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

Abstract:

[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 of L. pneumophila and non-L. pneumophila isolates.

Key words:Legionella, PCR, Restriction endonuclease