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Identification of nine species of the Chlamydiaceae using PCR-RFLP

Karin D. E. Everett and Arthur A. Andersen

Avian and Swine Respiratory Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, PO Box 70, Ames, IA 50010, USA

Author for correspondence: Karin D. E. Everett. Tel: +1 706 542 5823. Fax: + 1 706 542 5771. e-mail: keverett{at}calc.vet.uga.edu or kdeeverett{at}hotmail.com

ABSTRACT

The family Chlamydiaceae contains two genera and nine species. Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations. Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from >50 chlamydial strains representing all nin species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial species were reliably distinguished in the tests. The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria. The result was that a PCR method for species identification 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.

Key Words: Chlamydia • PCR-RFLP • rRNA • human pathogens • animal pathogens

Present address: Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602--7371, USA.

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