Institute of Disease Control and Prevention, Academy of Military Medical Sciences , Beijing , China.
National Institutes for Food and Drug Control , Beijing , China.
Front Public Health. 2015 Mar 5;3:39. doi: 10.3389/fpubh.2015.00039. eCollection 2015.
Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP) method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/μl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 B. pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.
百日咳博德特氏菌是一种重要的人类呼吸道病原体。在这里，我们描述了一种基于可视检测的用于快速检测临床样本中百日咳博德特氏菌的环介导等温扩增（LAMP）方法。LAMP 检测法在 60 分钟内检测到 BP485 靶序列，检测灵敏度比传统 PCR 提高了 10 倍。测试的 31 种非百日咳呼吸道病原体均未检测到 LAMP 反应，表明引物对百日咳博德特氏菌具有高度特异性。为了评估 LAMP 检测法在临床诊断中的应用，对中国疑似呼吸道感染患者采集的 105 份痰和鼻咽样本进行了检测，在基于 BP485 的监测中，LAMP 检测法共检测到 33 份阳性样本，其中有 12 株分离到百日咳博德特氏菌。值得注意的是，一个 4.5 个月大的婴儿和她的母亲同时被发现感染了百日咳博德特氏菌。所有分离株均属于不同的百日咳博德特氏菌多位点序列分型组，其毒力相关基因（包括 ptxA 的 4 个等位基因、prn 的 6 个等位基因、tcfA 的 4 个等位基因、fim2 的 2 个等位基因和 fim3 的 3 个等位基因）也各不相同。临床分离株中携带毒素基因的百日咳博德特氏菌多样性表明，百日咳博德特氏菌正在迅速不断进化。这与它的高流行率相结合，将使其难以控制。总之，我们开发了一种可视化检测 LAMP 检测法，它可能成为快速检测百日咳博德特氏菌的有用工具，特别是在资源匮乏和现场检测的情况下。
