Farook Saika, Bhattacharjee Arittra, Udoy Farhan Muhib, Rubel Robiul Islam, Rahman Fahmida, Barai Lovely, Islam K M Shahidul, Haq Jalaluddin Ashraful, Jilani Md Shariful Alam
Department of Microbiology, Ibrahim Medical College, Dhaka, Bangladesh.
Bioinformatics Division, National Institute of Biotechnology, Savar, Dhaka, Bangladesh.
Curr Res Microb Sci. 2025 Aug 26;9:100463. doi: 10.1016/j.crmicr.2025.100463. eCollection 2025.
, the causative agent of the potentially life-threatening infectious disease, melioidosis, is endemic in Bangladesh. The spatial clustering of clinical incidence and the fatal outcome of melioidosis necessitates unequivocal and rapid detection and identification. Culture constitutes the gold standard test for the diagnosis of melioidosis, but identification of by culture is time consuming. In the view of the above, the present study has been designed to develop a Loop Mediated Isothermal Amplification based assay (LAMP) for the rapid and accurate detection of . Twenty-two culture confirmed isolates and 18 non bacterial species were included in the study. DNA were extracted and amplified by in-house LAMP assay, utilizing a set of newly designed primers at optimized conditions. The LAMP product was visualized by various methods. The limit of detection (LOD) of LAMP was determined and detection of directly from sterile pre-inoculated urine by the LAMP technique was evaluated. All 22 were positive by the in-house LAMP while the 18 heterogeneous bacterial species were LAMP negative. The target sequence amplified by the LAMP outer primers showed the highest similarity with isolate UKMH10, confirming the specificity of the newly designed primers. The LOD of the in-house LAMP assay was found to be 0.78 pg, which is much lower than the LOD of PCR (0.78 ng). The in-house LAMP was also found positive for using direct sample such as pus and pre-inoculated urine. A closed tube LAMP assay using agar dye capsule was adopted to minimize contamination with LAMP amplification product and thus to increase the overall precision of the test. Rapid and accurate detection of was developed using LAMP-based assay with a set of in-house designed primers. This detection procedure can be established for a cost-effective diagnosis of melioidosis at the rural level.
作为一种可能危及生命的传染病——类鼻疽病的病原体，在孟加拉国呈地方性流行。类鼻疽病临床发病率的空间聚集性以及致命后果使得明确且快速的检测和鉴定成为必要。培养是诊断类鼻疽病的金标准检测方法，但通过培养鉴定[病原体名称]耗时较长。鉴于上述情况，本研究旨在开发一种基于环介导等温扩增法（LAMP）的检测方法，用于快速、准确地检测[病原体名称]。本研究纳入了22株经培养确认的[病原体名称]分离株和18种非[病原体名称]细菌物种。提取DNA并通过内部LAMP检测法进行扩增，在优化条件下使用一组新设计的引物。通过多种方法观察LAMP产物。确定了LAMP的检测限（LOD），并评估了通过LAMP技术直接从无菌预接种尿液中检测[病原体名称]的情况。所有22株[病原体名称]经内部LAMP检测均为阳性，而18种异源细菌物种LAMP检测为阴性。LAMP外部引物扩增的靶序列与[病原体名称]分离株UKMH10显示出最高的相似性，证实了新设计引物的特异性。内部LAMP检测法的LOD为0.78 pg，远低于聚合酶链反应（PCR）的LOD（0.78 ng）。使用直接样本如脓液和预接种尿液时，内部LAMP检测[病原体名称]也呈阳性。采用使用琼脂染料胶囊的封闭管LAMP检测法，以尽量减少LAMP扩增产物的污染，从而提高检测的整体精度。使用基于LAMP的检测法和一组内部设计的引物开发了快速、准确检测[病原体名称]的方法。该检测程序可用于在农村地区以具有成本效益的方式诊断类鼻疽病。
