Kyaw Soe Paing, Hanthamrongwit Jariya, Jangpatarapongsa Kulachart, Khaenam Prasong, Leepiyasakulchai Chaniya
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University Bangkok Thailand 73170
Clinical Pathology Laboratory, (1000) Bedded General Hospital Nay Pyi Taw Myanmar 15011.
RSC Adv. 2018 Oct 1;8(59):33674-33680. doi: 10.1039/c8ra06599c. eCollection 2018 Sep 28.
Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide. Early and accurate diagnosis of the disease is crucial to end the global TB epidemic. The current commercially available molecular tests are still unaffordable by most TB affected communities. Herein, we developed a novel rapid and sensitive diagnostic method to detect the IS sequence of () complex using PCR-magnetic bead ELISA. PCR amplification ofa 123 bp repetitive sequence of the IS gene was performed by using digoxigenin (DIG) and biotin-labelled primers. Streptavidin-conjugated magnetic beads were used to collect the dual-labelled amplicons and subsequently, colourimetric detection was done by using horseradish peroxidase (HRP)-conjugated anti-DIG antibody. This method is able to detect DNA down to 0.5 fg per reaction within 3 hours. The sensitivity of IS PCR detection by magnetic bead ELISA is 100 times higher than that of conventional agarose gel electrophoresis. The assay specificity was determined using a panel of DNA extracted from 10 common bacteria causing lower respiratory tract infections. No cross-reactivity was detected from those bacteria by IS PCR-magnetic bead ELISA. Thus, the novel highly sensitive and specific, reduced assay time and simplicity of this PCR-magnetic bead ELISA for the detection of the specific gene of complex makes it an attractive diagnostic tool for large-scale screening of tuberculosis in standard clinical laboratories.
结核病（TB）是全球传染病中的头号杀手。该疾病的早期准确诊断对于终结全球结核病流行至关重要。目前市面上的分子检测方法对于大多数受结核病影响的社区来说仍然难以承受。在此，我们开发了一种新型快速灵敏的诊断方法，即使用PCR-磁珠ELISA检测（）复合体的IS序列。通过使用地高辛（DIG）和生物素标记的引物对IS基因的123 bp重复序列进行PCR扩增。使用链霉亲和素偶联的磁珠收集双标记扩增子，随后，通过使用辣根过氧化物酶（HRP）偶联的抗DIG抗体进行比色检测。该方法能够在3小时内检测到每个反应低至0.5 fg的DNA。磁珠ELISA法检测IS PCR的灵敏度比传统琼脂糖凝胶电泳高100倍。使用从10种引起下呼吸道感染的常见细菌中提取的一组DNA来确定检测方法的特异性。通过IS PCR-磁珠ELISA未检测到这些细菌有交叉反应。因此，这种用于检测复合体特定基因的新型PCR-磁珠ELISA具有高灵敏度和特异性、检测时间缩短且操作简便的特点，使其成为标准临床实验室大规模筛查结核病的一种有吸引力的诊断工具。
