Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high-resolution melt analysis

Abstract

Mycoplasma pneumoniae is an important respiratory pathogen, accounting for up to 25% of community-acquired pneumonia, and is a common cause of hospitalized pneumonia in otherwise healthy adults and children. Mycoplasma pneumoniae isolates can be classified into two main genomic groups (type 1 and type 2) based on sequence variation within the gene encoding the major adhesion molecule P1. Although numerous publications have described real-time PCR assays for the detection of M. pneumoniae, none has been able to discriminate the two genomic types. Here, a real-time PCR assay that can distinguish each type of M. pneumoniae utilizing high-resolution melt-curve analysis is reported. Using this method, 102 isolates obtained from patients from 1965 to the present, including those from recent outbreaks, were typed along with reference strains M129 (type 1) and FH (type 2). The results show that 55 isolates (54%) can be classified as type 1 and 47 isolates (46%) as type 2, and 100% correlation was demonstrated when compared with a standard PCR-restriction fragment length polymorphism typing procedure. Typing of isolates obtained from recent outbreaks in the USA has revealed the presence of both types. This assay provides a rapid, reliable and convenient method for typing M. pneumoniae isolates and may be useful for surveillance purposes and epidemiological investigations, and may provide insight into the biology of M. pneumoniae distribution within populations.

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Although these preliminary typing results are promising, further studies that include a larger number of isolates are needed to confirm these findings. Moreover, MALDI-TOF MS allowed for not only the identification of M. pneumoniae isolates but also their adhesin P1 typing in a single step, without the need of additional molecular typing techniques (28,43,44). However, no direct correlation was found between MALDI-TOF MS and MLVA typing.…”

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint

“…The RAPD / AP-PCR method involves PCR amplification with a single arbitrary primer at low stringency, while AFLP method selectively amplifies restriction fragments from whole genome. These PCR-based genotyping techniques have allowed faster and reproducible typing of mycoplasmas for epidemiologic studies [Cousin-Allery et al, 2000;Geary & Forsyth, 1996;Grattard et al, 1995;Iverson-Cabral, et al, 2006;Kokotovic et al, 1999;Rawadi, 1998;Schwartz et al, 2009].…”

Identification of P1 Variants of Mycoplasma pneumoniae by Use of High-Resolution Melt Analysis

“…This assay requires amplification of five loci followed by capillary electrophoresis and analysis but provides a greater level of genomic resolution. We recently reported the development of a real-time PCR assay that is able to rapidly distinguish M. pneumoniae isolates into type 1 or type 2 categories (12). We used this technique to classify 102 isolates using real-time PCR followed by high-resolution melt (HRM) analysis of a variable region partially spanning the RepMP2/3 element of the P1 gene (10,12).…”

“…We recently reported the development of a real-time PCR assay that is able to rapidly distinguish M. pneumoniae isolates into type 1 or type 2 categories (12). We used this technique to classify 102 isolates using real-time PCR followed by high-resolution melt (HRM) analysis of a variable region partially spanning the RepMP2/3 element of the P1 gene (10,12). The current study reports the use of this assay to identify variants of each group based upon intratype sequence deviations.…”