Mycoplasma pneumoniae and viruses in acute respiratory tract infections in children were studied during the winter of 1992-1993 in Antwerp, Belgium. M. pneumoniae was diagnosed in nasopharyngeal aspirates by culture and polymerase chain reaction (PCR). For this, amplification of a fragment of the PI adhesin gene in samples prepared by two methods was compared in two laboratories, and in one laboratory, a fragment of the 16S rRNA gene was amplified. The sensitivity of culture versus PCR was 61.5%. Provided a specific internal control is used, sample preparation by freeze-boiling combined with PCR for the PI gene and amplicon detection by visual inspection of the electrophoresis gel can be recommended, although maximal results are obtained after hybridization. M. pneumoniae was present in 0.5% of patients <2 years old and 6.9% of patients >2. M. pneumoniae was second to respiratory syncytial virus or detected equally in lower respiratory infections.

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.

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacterjejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the

Polymerase chain reaction (PCR) amplification of specific regions in the genomes of a variety of lower eukaryotes permits rapid identification of these microorganisms. First, on the basis of the presence of both constant and variable regions in the small subunit (ssu) rRNA, a nested PCR for direct identification of various Candida species can be designed. Amplification of the entire ssu rRNA gene and subsequent reamplification of variable sequences within the V4 domains of these PCR products were combined with direct sequencing. This allows direct classification and identification of the microorganism present. MATERIALS AND METHODS Candida strains and DNA isolation. Candida species type strains were obtained from the Central Bureau voor Schimmelcultures, Baarn, The Netherlands. The following six 904