Carascal Mark B, Macalalad Lawrence S, Petronio-Santos Joy Ann, Destura Raul V, Rivera Windell L
Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines.
Clinical and Translational Research Institute, The Medical City, Ortigas Avenue, Pasig City 1605, Philippines.
Heliyon. 2024 Aug 5;10(15):e35653. doi: 10.1016/j.heliyon.2024.e35653. eCollection 2024 Aug 15.
The known intrinsic and polymorphic genes of were recently reported in other non- Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of genes. Specifically, an alignment-based primer design, primer screening, and assay confirmation were conducted. Both and results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3'-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel genes. Finally, it showed a superior limit of detection (as low as 10 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor genes in the clinical setting and provided important insights into LAMP primer design and screening.
近期在其他非革兰氏阴性病原体中报道了已知的内在基因和多态性基因。在临床环境中准确检测这种潜在可转移的碳青霉烯酶基因至关重要。本研究开发了一种针对该基因多个等位基因的环介导等温扩增（LAMP）检测方法。具体而言，进行了基于比对的引物设计、引物筛选和检测方法确认。实验结果均表明，LAMP检测方法对3'端引物区域以外多达五个引物 - 模板错配具有耐受性。在90分钟内，LAMP检测方法还检测到了其他具有已知和新型该基因的革兰氏阴性细菌中的基因靶点。最后，与聚合酶链反应相比，它显示出更高的检测限（低至10 CFU/mL），并且对非靶点具有高特异性。本研究开发了一种高度适应性的LAMP检测方法，用于在临床环境中监测该基因，并为LAMP引物设计和筛选提供了重要见解。
