细胞培养中支原体污染的PCR检测

DETECTION OF MYCOPLASMA CONTAMINATION IN CELL CULTURES BY POLYMERASE CHAIN REACTION

根据支原体16s rDNA序列,选择RemyTeyssou设计的三条寡核苷酸链,组成两套引物:P_(1-2a)能检测出细胞培养中常见的各种支原体,P_(1-2b)能检出无胆甾原体。反应可检出体系中10CFV的菌体。此法先用于对实验室人为污染支原体Vero细胞的检测,后与DNA 染色法和培养法比较,检测了49份生物样品,其中24份传代细胞,PCR检测的阳性率为58％,DNA染色法为42％,培养法为33％;三者的灵敏性比较,PCR可检出10^(-3)稀释度的阳性样品,高于其他两种方法。此PCR方法快速、灵敏、特异,适用于细胞培养中支原体污染的检测。

According to 16 s rDNA sequences, three oligonucleotides designed by Remy Teyssou were synthetized to form two sets of primers. Primer 1-2 a is able to promote amplification of all Mycoplasma and Primers 1-2 b to Acholeplasma species examined. In the study of sensitivity, 10 CFV of mycoplasma can be detected. In this study, the PCR method, first applied to the detection of experimentally infected Vero cell lines, was then evaluated for the detection of mycoplasma in 49 biological samples and compared with DNA staining and culture methods. The positive rates of mycoplasma contamination in 24 continuous cell lines obtained by PCR. DNA staining or culture were 58%,42% and 33% respectively Furthermore, in dilution expe-riments. PCR detected the infecting mycoplasma at the lowest level of 10-3, whercas the other assays were less scnsitive. It is conclutled that this PCR system is a simple, rapid, specific and high sensitive method suitable for the detection of roycoplasma contarnination in cell cultures.