用滚环扩增技术检测污染细胞的5种支原体  

Detection of five common cell culture contaminant Molllcutes using rolling circle amplification

作　　者：王辉[1] 周小勇[1] 孔繁荣[1] 王玮蓁[1] 段逸群[1] 

机构地区：[1]湖北省武汉市第一医院皮肤科湖北省感染性皮肤病研究中心,430022

出　　处：《中华微生物学和免疫学杂志》2010年第10期949-952,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的 建立滚环扩增技术检测5种常见的细胞污染支原体（猪鼻支原体、发酵支原体、口腔支原体、精氨酸支原体、莱氏无胆甾原体）的方法.方法 使用细胞污染支原体16S-23SrRNA间隔区公共引物（SPS1,SPA2）扩增间隔区靶基因.设计每一种支原体特异性锁式探针,锁式探针与扩增的靶基因杂交结合形成环化单链分子,加入特异性引物在Corbett RotorGeneTM 6000扩增仪滚动扩增环化单链分子,观察结果.结果 滚环扩增技术能敏感和特异的检测上述5种支原体标准株,并能敏感的检测10个拷贝的靶基因.在62个细胞培养物样本的检测中,37个样本检测出一种支原体,14个样本检测出两种支原体,11个样本为支原体阴性.滚环扩增方法检测结果与支原体种特异性PCR检测结果一致.结论 滚环扩增作为一种新的扩增技术能敏感和特异的检测细胞污染支原体.Objective To develop a novel padlock probe and rolling circle amplification（RCA） to detect 5 common cell culture contaminant Mollicute（ Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ）. Methods ＂Universal＂ primers （ SPS1, SPA2 and SPS2, SPA1） were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.

关 键 词：滚环扩增 锁式探针 

分 类 号：R440[医药卫生—诊断学]