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Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

ABSTRACT: The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human ?-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.

SUBMITTER: Nummi M

PROVIDER: S-EPMC4641141 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

Nummi Maaret M Mannonen Laura L Puolakkainen Mirja M

SpringerPlus 20151110

The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pn ...[more]

PMID: 26576327

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Project description:Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.

Dataset Information

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

Publications

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

Similar Datasets

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