Single-run, parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain reaction

Abstract

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.

Figure 1

DNA fragment including the specific forward and reverse primer sequences of Chlamydia pneumoniae (CP)-, Legionella pneumophila (LP)-, and Mycoplasma pneumoniae (MP)-specific real-time PCRs. The center box represents the heterologous DNA derived from the neomycin phosphotransferase gene (neo), which is detected with one pair of fluorescence resonance energy transfer hybridization probes. The composite primers for the preparative PCRs are shown as horizontal lines from bottom up in the order of their use. Primer sequences are listed in Table 1.

Figure 2

Fluorescence versus cycle number plots of 10-fold dilutions of a bronchoalveolar lavage specimen spiked with standards of the Mycoplasma pneumoniae strain (5000, 500, 50, and 5 CFU per PCR reaction) and repeat tested showing Mycoplasma-specific amplification products (channel F2/backF1; a) and the corresponding internal control-specific amplification products (channel F3/backF1; b).