A new amplification target for PCR-RFLP detection and identification of Chlamydiaceae species.
The family Chlamydiaceae contains nine species pathogenic to humans and animals, but their routine identification is hampered by inadequate detection methods. In an attempt to find a new region for PCR detection and discrimination of the Chlamydiaceae species, the 3' end of the omp2 gene of Chlamydiaceae has been examined. Since sequence data for this part of the genes of Chlamydophila felis and Chlamydia suis had not been available, the near full length of the omp2 genes of these species were cloned and sequenced. Consensus primers enabling amplification of a previously untargeted region spanning 1,030 bp at the 3' end of the gene were designed. Discrimination of all nine Chlamydiaceae species was achieved via RFLP analysis of the amplicons with RsaI and HinfI or RsaI and TaqI endonucleases or via electrophoretic mobility analysis of the RsaI restriction fragments in agarose gel with bisbenzimide-PEG. Intraspecies uniformity of the RFLP patterns was evaluated by the typing of reference strains, isolates of human and animal origin from culture collections, and clinical specimens, and by computer analysis of GenBank sequences. The 3' end of the omp2 gene was shown to be an appropriate marker region suitable for rapid identification of Chlamydiaceae species and can be used for characterization of collection strains and new isolates in taxonomic, epidemiological, and clinical purposes.
Demkin
Vladimir V
VV
Laboratory of Molecular Diagnostics, Institute of Molecular Genetics RAS, Kurchatov Sq. 2, Moscow 123182, Russia. vdemkin@img.ras.ru
Zimin
Andrey L
AL
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002693:Chlamydiaceae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C063931:Omp2 protein, bacteria; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases; C040158:GTAC-specific type II deoxyribonucleases; C027046:TCGA-specific type II deoxyribonucleases