A snapback primer mediated one-step PCR assay for the identification of Legionella and Legionella pneumophila strains

Abstract

Objective To test a snapback primer for the identification of Legionella and Legionella pneumophila (L. pneumophila) strains in a one-step real-time PCR assay. Methods A novel primer was designed with a pair of genus-specific primers of Legionella strains. The species-specific probe sequences of L. pneumophila strains were linked at the 5′ end of the reverse primer. The sensitivity and specificity of the novel PCR assay were tested with 43 types of Legionella and non-Legionella strains. The established PCR assay was used to identify 186 wild Legionella strains isolated from 11 provinces of China and 15 environmental water samples. Results The amplicon melting peak of Legionella strains was detected at 85-86℃. The snapback melting peak of L. pneumophila was detected at 71℃. No melting peak of non-Legionella strains was detected. The sensitivity of the standard strains and simulated water samples were 1 ng/μl DNA templates and (1×103-1×104)/ml, respectively. 186 Legionella strains and 44 L. pneumophila strains isolated from environmental water samples were successfully identified with the snapback primer. Twealve Legionella strains and 4 L. pneumophila strains were identified from 15 environmental water samples with the snapback primer as compared with 8 Legionella strains identified with the culture method. Conclusion The snapback primer mediated one-step PCR assay could be used for the identification of Legionella and L. pneumophila strains with the advantages of high specificity and sensitivity. Key words: Legionella; Legionella pneumophila; Snapback primer; Real-time PCR