Polymerase chain reaction amplification of genes encoding P1 protein and 16S rRNA for detection of Mycoplasma pneumoniae

Abstract

M. pneumoniae is a causative agent of human respiratory tract, especially in children and young adults. Clinical diagnosis cannot be differentiated from infection caused by other bacteria or viruses. The rapid method for diagnosis of M. pneumoniae infection is important for effective treatment and the prevention of severe complications. This study was designed to evaluate nested polymerase chain reaction (nested-PCR) assay by the use of two sets of primers for detection of M. pneumoniae in clinical samples. Primers were selected from the gene encoding P1 protein (MP-primer) which is responsible for cell adherence, and a fragment of 16S rRNA gene (16S rDNA primer) which is highly conserved in cell. The false-positive due to amplicon carryover was prevented by the enzymatic degradation procedure with the use of uracil-N-glycosylase (UNG). The sensitivity for detection of purified M. pneumoniae DNA for MP-and 16S rDNA-nested PCR was 1 fg and 0.1 fg, respectively. Throat swab specimens obtained from 100 patients with respiratory complaints and 100 healthy volunteers were subjected to nested-PCR. The results of PCR were compared to those obtained by culture, and serologic testing by micorparticle agglutination. In patients, PCR detected M. pneumoniae DNA in 4 adults and 3 children who were all seropositive, except in one adult. This patient had seronegative result in the first serum but the second serum was not available for detecting rising in antibody titer. M. pneumoniae DNA was not detected in healthy volunteers. Antibody titers of <1:40, 1:40 and 1:80 were found in 81, 9, and 10 healthy volunteers, respectively. PCR inhibitors were detected in 20% of all samples : after dilution of these samples, all samples were reactive. Colonies of live M. pneumoniae were not detected on modified Hayflick agar plate by culture. The results suggest that MP- and 16S rDNA-nested PCR were sensitive, reliable, and suitable for rapid detection of M. pneumoniae in respiratory tract samples of patients with respiratory complaints