PCR-酶切技术快速鉴定军团菌

Rapid identification of Legionella isolates using PCR combined with restriction endonuclease digestion technology

【目的】建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值。【方法】根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法。对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序。【结果】该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的HinfⅠ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致。【结论】PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测。

[Objective] To develop a new method based on PCR amplification and restriction endonuclease digestion of a 557 bp fragment of the 16S rRNA gene for identification of Legionella isolates. [Methods] The primers were designed according to sequences of 16S rRNA gene of Legionella. Pure culture microbial strains were used as templates. The reaction conditions were optimized and the specificity were verified by 16 L. pneumophila strains, 22 non-L. pneumophila strains and 12 other bacteria strains. One hundred sixty-nine isolates were identified by PCR-enzymatic digestion, and 16S rDNA and mip gene sequencing analysis. [Results] The results showed that 16 L. pneumophila strains, 22 non-L, pneumophila strains and 12 other bacteria strains could be correctly identified and differentiated by this scheme. 169 isolates examined by the scheme, 79 L. pneumophila strains and 81 non-L, pneumophila strains were identified by PCR-enzymatic digestion, and the results were consistent with 16S rDNA and mip gene sequencing analysis among them. [Conclusion] PCR combined with restriction endonuclease digestion technology is a simple and specific method for rapid identification and differentiation ofL. pneumophila and non-L, pneumophila isolates.