Novel PCR for Mycoplasma pneumoniae detection in specimens from patients with various types of respiratory infections

Abstract

INTRODUCTION: M. pneumoniae is the causative agent of primary atypical pneumonia and causes 20-40% of community acquired pneumonia. Patients mount IgM and IgG antibody responses, which provide useful diagnostic markers. IgM antibodies are not always produced in adults upon reinfection. Specific IgG antibodies increase slowly during the course of illness. Hence, test interpretation needs paired-serum which is not user friendly. Use of molecular diagnostic methods will overcome these. OBJECTIVE: To develop novel PCR primers to detect M. pneumoniae. METHODOLOGY: New forward and reverse primers which exclusively amplify M. pneumoniae-DWk encoding P1 adherent protein were developed. Master mix consisted of distilled water, 25mM-Mgcl2, 10X-PCR-Buffer, 10mM-dNTPS, two primers (10-p.M-Mpn-S (0.50p.M), 10-y.M-Mpn-RS (0.50|iM)) and Taq-Gold (5U/pJ). Purified M. pneumoniae- DNA (M129-B7-ATCC-29342) (20pg/ I) was used to determine PCR sensitivity. Detection limit was expressed as M. pneumoniae-DNA copy number. Each test had positive and negative controls. Specificity of PCR was evaluated using blast search. In addition, specificity was checked in the laboratory by doing the M. pneumoniae PCR with S. pneumoniae, H. influenzae and S. aureus (common respiratory pathogens causing pneumonia) and no positive reactions were observed among them. RESULTS: Limit of detection of M.pneumoniae-PCR was 400 fg of DNA which is equivalent to 10 copies per45pl of reaction mix. Specificity of the designed primer sequences was 100% with GenBank blast search and no cross reactions were observed with other respiratory-pathogens. M.pneumoniae-DNfltwas detected in 52% (13/25) of sero¬logy confirmed (positive IgM +/ IgG seroconversion) cases. CONCLUSION: Novel M. pneumoniae PCR has a sensitivity of 52% when tested with serology confirmed cases and a specificity of 100% when tested against other common respiratory pathogens. Detection limit was 10 copies / 45 pi of reaction mix.