特异性扩增带，敏感性为１００～１０００个支原体细胞，且常见的６种细胞培养污染支原体的扩增片段均能被ＨｉｎｄⅢ切成２８０与３４０ｂｐ左右的２条带。理论上推测这对引物可扩增所有支原体１６ＳｒＤＮＡ。但对大肠杆菌、变形杆菌及无支原体污染的组织培养细胞等未见有扩增带出现。在对３３份细胞培养标本的检测中，除ＰＣＲ和分离培养同时检出２份阳性外，前方法还另检出阳性３份。５份ＰＣＲ扩增物均被ＨｉｎｄⅢ切出２条带。结论：ＰＣＲ较分离培养敏感性高，可提高细胞培养污染支原体的检出率，防止出现假阴性结果；如做好质量控制，对于一般实验室检测细胞培养支原体污染。
Objective: To detect mycoplasmas contaminations in cell cultures and establish a mycoplasma group specific PCR technique. Methods: A pair of 16S rRNA based mycoplasmas (Mollicute) group specific primers were designed and synthesized. The established PCR method was investigated by testing 12 Mycoplasma and Acholeplasma species preserved in our laboratory as well as E.coli, P.vulgaris and culture cell lines not carrying mycoplasma contaminants. Thirty three cell cultures were examined by using PCR and microbiological culture. Results: About 620 basepairs specific fragments from all 12 Mycoplasma and Acholeplasma species were amplified by this PCR. The sensitivity of mycoplasma detection ranged between 100 and 1 000 cells. PCR products from 6 common contaminants of cell cultures could be digested by Hin d Ⅲ to produce 2 fragments of each of 280 bp and 340 bp size. It was inferred that 16S rDNA of all mycoplasma (Mollicute) species may be detected by this PCR method. DNA from E.coli, P.vulgaris and normal culture cell lines were not amplified by the PCR. Two of 33 cell cultures were found positive by both PCR and culture. Additional 3 samples were positive by PCR but negative by culture. All 5 amplified products could be digested by Hin d Ⅲ resulting in two fragments. Conclusion: As a more sensitive method than culture, PCR might increase detection rate of mycoplsma contaminants and avoid false negative results. PCR will serve as a major diagnostic tool in the detection of mycoplasmas contamination in cell cultures if quality control of the method is implemented.
出处
《中山医科大学学报》
CSCD
1999年第2期151-154,共4页
Academic Journal of Sun Yat-sen University of Medical Sciences
关键词
细胞培养
支原体污染
聚合酶链反应
cells, cultured
mycoplsma
contamination
16S rRNA
polymerase chain reaction
