Sung Hyeran, Kang Seung Hye, Bae Yoon Jin, Hong Jin Tae, Chung Youn Bok, Lee Chong-Kil, Song Sukgil
College of Pharmacy, Chungbuk National University, 12 Gaeshindong, Cheongju, Chungbuk, Republic of Korea.
J Microbiol. 2006 Feb;44(1):42-9.
In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.
在本研究中，我们描述了一种新开发的灵敏的两阶段PCR程序，用于检测13种常见的支原体污染物（关节炎支原体、牛支原体、发酵支原体、生殖支原体、人型支原体、猪鼻支原体、溶神经支原体、口腔支原体、梨支原体、肺炎支原体、肺支原体、唾液支原体、解脲脲原体）。对于初次扩增，使用通用支原体引物靶向13种支原体16S和23S rRNA基因所在的DNA区域。然后，将初次PCR产物进行二次巢式PCR，使用通过对13种支原体物种获得的核苷酸序列进行多重比对设计的两组不同引物对。巢式PCR产生165 - 353 bp的DNA片段，能够检测到1 - 2个拷贝的目标DNA，并且证明与相关微生物或人类细胞系的基因组DNA无交叉反应，从而证实了所用引物的敏感性和特异性。通过进行限制性片段长度多态性（RFLP）结合Sau3AI酶切来鉴定污染的物种。本研究获得的结果提供了证据，表明所采用的检测系统是诊断细胞培养系统中支原体污染的有效工具。
