摘要
本文报告两步ＰＣＲ法快速检测污染动物细胞培养物的支原体（Ｍｙｃｏｐｌａｓｍａ）．外部引物ＰＦ１，ＰＲ１和内部引物ＰＦ２，ＰＲ２选自ｒＲＮＡ操纵子１６ＳｒＲＮＡ，２３ＳｒＲＮＡ空间保守区域，第一步ＰＣＲ产物长度在３０９～５２７ｂｐ之间，第二步ＰＣＲ产物长度在１１０～３０９ｂｐ之间，此方法能扩增污染细胞常见的七种支原体，灵敏度达到２０３ｆｇ／ｍｌ，不能扩增常污染细胞的ＣＭＶ、Ｅ．ｃｏｌｉ以及ＳＰ２／０细胞、Ｈｅｌａ细胞的ＤＮＡ．５３株细胞培养物，ＰＣＲ检测２０株（３７．７％）为阳性，直接培养法检测１３株（２４．５％）为阳性，ＤＮＡ荧光染色法检测１４株（２６．４％）为阳性，其中动物细胞２３株，ＰＣＲ检测９株（３９．１％）为阳性，直接培养法检测７株（３０％）为阳性，ＤＮＡ荧光染色法检测８株（３４．８％）为阳性，不计ＡＴＣＣ提供的４株阴性细胞，阳性率分别为４７．４％，３６．８％，４２．１％，三种方法检测１８株动物细胞结果一致（１２株阴性，６株阳性）．３株ＰＣＲ检测阳性，其他方法检测阴性；Ｖｅｒｏ４１ＤＮＡ荧光染色法检测阳性，其他方法检测阴性：Ｊ－１１１ＰＣＲ检测阴性，其他方法检测阳性。ＰＣＲ法与常规法相比，灵敏度提高?
Two-step PCR has been applied to detect the contamination of Myco-Plasmas in mammalian cell cultures. Two sets of universal primers wereselected from the conserved regions between 16S and 23S intergenic spacesof these Mycoplasma specias.The size of the first PCR products was from 309 to 527bp,the size of the second PCR products was from from to 309bp.The PCR could detect 7 species of Mycoplasmas, but not CMV,E.coli SP2/0cell and Hela cell.The sensitivity is 203fg/ml.53 cell cultures were detectedby PCR,direct culture test and DNA fluorescent staining, the positive ratewas 20/53( 37 .7 % ) ,13/53 (24.5%) and 14/53 (26.4% ) respectively.There were 23mammalian call cultures, three methods were used to detect showing 9/23 (39 . 1 %) ,7/23(30%) and 8/23(34 . 8 %) showing positive raspectively .The resultswere in agreement in 18 mammalian cells detected by the three methods ( 12negative,6 positive ) .3 cases were positive by PCR but negative by cultureand staining.Vero 41 DNA was positive by staining but negative by PCR andculture, J-111 was negative by PCR but positive by culture and staining.Compared with the routine methods,the PCR was sensitive,specific ,simple andrapid, The volume was 10l ,the time of 30 cycles was about 20 minutes, soPCR was useful for detecting plenty of samples.
出处
《中国实验动物学报》
CAS
CSCD
1994年第2期81-87,共7页
Acta Laboratorium Animalis Scientia Sinica
