Optimization of Loop-Mediated Isothermal Amplification (LAMP) reaction mixture for biosensor applications

Abstract

Genetically Modified (GM) foods are becoming the future of agriculture on surviving global natural disasters and climate change by their enhanced production efficiency and improved functional properties. On the other hand, their adverse health and environmental effects, ample evidence on transgene leakage of Genetically Modified Organisms (GMOs) to crops have raised questions on their benefits and risks. Consequently, low-cost, reliable, rapid, and practical detection of GMOs have been important. GMO-detection platforms should be capable of stably storing detection reagents for long-delivery distances with varying ambient temperatures. In this study, we developed an event-specific, closed tube colorimetric GMO detection method based on Loop-Mediated Isothermal Amplification (LAMP) technique which can be integrated into GMO-detection platforms. The entire detection process optimized to 30 min and isothermally at 65 °C. The durability of the LAMP mixture in the test tubes showed that the LAMP reaction mixture, in which Bst polymerase and DNA sample was later included, yielded DNA amplicons for 3 days at room temperature, and for 6 days at 4 °C. Simple, stable, and cheap storage method of LAMP reaction mixture for GMO-detection technologies. GMO-detection platforms can stably store detection reagents for long-delivery distances with varying ambient temperatures. Any DNA sample can be used in the field or resource-limited setting by untrained personnel.

Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen

“…Most previous studies conducted with the LAMP technique employed a concentration of 8 U of Bst DNA polymerase per reaction. 10 , 23 , 24 , 25 To reduce costs, the reduction of Bst DNA polymerase concentration in the LAMP reaction was investigated. In Fig.…”

A low‐cost, portable, and practical LAMP device for point‐of‐diagnosis in the field

“…In most studies, the focus is developing microfluidic devices for LAMP‐based assays. However, microfluidic devices are expensive to fabricate, complex to use, and require trained personnel and stable operation conditions, which makes this technology not feasible for field‐deployable real‐time diagnosis (Almassian et al, 2013, de Oliveira et al, 2021, El‐Tholoth et al, 2021, Ganguli et al, 2020, Kaymaz & Elitas, 2021, Ma et al, 2018, Mori et al, 2001, Wan et al, 2017, Wan et al, 2019). Therefore, portable, rapid, cost‐effective, automated, and remotely controllable LAMP platforms with colorimetric readouts will be the future of field‐ready modern nucleic‐acid‐based detection platforms (Kaygusuz et al, 2019, Ludwing et al, 2021, Puri et al, 2021, Xun et al, 2021).…”

“…Moreover, versatility of our platform can be enhanced when different isothermal conditions can be set by the users to perform various LAMP reactions. Besides, the naked-eye color difference is still prone to introduce some errors to determine the results of the detection (Kaymaz & Elitas, 2021). Hence, digitally F I G U R E 3 Interface of the LAMP device through the server displaying the readouts of the reactions will help for rapid and reliable diagnosis.…”

Molecular Point‐of‐Care Assay Development: Design and Considerations

“…Storage, shipping conditions, and shelf life of reagents are important considerations when choosing an assay for POC testing. For conventional LAMP assay primers, dNTPs and Bst DNA polymerase enzyme should be stored at –20°C; however, Kaymaz and Elitas (2021) proved that the reaction mixture of LAMP can be stored at 4°C for ∼6 days and at room temperature for 3 days without the addition of DNA template and Bst polymerase enzyme. The lyophilized LAMP reagent mixture remains stable at 4°C for ∼24 months and at room temperature for 28 days without losing sensitivity.…”