FARRELL DJ, MCKEON M, DAGGARD G, MUKKUR TK.
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 225 (abstract no. 1569).Queensland Health Pathology Service, Toowoomba, AUSTRALIA
BACKGROUND: Although many PCR methods have been reported, none has yet fulfilled the consensus recommendations for the use of PCR in the diagnosis of B. pertussis (BP) infections (Meade & Bollen, J Med Microbiol 1994; 41:51) and hence a standardised method has not been forthcoming. The aim of this study was to develop such a method.METHODS: A rapid cycle PCR method with a microwell format/probe hybridisation detection step was developed using novel oligonucleotides targeted at the pertussis toxin operon. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. A novel hybridisation stringency ratio (HSR) was developed to differentiate BP from B. parapertussis (BPP) and B. bronchiseptica (BB). Contamination was minimised by the use of heat labile uracil N-glycosylase. Specimens were treated by simply diluting 1:10 in H[2]O and then heating for 20 minutes at 99.9[o]C. Analytical sensitivity and specificity were ascertained using 35 strains of Bordetella species and 30 strains of common respiratory pathogens and commensal organisms. Clinical sensitivity and specificity were ascertained using 79 specimens tested using a nested PCR method targeting the insertion sequence IS481 (Farrell et al., J Clin Microbiol 1999; 37:606).RESULTS: Analytical specificity was 100%. Analytical sensitivity was comparable to nested IS481 PCR (1 organism per reaction). 35/36 nested PCR positive specimens were positive - inhibitory substances being detected in the remaining specimen (which became positive after DNA purification). 2 specimens that fulfilled a clinical definition of pertussis were positive by the new method/negative by IS481 PCR. 41 specimens were negative by both methods. All Bordetella species and clinical specimens were identified correctly to the species level using HSR.CONCLUSION: The following were successfully developed: 1) A PCR method which fulfils all recommendations with the added benefit of being rapid (4 hours) and reproducible. 2) A novel, simple mechanism (HSR) to allow differentiation between BP and BPP/BB post-amplification.
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http://gateway.nlm.nih.gov/MeetingAbstracts/102245122.html
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