Rahman Ahmad Mukhlis Abdul, Ransangan Julian, Subbiah Vijay Kumar
Biotechnology Research Institute, Universiti Malaysia Sabah, Jln UMS, Kota Kinabalu 88400, Sabah, Malaysia.
Faculty of Chemical Engineering & Technology, Uniciti Alam Campus, Universiti Malaysia Perlis, Sg. Chuchuh, Padang Besar 02100, Perlis, Malaysia.
Microorganisms. 2022 Nov 27;10(12):2346. doi: 10.3390/microorganisms10122346.
The common methods that are presently used to identify include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the gene sequence of . The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with and non- samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of contamination both in laboratories and in the field.
目前用于鉴定的常用方法包括显微镜检查以及基于生化、免疫和聚合酶链反应（PCR）的检测方法。这些方法需要专业技术知识，且可能耗时较长。对于大量样品的高通量筛选，需要一种快速的方法。因此，我们基于DNA的环介导等温扩增（LAMP）开发了一种快速、简单但灵敏且特异的检测方法。基于对一大组39株[具体基因名称]基因序列的计算机分析，设计了一组六种引物，即两条外引物、两条内引物和两条环引物。环引物的加入使LAMP反应时间缩短了一半以上。此外，通过应用SYBR Green，无需凝胶电泳，10至15分钟内即可快速获得结果。然后通过对[目标样本名称]和非[目标样本名称]样本进行LAMP反应来确定该方法引物的特异性。LAMP比PCR反应具有更高的灵敏度。PCR的灵敏度为0.6 pg浓度的[重组质粒DNA标准名称]重组质粒DNA标准，而LAMP即使在0.6 fg时也能检测到更低的量。LAMP检测方法的开发将为实验室和现场[目标样本名称]污染的高通量快速检测提供有价值的工具。
