Banger Swati, Pal Vijai, Tripathi N K, Goel A K
Bioprocess Technology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India.
Mol Biotechnol. 2021 Aug;63(8):702-709. doi: 10.1007/s12033-021-00335-6. Epub 2021 May 12.
Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 10 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.
炭疽芽孢杆菌是炭疽病的病原体，是最具杀伤力的法定生物战剂之一。其传统的微生物检测方法 labor intensive 且耗时，而分子检测方法快速、灵敏且特异。聚合酶链反应（PCR）是分子生物学中最可靠的诊断工具之一。PCR 与侧向流动试纸条相结合可缩短诊断/检测时间。它是凝胶电泳的一种替代方法，且结果解读简便明了。在本研究中，已开发出一种针对炭疽芽孢杆菌 pXO1 质粒上存在的 cya 基因的 PCR 侧向流动（PCR-LF）检测方法。正向引物和反向引物分别在 5' 端用 6-羧基荧光素（6-FAM）和生物素进行了标记。使用本研究开发的侧向流动（LF）试纸条检测双标记的 PCR 产物。该 PCR-LF 检测方法能够检测到≥5 pg 的基因组 DNA 以及重组质粒中≥500 拷贝的靶标 DNA。该检测方法分别在富集 6 小时和 24 小时后，能够检测到掺入人血中的低至 10 和 10 CFU/mL 的炭疽芽孢杆菌斯terne 株细胞。 （注：原文中“10 and 10 CFU/mL”表述似乎有误，可能是想表达“10^1 and 10^2 CFU/mL”之类的意思，翻译时保留了原文错误表述）
