We tested two commercial and three in-house PCR assays under standardized conditions to detect Mycoplasma pneumoniae. All five procedures were able to demonstrate M. pneumoniae DNA in a concentration comparable to 1 CFU/l, but the mean crossing points resulted in differences in the concentration of the genome copies of a factor of 20.Recent studies have shown that the cell wall-less mollicute species Mycoplasma pneumoniae is the causative agent of about 5 to 20% of all cases of community-acquired pneumonia in humans (2). In closed populations like those in army bases or in periods showing epidemic peaks of M. pneumoniae infections (occurring every 3 to 7 years) (8, 14), the incidence of infections might increase up to 50% (2). The primarily respiratory tract infection can be followed by extrapulmonary complications (22). The specific antibiotic treatment of the disease, restricted to macrolides, tetracyclines, or fluoroquinolones, accounts for the search for sensitive, specific, and fast methods for the detection of M. pneumoniae infections in routine bacteriological practice. Cultivation is time consuming (up to 3 weeks), insensitive, and requires confirmation that the colonies grown are M. pneumoniae bacteria. Serological methods, such as the complement fixation test, enzyme-linked immunosorbent assay, and Western blot, are commonly used in the clinical laboratory but have practical limitations. The age-and timedependent formation of specific immunoglobulin A and immunoglobulin M antibodies, the persistence of detectable antibody levels resulting from previous contacts with M. pneumoniae, and the variable specificity and sensitivity of the test kits used influence the significance of the results of serodiagnosis (2). Therefore, PCR approaches extended the spectrum of available routine methods. Recent results reported the superiority of PCR over serology for the confirmation of a M. pneumoniae infection in the clinically important first week after the onset of pneumonia symptoms (17).In recent years, different PCR systems targeting a wide range of genes of M. pneumoniae (e.g., P1 adhesin, 16S rRNA, and ATPase operon gene) have been developed (16). Real-time PCR especially is characterized by rapidity, practicability, reduced risk of contamination, and high specificity and sensitivity of detection (3,7,9,13,15,19,23,24,25). However, the increasing number of real-time PCR approaches stresses the need for comparative validation of the performance of the different test procedures. Up to now, studies dealing with the evaluation of more than a single quantitative PCR system to detect M. pneumoniae have been very rare (7, 25) and have not included commercial test kits. To our knowledge, commercial quantitative PCR systems for the detection of M. pneumoniae are not available in the United States (2), whereas kits from different manufacturers are widely used in Europe. The aim of the present study was to investigate the sensitivities of different commercial and in-house real-time PCR approaches for the detection of M. p...