Identification of nine species of the Chlamydiaceae using PCR-RFLP

The family Chlamydiaceae contains two genera and nine species. Rapid and ea sy identification of these species is essential for taxonomic, epidemiologi cal and clinical determinations. Currently, DNA sequence analysis is the on ly accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laborat ory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 1 6S and 23S rRNA sequence data from >50 chlamydial strains representing all nine species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial spec ies were reliably distinguished in the tests. The procedure was optimized b y adjusting the annealing temperature using both a standard and a heat-acti vated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas an d other bacteria. The result was that a PCR method for species identificati on of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created. This method can be used to rapidly identify known species of the family Chlamydiaceae.