两步PCR法检测细胞培养物中支原体污染的初步结果  

DETECTION OF MYCOPLASMAS IN CELL CULTURES BY USING TWO-STEP POLYMERASE CHAIN REACTION

作　　者：魏红梅[1] 袁曾麟[1] 陈天寿[1] 

机构地区：[1]中国药品生物制品检定所

出　　处：《中华微生物学和免疫学杂志》1994年第2期131-134,共4页Chinese Journal of Microbiology and Immunology

摘　　要：本文报道用两步ＰＣＲ法快速检测细胞培养物中污染的支原体（Ｍｙｃｏｐｌａｓｍａ）。两套引物选自１６ＳｒＲＮＡ和２３ＳｒＲＮＡ保守区域，外部引物ＰＦ１，ＰＲ１扩增的ＤＮＡ片段在３６０～５００ｂｐ之间，内部引物ＰＦ２，ＰＲ２扩增的ＤＮＡ片段在１４０～２２０ｂｐ之间。试验表明，两步ＰＣＲ法能检出污染细胞培养物七种常见的支原体。１７份待检细胞培养物，两步ＰＣＲ法检测，９份（５２．９％）为阳性，直接培养法检测，５份（２９．４％）为阳性。采用快速热循仪，使ＰＣＲ反应时间大大缩短，３０次循环仅需２０分钟。反应液的用量也仅１０μｌ，提高了检测效率，节省了材料，降低了试验用费。本文就两步ＰＣＲ的灵敏度等特性也作了初步测试。es.Two sets ofuniversal primers were selected from the conserved regions betweem 16S/23S intergenic spaces of these Mycoplas-ma species.The first PCR produced fragments of 360 to 500bp ,the second PCR products were from 140 to 220bp.The test showed that the two-step PCR could detect 9 species of Mycoplasmas ,compared with the direct culturetest that was able to detect 5 cell cultures positive from 17 samples ,the two-step PCR could detect 9 positive.Itshowed that the PCR is more sensitive.We used the 1605 Air thermo-cycler ,for shortening the amplification timefrom several hours to 20 minutes for 30 cycles ,lessening the reaction volume to 10μl,and increasing work efficien-cy.

关 键 词：聚合酶链反应 支原体 细胞培养物 

分 类 号：R446.5[医药卫生—诊断学]