In this paper, we have described a mycoplasma genus-specific primer set (primers GPO-1 and MGSO), which specifically amplifies a 715-bp fragment with all mycoplasmal species investigated but not with other species. The specificity of this primer set, as is demonstrated in Fig. 7 in our article, was proven on the rDNA level. Very recently we have found that when the mycoplasma genus-specific primer set is used for the amplification of rRNA sequences, predominantly a 350-bp product is formed instead of the 715-bp product. Since this unusual phenomenon is observed only on the rRNA level, not on the rDNA level (as can be seen in Fig. 7), it is most probably due to some unusual features or (length) restrictions of the reverse transcription step. To avoid this problem, we have replaced primer GPO-1 with a new 5' primer (GPO-3), which is closer to the 3' primer MGSO. We have demonstrated that amplification with primer GPO-3 in conjuction with primer MGSO results in a polymerase chain reaction product of 270 bp on both the rDNA and rRNA level (see Fig. 1 in our article) and that these primers display the same specificity as was described previously for the primers GPO-1 and MGSO (see Fig. 2 in our article). For confirmation of the polymerase chain reaction with the primers GPO-3 and MGSO, we have used probe GPO-4. Thus, for amplification of rRNA sequences with the genus-specific primer set, we advise replacement of 5' primer GPO-1 and probe GPO-2 with the following oligonucleotides, GPO-3 (new 5' primer) and GPO-4 (new probe).

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus-and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Achokplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, LactobaciUlus, BaciUlus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.

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