Detection of Mycoplasma pneumoniae by using the polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) technique was used to detect Mycoplasma pneumoniae. A specific DNA sequence for M. pneumoniae was selected from a genomic library, and two oligonucleotides were chosen in this sequence to give an amplified fragment of 144 base pairs. We show that DNA from different M. pneumoniae strains can be detected by PCR, with DNA from other Mycoplasma species giving negative results. Analysis of biological samples (throat swabs) obtained from hamsters that were experimentally infected with M. pneumoniae showed that PCR was more sensitive and reliable than conventional culture techniques for the detection of M. pneumoniae. Initial experiments on artificially seeded human bronchoalveolar lavages showed that PCR can be used to detect 102 to 103 organisms.Mycoplasma pneumoniae is responsible for a wide spectrum of respiratory diseases. Most of them are mild infections. However, this pathogen is an important cause of primary atypical pneumonia; thus, the development of a highly sensitive microbiological diagnostic method is worthwhile. Two methods are currently used for this purpose: serological procedures or isolation of the organism by culture (3, 5). The cultivation of M. pneumoniae, a fastidious microorganism, is time-consuming and may require 3 weeks for results. Serological procedures are the most widely used and require the demonstration of a rise in antibody titer. It takes too long for results of both of these diagnostic methods to be obtained to allow for the rapid application of an effective treatment.Hybridization with DNA probes has been proposed as a rapid and specific procedure to replace the culture method for detecting M. pneumoniae (7, 10). Unfortunately, this procedure lacks sensitivity. Furthermore, most of the probes use radioactive labeling, a technique that is unapplicable for diagnosis on a widespread basis.Recently, an in vitro technique was developed to amplify short segments of DNA. This is referred to as the polymerase chain reaction (PCR) (18). Two primers that flank the region to be amplified are used with this technique, which is based on repeated cycles of denaturation, annealing of primers, and primer extension by a DNA polymerase. This results in the exponential accumulation of the target sequence copies. This technique has already been used for the diagnosis of genetic diseases (17) and viral infections (16). It is also an effective tool for bacteriological diagnosis (6, 15).We report here the enzymatic amplification by PCR of a specific DNA fragment of M. pneumoniae. Enzymatic amplification was performed under different conditions and was applied to several M. pneumoniae isolates and to dilutions of the organism in bronchoalveolar lavages (BALs). Furthermore, PCR was compared with the classical culture method for the detection of M. pneumoniae in experimentally infected animals. * Corresponding author. MATERIALS AND METHODSOrganisms and clinical specimens. M. pneumoniae FH and PI 1428, five M. pneumoniae clinical isolates, Mycoplasma hominis PG21, Mycoplasm...