Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia

Abstract

Background & objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP). The conventional detection methods (culture and serology) lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR) method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were used to detect M. pneumoniae infection in cases of pneumonias and findings compared.

Methods: A total of 134 samples consisting of blood (for serology) and respiratory secretions (for PCR and qRT-PCR) from 134 patients were collected. The blood samples were tested for IgG, IgM and IgA using commercially available kits. For standardization of PCR of M. pneumoniae P1 gene was cloned in pGEMTEasy vector. Specific primers and reporter sequence were designed and procured for this fragment. The qRT-PCR assay was performed to prepare the standard curve for M. pneumoniae positive control DNA template and detection in patient samples.

Results: Of the 134 patients, 26 (19%) were positive for antibodies against M. pneumoniae. IgG was positive in 14.92 per cent (20) cases, IgM in 4.47 per cent (6) and IgA was positive in 5.22 per cent (7) cases. In the qRT-PCR assay 19 per cent (26) samples were positive. Of the 26 qRT-PCR positive samples, nine could be detected by serology. PCR was positive for 25 samples. An extra sample negative by PCR was detected by qRT-PCR. Thus, real-time PCR assay, PCR and serology in combination could detect M. pneumoniae infection in 43 patients.

Interpretation & conclusions: The study shows that 17 patients were detected by serology alone, 17 were detected by qRT-PCR only and nine patients were positive by both serology and real-time PCR. Of the 134 samples tested, 25 were positive by conventional PCR, but qRT-PCR could detect one more sample that was negative by PCR and serology. These results suggest that a combination of two or three methods may be required for reliable identification of CAP due to M. pneumoniae.

Fig.

a. PCR amplification of P1 gene fragments for positive control preparation. Lane M: 1 kbp ladder, lane 1: P1 gene 543 bp positive PCR, lane 2: no template control. b. Restriction digestion of positive clones. Lane M: 1 kbp ladder, lanes 1-4: positive clones.