Bacteriology
Detection of Mycoplasma pneumoniae P1 subtype variations by denaturing gradient gel electrophoresis

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Abstract

There were several methods to detect p1 gene variations in Mycoplasma pneumoniae. In this study polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) assay was performed to establish a rapid and precise detection method for identifying M. pneumoniae p1 gene variations. We detected p1 gene variations in 109 M. pneumoniae clinical isolates from Shanghai, China, which were collected from 2009 to 2011 by DGGE, and compared this method with the PCR-based restriction fragment length polymorphism assay and sequencing. By PCR-DGGE method, among the 109 M. pneumoniae isolates, 101 (92.7%) isolates were classified into type I, and 8 (7.3%) were classified into type II. Seven (6.9%) type I variations and 8 (100%) type II variations were identified. The match rate of p1 gene variation detected by DGGE reached 100% when compared to DNA sequencing and was more sensitive than restriction fragment length polymorphism. One new type II variant, designated as V2d, was found in this study. The sequence of the new variant was characterized. Our results indicated that PCR-DGGE is a rapid and reliable bio-technique for direct detection of p1 gene variations.

Introduction

Mycoplasma pneumoniae is the most common pathogen of community-acquired respiratory tract infections (Waites and Talkington, 2004). Epidemiological studies have shown that M. pneumoniae is responsible for 20–50% of community-acquired pneumoniae (Atkinson et al., 2008). Strains typing or subtyping by molecular methods is a powerful tool for surveillance and outbreak investigation. Several molecular typing methods have been developed. Historically, typing schemes of M. pneumoniae were based upon restriction fragment length polymorphism (RFLP) analysis (Cousin-Allery et al., 2000), multiple-locus variable-number tandem-repeat analysis assay (Dégrange et al., 2009, Dumke and Jacobs, 2011, Liu et al., 2012), pyrosequencing (Spuesens et al., 2010, Spuesens et al., 2012) and sequencing (Zhao et al., 2011). Among them, restriction fragment length polymorphism (RFLP) analysis of P1 gene (the gene encoding for the major adhesin of M. pneumoniae), which contains copies of repetitive elements (repMp4 and repMp2/3), is the most common genotyping method for M. pneumoniae molecular typing and identification of variants of each subtype (Cousin-Allery et al., 2000, Kenri et al., 1999). However, clinical isolates are poorly differentiated by PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis as M. pneumoniae is a genetically homogeneous species (Dégrange et al., 2009).

Denaturing gradient gel electrophoresis (DGGE) was used to detect single base mutations in DNA products (Su et al., 2012). This method is based on the different level of migration of DNA fragments following strand separation caused by chemical denaturants. DGGE was now applied to investigate the fingerprint of samples (Matussek et al., 2011, McAuliffe et al., 2003, Muyzer, 1999, Oates et al., 2012) and adopted for the investigation of the microbial diversity of food-contact surfaces (Koo et al., 2013).

The aim of this study was to determine the P1 variants of M. pneumoniae in DNA samples from 109 isolates commonly associated with respiratory tract infections (RTI) collected during 2009–2011 in Shanghai, China, using a novel and rapid PCR-based denaturing gradient gel electrophoresis (DGGE) method.

Section snippets

M. pneumoniae clinical isolates and DNA preparation

One hundred nine unique M. pneumoniae clinical isolates, which had never been described before, were obtained from bronchial aspirations with low respiratory infections (each specimen collected from one patient) from January 2009 to march 2011 in Shanghai. Culture and identification of M. pneumoniae was carried out as described previously (Liu et al., 2009). The Genomic DNAs of each isolate were extracted using TIANamp Bacteria DNA kit. Two reference strains of M. pneumoniae M129 (ATCC 29342,

Results of p1 gene subtyping by PCR-RFLP

The p1 gene PCR generated fragments of approximately 1686 bp (primers RepMp2/3 F and RepMp2/3R) and 1877 bp (primers RepMp4F and RepMp4R) from the 109 clinical isolates (data not shown). After digestion of these fragments with Rsa I/Taq I, 101 (92.7%) clinical isolates were classified into type I, and 8 clinical isolates (7.3%) were classified into type II (Fig. 1). Among these strains, 4 (4.0%) type I variants and 7 (87.5%) type II variants were identified.

Results of p1 gene variation by PCR-DGGE

Results of parallel gel electrophoresis

Discussion

PCR-based molecular techniques such as DGGE (Oates et al., 2012) and RFLP (Laguerre et al., 1994) have been widely used for study microbial communities. PCR-RFLP typing in P1 gene has played an important role in the subtyping of M. pneumoniae for a long time. However, now the concept has to be accepted that the power of discrimination for PCR-RFLP is limited. In this assay and previous assays, M. pneumoniae clinical isolates could only be divided into two types and the variations could not be

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81000753 and 81072418).

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    Contributed equally to this work.

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