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PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

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Abstract

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.

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Acknowledgements

This work was supported by grants from the National High-Tech R&D Program of China (863 Program) (Grant Nos. 2006AA020703, 2006AA06Z409 and 2009AA06Z403), and the National Key Programs for Infectious Diseases of China (Grant Nos. 2008ZX10004-002, 2008ZX10004-009, 2009ZX10004-108 and 2008ZX10003-005). We would like to thank Ms. Gangyi Wang for supplying the condensed water samples.

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Authors and Affiliations

  1. Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, College of Life Sciences, Nankai University, Tian**, 300071, China

    Guangpeng Zhou, Boyang Cao, Yan Dou, Yanwei Liu, Lu Feng & Lei Wang

  2. TEDA School of Biological Sciences and Biotechnology, Nankai University, 23# HongDa Street, TEDA, Tian**, 300457, China

    Guangpeng Zhou, Boyang Cao, Yan Dou, Yanwei Liu, Lu Feng & Lei Wang

  3. Tian** Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tian**, 300457, China

    Guangpeng Zhou, Boyang Cao, Yan Dou, Yanwei Liu, Lu Feng & Lei Wang

  4. Tian** Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, 23# HongDa Street, TEDA, Tian**, 300457, China

    Guangpeng Zhou, Boyang Cao, Yan Dou, Yanwei Liu, Lu Feng & Lei Wang

  5. Tian** Biochip Corporation, 23# HongDa Street, TEDA, Tian**, 300457, China

    Lei Wang

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  1. Guangpeng Zhou
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  2. Boyang Cao
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  3. Yan Dou
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  4. Yanwei Liu
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Correspondence to Lei Wang.

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Zhou, G., Cao, B., Dou, Y. et al. PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB . Appl Microbiol Biotechnol 91, 777–787 (2011). https://doi.org/10.1007/s00253-011-3283-6

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  • DOI: https://doi.org/10.1007/s00253-011-3283-6

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