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Literature reviews > Articles for review > Tabrizi et al. Evaluation of opa-based real-time PCR... |
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A real-time PCR assay using an opa-based target can be used as an accurate and rapid assay for detection of N. gonorrhoeae in clinical specimens. Evaluation of opa-based real-time PCR for detection of Neisseria gonorrhoeae. Question What is the sensitivity and specificity of a N. gonorrhoeae real-time PCR assay targeting the opa gene? Design This article describes the evaluation of the analytical and clinical sensitivity and specificity of a real-time 5' nuclease PCR assay targeting a conserved 90 bp region of the multicopy opa gene for detection of N. gonorrhoeae by testing a panel of microorganisms and clinical samples previously evaluated by 4 nucleic acid amplification tests (NAAT). Participants A microorganism panel of 173 ATCC strains and clinical isolates, including 100 N. gonorrhoeae, 10 N. meningitidis, 15 N. subflava, 5 N. lactamica, 6 other Neisseria species, and 37 non-Neisseria specimens commonly isolated from the urogenital area were tested. The N. gonorrhoeae isolates included 20 clinical strains that were negative by PCR targeting the cppB gene. One hundred thirty-five clinical samples previously evaluated by 4 NAAT (COBAS, 16S, and cryptic plasmid (cppB gene) PCRs and LCx ligase chain reaction), including 50 that were N. gonorrhoeae positive by all 4 NAAT, 50 that were negative by all 4 NAAT, and 35 that were positive by COBAS PCR and negative by confirmation cppB PCR were tested. Description of Tests and Diagnostic Standard DNA was extracted from cultured bacteria and clinical samples using the MagNA Pure LC instrument (Roche Diagnostics) and the DNA Isolation Kit I (Roche). Ten-fold serial dilutions of quantitated N. gonorrhoeae DNA were tested to assess sensitivity. DNA was tested using a real-time PCR assay that targeted a conserved region of the N. gonorrhoeae multicopy cell-surface opacity protein gene (opa). The reactions were performed in a LightCycler (Roche) using primers GCopaF-LNA and GCopaR-LNA. The 90 bp amplicon was detected using the TaqMan FAM and TAMRA labeled locked nucleic acid probe GCopa-LNA.The DNA from the clinical samples was also tested by real-time amplification of the human β-globin gene to assess presence of adequate amplifiable DNA. Main Outcome Measures The analytical and clinical sensitivity and specificity of the opa gene real-time PCR assay were determined. Main Results The opa gene PCR was positive for all 100 N. gonorrhoeae isolates tested and negative for all other Neisseria species and bacterial species tested. The analytical sensitivity was 1 gene copy per PCR reaction. The opa gene PCR results of the 135 previously tested clinical specimens are shown in the table. One of the 35 specimens that were positive by the COBAS PCR assay and negative by a confirmatory cppB gene assay was positive by the opa gene PCR. This one specimen was confirmed positive by the 16S PCR assay. Results of opa gene PCR for detection of N. gonorrhoeae among 135 clinical specimens by results of 4 other NAAT
Authors' Conclusions The opa gene PCR is a sensitive and specific gonococcal real-time PCR that can be used for screening and also as a supplemental assay for confirmation of positive specimens tested by commercial NAAT. Confirmation by the opa gene PCR may be more accurate in populations where N. gonorrhoeae strains without plasmids are circulating. Source of funding: None given For correspondence: Sepehr Tabrizi, Department of Molecular Microbiology, The Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia.E-mail address: sepehr.tabrizi@wch.org.au |
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