Guo Mingjing, Feng Pan, Zhang Liqun, Feng Chunfeng, Fu Jie, Pu Xiaoyun, Liu Fei
Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University, No. 183 Xinqiao Main St, Shapingba District, Chongqing 400037, P.R. China.
J Microbiol Biotechnol. 2022 Jan 28;32(1):91-98. doi: 10.4014/jmb.2109.09022.
Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by . is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of . Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.
破伤风是一种由 产生的神经毒素导致的潜在致命性公共卫生疾病。 不易培养，从感染伤口培养相关细菌在诊断中很少有用；基于聚合酶链反应（PCR）的检测只能在高度先进的实验室进行。因此，构建了一种实时重组酶聚合酶扩增检测法（Exo-RPA）来鉴定 的神经毒素基因片段。设计了靶向保守区域的引物和外切核酸酶探针，所得扩增子可在不到20分钟内检测到，检测限为20拷贝/反应。RPA检测显示出良好的选择性，与穿透伤中常见的其他感染性细菌无交叉反应。对添加靶标的血清和脓液提取物的检测表明，RPA对干扰因素具有较强的耐受性，在生物样本分析方面具有很大的进一步开发潜力。该方法已被证实对于区分有毒和无毒 菌株是可靠的。RPA检测通过简化设备架构显著提高了诊断效率，是破伤风检测中实时PCR的一种有前景的替代方法。
