Centers for Disease Control and Prevention, Mailstop D11, Atlanta, GA 30333, USA.
J Clin Microbiol. 2011 Dec;49(12):4059-66. doi: 10.1128/JCM.00601-11. Epub 2011 Sep 21.
A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.
一种新型的多靶实时 PCR（RT-PCR）检测方法，用于快速鉴定百日咳博德特氏菌、副百日咳博德特氏菌和霍姆斯博德特氏菌，该方法结合了多拷贝插入序列（ISs）和百日咳毒素亚单位 S1（ptxS1）单重检测法。多重检测的 RT-PCR 靶标包括常见于百日咳博德特氏菌和霍姆斯博德特氏菌的 IS481；副百日咳博德特氏菌的 IS1001；以及霍姆斯博德特氏菌的 IS1001 样序列。总的来说，402 株博德特氏菌物种和 66 株非博德特氏菌物种分离株在多靶检测中进行了测试。交叉反应仅在 5 株支气管败血博德特氏菌分离株中发现，这些分离株与副百日咳博德特氏菌的 IS1001 呈阳性。多靶检测的最低检测限（LLOD）与单个检测格式中每个靶标的 LLOD 相似，即所有靶标每反应约为 1 个基因组当量。共使用了在咳嗽疾病暴发调查期间获得的 197 个人类临床标本来评估多靶 RT-PCR 检测。将 87 份临床标本的多靶 RT-PCR 结果与单独的 RT-PCR 检测和培养结果进行比较。多靶检测可作为一种诊断工具，用于确认百日咳感染，并快速鉴定其他博德特氏菌物种。总之，这种多靶 RT-PCR 方法的使用提高了特异性，同时减少了 RT-PCR 用于准确诊断类百日咳疾病所需的时间、试剂和标本量。
