Templeton Kate E, Scheltinga Sitha A, van der Zee Anneke, Diederen Bram M W, van Kruijssen Alida M, Goossens Herman, Kuijper Ed, Claas Eric C J
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
J Clin Microbiol. 2003 Sep;41(9):4121-6. doi: 10.1128/JCM.41.9.4121-4126.2003.
PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.
聚合酶链反应（PCR）越来越多地被用作检测百日咳博德特氏菌和副百日咳博德特氏菌DNA的诊断试验，因为与传统技术相比，它具有更高的灵敏度和特异性。本文所述的检测方法分别使用插入序列IS481和IS1001来检测百日咳博德特氏菌和副百日咳博德特氏菌，并通过分子信标进行检测。针对IS481的实时PCR可检测百日咳博德特氏菌和霍氏博德特氏菌，针对IS1001的实时PCR可检测副百日咳博德特氏菌和霍氏博德特氏菌。通过同时进行这两种检测，可区分百日咳博德特氏菌和副百日咳博德特氏菌。在两种检测中，百日咳博德特氏菌的灵敏度为1至10 CFU/ml，副百日咳博德特氏菌为10 CFU/ml，霍氏博德特氏菌为10 CFU/ml。百日咳博德特氏菌检测的临床灵敏度不受与内部对照PCR双重检测的影响。对57份临床样本进行了实时PCR、传统PCR和培养检测。57份样本中有8份（14%）培养呈阳性，57份中有19份（33%）传统PCR呈阳性，57份中有22份（39%）实时PCR呈阳性。有1份样本具有抑制作用。当将百日咳博德特氏菌检测与百日咳博德特氏菌感染的临床标准进行比较时，培养、传统PCR和实时PCR的灵敏度分别为38%、83%和100%，特异性分别为100%、97%和97%。针对百日咳博德特氏菌和副百日咳博德特氏菌设计的实时PCR为百日咳博德特氏菌和副百日咳博德特氏菌感染提供了灵敏且特异的诊断，因此适用于诊断实验室。
