A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
| Autor: | Vegard Skogen, Merethe Elise Olsen, Johanna U. Ericson Sollid, Håkon Haaheim, Stig Ove Hjelmevoll, Magnus Unemo |
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| Rok vydání: | 2006 |
| Předmět: |
DNA
Bacterial Specific detection Pseudogene Porins Biology medicine.disease_cause Genome Polymerase Chain Reaction Sensitivity and Specificity Pathology and Forensic Medicine law.invention Microbiology Specimen Handling chemistry.chemical_compound Gonorrhea law medicine Polymerase chain reaction Neisseria meningitidis Virology Neisseria gonorrhoeae Real-time polymerase chain reaction chemistry Molecular Medicine DNA Pseudogenes Regular Articles |
| Zdroj: | The Journal of molecular diagnostics : JMD. 8(5) |
| ISSN: | 1525-1578 |
| Popis: | Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. |
| Databáze: | OpenAIRE |
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