Guiver M, Borrow R, Marsh J, Gray S J, Kaczmarski E B, Howells D, Boseley P, Fox A J
Meningococcal Reference Unit, Withington Hospital, Nell Lane, Manchester, UK.
FEMS Immunol Med Microbiol. 2000 Jun;28(2):173-9. doi: 10.1111/j.1574-695X.2000.tb01473.x.
In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.
在英国培养确诊病例比例一直在稳步下降的时期，聚合酶链式反应（PCR）诊断法已被用于增加确诊病例数量并提供更多流行病学数据。本报告对使用珀金埃尔默应用生物系统公司7700型自动序列检测系统的基于荧光探针的5′核酸外切酶测定法（Taqman法）与先前报道的用于检测脑脊液、血浆和血清样本中脑膜炎球菌DNA的聚合酶链反应酶联免疫吸附（PCR ELISA）测定法进行了比较评估。所开发的Taqman测定法基于检测脑膜炎球菌荚膜转移基因（ctrA）、插入序列IS1106和用于确定B群和C群的唾液酸转移酶基因（siaD），并与PCR ELISA形式的类似测定法进行比较。Taqman ctrA测定法对脑膜炎奈瑟菌具有特异性，然而IS1106测定法对一些非脑膜炎球菌分离株产生了假阳性反应。检测培养确诊病例样本时，Taqman ctrA、IS1106和siaD测定法的灵敏度分别为64%、69%和50%，而相应的PCR ELISA测定法的灵敏度分别为26%、67%和43%。与培养确诊病例相比，DNA提取程序的改进已将TaqMan ctrA和siaD测定法的灵敏度分别提高到93%和91%。自引入Taqman PCR以来，与仅通过培养确诊的病例相比，实验室确诊的脑膜炎球菌病病例增加了56%。所开发的用于诊断脑膜炎球菌病的Taqman测定法能够实现高通量、样本快速周转，且污染风险大幅降低。
