Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China; Hebei General Hospital, Shijiazhuang, 050051, Hebei, China.
Hebei General Hospital, Shijiazhuang, 050051, Hebei, China.
Int J Infect Dis. 2020 Apr;93:224-230. doi: 10.1016/j.ijid.2020.01.053. Epub 2020 Feb 8.
Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity.
This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison.
Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%.
This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.
百日咳博德特氏菌是一种高度传染性的呼吸道病原体，也是百日咳的病原体，主要影响儿童。目前针对该病原体的诊断技术存在多种局限性，包括培养时间长、细菌载量低以及缺乏特异性。
本文报道了一种使用锁核酸（LNA）技术（LNA-OTN-q-PCR）的单管嵌套定量实时 PCR 检测方法的开发，该方法针对 BP485 基因，采用简单且廉价的提取方法。共检测了来自中国河北儿童医院 130 例临床疑似百日咳患者的临床样本，采用 LNA-OTN-q-PCR 检测法、RT-PCR 和两步半嵌套 PCR 检测法进行平行比较。
LNA-OTN-q-PCR 检测法仅鉴定出百日咳博德特氏菌菌株为阳性，而所有其余菌株均被适当鉴定为阴性。LNA-OTN-q-PCR 检测法可检测到单个拷贝/反应。此外，该方法的灵敏度比 RT-PCR 检测法高 100 倍（反应中 100 个拷贝）。在 130 例临床样本中，63 例经 LNA-OTN-q-PCR 检测法检测为阳性；相比之下，RT-PCR 仅能检测到 41 例阳性样本。随后，两步半嵌套 PCR 鉴定所有 63 例样本均为阳性。与两步半嵌套 PCR 检测法相比，使用纯化 DNA 和粗提物的 LNA-OTN-q-PCR 检测法的特异性和灵敏度均为 100%。
该检测法能够高灵敏度和特异性地检测百日咳博德特氏菌感染。该试验在临床实验室和公共卫生环境中作为检测百日咳博德特氏菌的一种很有前途的技术具有很大的潜力。
