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The Journal of the Japanese Society for Clinical Microbiology |
Biblioraphy Information
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[Vol.28 No.2 contents] Japanese / English |
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| ArticleTitle | Construction and evaluation of a method for detecting genetic mutations for macrolide resistance in Mycoplasma pneumoniae with an auto gene analyzer, GENECUBE |
| Language | J |
| AuthorList | Yosuke Kawashima1), Yoshiko Uekura2), Keita Yamashita3), Shigeyuki Notake3), Koji Nakamura3), Shinsuke Kimata2), Yoshihiro Soya1), Hiromichi Suzuki4,5) |
| Affiliation |
1) Diagnostic System Department, TOYOBO CO., LTD. 2) Tsuruga Institute of Biotechnology, TOYOBO CO., LTD. 3) Department of Clinical Laboratory, Tsukuba Medical Center Hospital 4) Department of Clinical Laboratory Medicine, Tsukuba Medical Center Hospital 5) Division of Infectious Diseases, Department of Medicine, Tsukuba Medical Center Hospital |
| Publication | J.J.C.M.: 28 (2), 98-105, 2018 |
| Received | July 18, 2017 |
| Accepted | October 19, 2017 |
| Abstract | The incidence of macrolide-resistant M. pneumoniae has increased in recent years. Mutations in the 23S rRNA gene are known to cause changes in the ribosome structure, which are related to macrolide resistance. Therefore, detection of mutations in the 23S rRNA gene using direct sequencing or melting curve analysis is the method of choice for confirming macrolide resistance in M. pneumoniae samples. In this study, we developed a detection method using the fully automated gene analysis GENECUBE and the PCR-Quenching Probe (QProbe). This method can be used for detecting the 23S rRNA gene of M. pneumoniae and identifying 2063 and 2064 base mutations, which are the main mutations responsible for macrolide resistance. The presence or absence of mutations at the position 2063 or 2064 of 23S rRNA gene can be clearly distinguished using the melting curve analysis. Utilizing this method, we examined 832 posterior pharyngeal wall samples and found that 261 were M. pneumoniae-positive and 571 were negative. We analyzed mutations in the 23S rRNA gene by direct sequencing of the 217 samples that were preserved from the extracted M. pneumoniae-positive samples. Among the 173 samples that were successfully analyzed by direct sequencing, 82 were found to be wild-type by direct sequencing. The mean fluorescence peak temperature of these samples was 57.5±1.16°C (mean±2SD). The remaining 91 samples were found to be mutants. The mean fluorescence peak temperature of these samples was 47.8±1.85°C (mean±2SD). The genotype obtained using our detection method was 100% consistent with the results obtained by direct sequencing of the 23S rRNA gene. |
| Keywords | Mycoplasma pneumoniae, PCR, macrolide resistance, GENECUBE, QProbe |
| Copyright © 2002 The Japanese Society for Clinical Microbiology All rights reserved. |