Amplification of p1-gene by polymerase chain reaction for detection of mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is the causative agent of respiratory and lung infection, especially in children and young adults. Severe complications frequently occur when appropriate antibiotic treatment is not administered. Correct diagnosis is, therefore, important effective treatment and the prevention of complications. Laboratory diagnostic methods such as culture and serological tests are time-consuming, insensitive and nonspecific. With the advent of DNA technology, polymerase chain reaction (PCR) was applied in the detection of microorganisms in clinical specimens and demonstrated to provide rapid, sensitive and specific results. Many studies for detecting M. pneumoniae by PCR have been reported but still have limitation of using radioisotope and the amplicon carryover. In order to apply PCR for detection of M. pneumoniae, primers were selected from the gene encoding P1 protein which is responsible for cell adherence. PCR conditions were determined. When the amplified product was analysed by gel electrophoresis, the PCR had a sensitivity of 10 fg purified M. pneumonia DNA. The sensitivity of the PCR was increased to 1 fg when the amplified products were analysed by fluorescein-labelled probes with dot blot hybridization or nested PCR. The specificity of PCR was determined by using DNAs from other Mycoplasma species, bacterial isolates from respiratory infected patients, and also human leukolytes. It was found that no amplification occurred. The false-positive due to amplicon carryover was prevented by the incorporation of dUTP instead of dTTP and adding the uracil-N-glycosylase(UNG) in reaction mixture prior to PCR. The efficiency of PCR for detection of M. pneumoniae in simulated specimens was determined and found that sensitivity was 100 fg of spiked M. pneumoniae DNA. Considering of speed, sensitivity, specificity, the use of nonradioisotope-labelled probe and the use of enzymatic pre-PCR sterilization, the developed PCR-based protocol was more suitable and reliable for laboratory diagnosis of M. pneumoniae.