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. 2016 Apr;84(4):298-303.
doi: 10.1016/j.diagmicrobio.2016.01.007. Epub 2016 Jan 12.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay

Alvaro J Benitez  1 , Jonas M Winchell  2
Affiliations

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay

Alvaro J Benitez et al. Diagn Microbiol Infect Dis. 2016 Apr.

Abstract

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources.

Keywords: Detection; HRM Analysis; Legionella spp.; Real-time PCR; Typing.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Multiplex assay for detection and identification of 9 clinically relevant nonpneumophila Legionella isolates. (A) L. micdadei, (B) L. dumoffii, (C) L. feeleii, (D) L. longbeachae, L. sainthelensis sg 1 and sg 2, and (E) L. bozemanii, L. anisa, L. parisiensis, and L. tucsonensis sg 1 and sg 3. All samples were run in duplicate.
Fig. 2.
Fig. 2.
Real-time PCR-HRM assay for discrimination of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and sg 3 isolates targeting the ssrA gene.
Fig. 3.
Fig. 3.
HRM profiles for subtyping of Legionella spp., targeting nucleotide differences in the mip gene. (A) Subtyping of 26 L. bozemanii isolates, (B) subtyping of 77 L. longbeachae isolates, and (C) subtyping of 11 L. feeleii isolates.
See this image and copyright information in PMC

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