A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
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DOI：
10.2353/jmoldx.2006.060024
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发表时间：
2006-11-01
影响因子：
4.1
通讯作者：
Skogen, Vegard
中科院分区：
文献类型：
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作者：
Hjelmevoll, Stig Ove;Olsen, Merethe Elise;Skogen, Vegard
自从分子方法出现以来，淋病奈瑟菌的诊断与培养相比一直受到假阴性和假阳性结果的困扰。共生奈瑟菌属种和脑膜炎奈瑟菌与N.淋病，并且当使用包含太低特异性的分子测试时可能交叉反应。我们设计了一种实时聚合酶链反应（PCR），包括一个内部扩增控制，目标是N。淋病porA假基因在BioRobot M48上自动分离DNA。我们随后的PCR方法扩增了所有不同的N.淋病国际参考株（n = 34）和淋病奈瑟菌（N.淋病临床分离株（n = 176），但不是我们测试的13种不同的非淋球菌奈瑟菌属的分离株（n = 68）。此外，一组革兰氏阴性细菌（n = 18）、革兰氏阳性细菌（n = 23）、真菌（n = 1）和病毒（n = 4）以及人DNA均未扩增。检测限被确定为小于7.5基因组当量/PCR反应。最后，N.本研究建立的淋病porA假基因实时荧光定量PCR方法具有高度的敏感性、特异性、稳定性、快速性和重复性。适合于N.淋病感染
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.