Ckumdee Jutturong, Chamnanphom Monpat, Wiwattanakul Supaporn, Santiwatanakul Somchai, Onruang Kwanchai, Kaewphinit Thongchai
Department of Clinical Pathology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand.
Center of Excellence in Medical and Environmental Innovation Research (CEMEIR), Srinakharinwirot University, Bangkok 10110, Thailand.
Diagnostics (Basel). 2025 Aug 28;15(17):2183. doi: 10.3390/diagnostics15172183.
Tuberculosis (TB) remains a primary global health concern, despite the widespread availability of effective chemotherapeutic interventions. The emergence and dissemination of drug-resistant strains of , particularly those exhibiting resistance to rifampicin, present significant obstacles to the success of TB control programs. Consequently, there is an urgent need for rapid, sensitive, and specific molecular diagnostic tools to inform timely clinical decision-making and reduce the transmission of disease. Loop-mediated isothermal amplification (LAMP) has gained attention as a promising alternative to conventional polymerase chain reaction (PCR) techniques. This method, which facilitates DNA amplification under constant temperature conditions, offers advantages including high specificity, rapid turnaround time, and operational simplicity-features that render it especially suitable for implementation in resource-limited settings. In this study, a LAMP assay targeting the gene was developed, with particular focus on detecting the codon 531 C→T mutation associated with rifampicin resistance. A set of four to six primers was designed to recognize six distinct regions of the target sequence. Allele-specific amplification was achieved by incorporating a deliberate single nucleotide mismatch at the 3' terminus of the B2 primer to enable precise discrimination between wild-type and mutant alleles. The assay was conducted at an optimized temperature of 61 °C for 60 min, followed by visual detection using a lateral flow dipstick (LFD) within five minutes. The LAMP-LFD assay demonstrated 100% concordance with drug susceptibility testing (DST) and DNA sequencing. No cross-reactivity with wild-type strains was observed, underscoring the assay's high specificity. This platform offers a robust, field-deployable solution for detecting the codon 531 C→T mutation associated with rifampicin resistance in low-resource settings.
尽管有效化疗干预措施已广泛可得，但结核病仍然是全球主要的健康问题。耐药菌株的出现和传播，尤其是对利福平耐药的菌株，给结核病控制项目的成功带来了重大障碍。因此，迫切需要快速、灵敏且特异的分子诊断工具，以便及时做出临床决策并减少疾病传播。环介导等温扩增技术（LAMP）作为传统聚合酶链反应（PCR）技术的一种有前景的替代方法而受到关注。该方法在恒温条件下促进DNA扩增，具有高特异性、快速周转时间和操作简单等优点，这些特性使其特别适合在资源有限的环境中应用。在本研究中，开发了一种针对 基因的LAMP检测方法，特别侧重于检测与利福平耐药相关的密码子531 C→T突变。设计了一组四到六条引物，以识别靶序列的六个不同区域。通过在B2引物的3'末端引入一个特意的单核苷酸错配来实现等位基因特异性扩增，从而能够精确区分野生型和突变型等位基因。该检测在61°C的优化温度下进行60分钟，随后在五分钟内使用侧向流动试纸条（LFD）进行视觉检测。LAMP-LFD检测与药物敏感性试验（DST）和DNA测序显示出100%的一致性。未观察到与野生型菌株的交叉反应，突出了该检测方法的高特异性。该平台为在资源有限的环境中检测与利福平耐药相关的密码子531 C→T突变提供了一个强大的、可现场部署的解决方案。
