Koryukov Maxim A, Oscorbin Igor P, Gordukova Maria A, Turina Irina E, Aminova Alfiya I, Filipenko Maxim L
The Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia; Department of Natural Sciences, Novosibirsk State University, 2 Pirogov Street, Novosibirsk 630090, Russia.
The Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.
Methods. 2025 Aug;240:63-72. doi: 10.1016/j.ymeth.2025.04.005. Epub 2025 Apr 9.
Whooping cough, or pertussis, is a highly contagious disease caused by several Bordetella species and continues to pose a significant global public health concern. The rising incidence of pertussis highlights the urgent need for effective public health strategies to address Bordetella infections. Rapid and species-specific diagnostic tools are essential for preventing Bordetella transmission and are vital components of anti-infective measures. This study aimed to develop novel loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays for the detection of four Bordetella species responsible for human respiratory tract infections: B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii. The qPCR assay demonstrated a low limit of detection (LoD), reliably identifying up to 5 copies of target DNA per reaction. The LAMP assays were approximately three times faster than qPCR (30 min) but had higher LoDs. Notably, qLAMP had a limit of detection of 25 copies per reaction for all four Bordetella species. In contrast, vLAMP had a LoD of 25 copies per reaction for B. pertussis and B. parapertussis; and a LoD of 50 copies per reaction for B. holmesii and B. bronchiseptica. We validated the assays using nasal swab samples from patients with respiratory tract infections, analyzing a total of 651 samples with qPCR and 145 samples with LAMP. Both assays exhibited no cross-reactivity with common viral and bacterial respiratory pathogens. The concordance rate between qPCR and LAMP was 94.5%, underscoring the reliability of both methods for clinical application. These findings suggest that the developed qPCR and LAMP tests can be successfully integrated into clinical practice for the detection and management of Bordetella infections.
百日咳，又称pertussis，是由多种博德特氏菌引起的一种高度传染性疾病，仍然是全球公共卫生的重大关注点。百日咳发病率的上升凸显了制定有效公共卫生策略以应对博德特氏菌感染的迫切需求。快速且具有种特异性的诊断工具对于预防博德特氏菌传播至关重要，是抗感染措施的重要组成部分。本研究旨在开发用于检测导致人类呼吸道感染的四种博德特氏菌的新型环介导等温扩增（LAMP）和定量聚合酶链反应（qPCR）检测方法：百日咳博德特氏菌、副百日咳博德特氏菌、支气管败血博德特氏菌和霍氏博德特氏菌。qPCR检测方法显示出较低的检测限（LoD），每个反应能够可靠地鉴定出多达5个目标DNA拷贝。LAMP检测方法比qPCR快约三倍（30分钟），但检测限更高。值得注意的是，qLAMP对所有四种博德特氏菌的检测限为每个反应25个拷贝。相比之下，vLAMP对百日咳博德特氏菌和副百日咳博德特氏菌的检测限为每个反应25个拷贝；对霍氏博德特氏菌和支气管败血博德特氏菌的检测限为每个反应50个拷贝。我们使用呼吸道感染患者的鼻拭子样本对这些检测方法进行了验证，共分析了651份样本进行qPCR检测，145份样本进行LAMP检测。两种检测方法均未与常见的病毒性和细菌性呼吸道病原体发生交叉反应。qPCR和LAMP之间的一致性率为94.5%，强调了两种方法在临床应用中的可靠性。这些发现表明，所开发的qPCR和LAMP检测方法可以成功地整合到临床实践中，用于博德特氏菌感染的检测和管理。
