Room-temperature stable loop-mediated isothermal amplification (LAMP) reagents to detect leptospiral DNA

Abstract

Background Loop-mediated isothermal amplification (LAMP) is one of the most promising tools for rapidly detecting Leptospira spp. However, LAMP is hampered by cold storage to maintain the enzymatic activity of Bst DNA polymerase. Objective To overcome the drawback of cold storage requirement for LAMP reagents we modified the reagents by adding sucrose as stabilizer. We then sought to determine the stability at room temperature of the premixed LAMP reagents containing sucrose. Method Premixed LAMP reagents with sucrose and without sucrose were prepared. The prepared mixtures were stored at room temperature for up to 60 days, and were subjected to LAMP reactions at various intervals using rat kidney samples to detect leptospiral DNA. Results The premixed LAMP reagents with sucrose remained stable for 45 days while sucrose-free premixed LAMP reagents showed no amplification from day 1 of storage at room temperature up to day 14. Conclusion The LAMP reagent system can be refined by using sucrose as stabilizer, thus allowing their storage at room temperature without the need for cold storage. The modified method enables greater feasibility of LAMP for field surveillance and epidemiology in resource-limited settings.

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

“…Other studies reported stability of up to 15 days at 25 or 37 °C 49 and up to 45 days at room temperature. 50 For the RTs, both CQMED HIV -RT and CQMED GO -RT maintained their activity, with little (room temperature) or no loss of function (4 °C) for up to 60 days of adverse storage, the longest tested condition.…”

“…A series of experiments were conducted to assess the quality of the enzymes produced in-house and to determine their effectiveness, even under adverse storage conditions, based on the guidelines established in EP25-A (Evaluation of Stability of In vitro Diagnostic Reagents) 47 with modifications, and results from previous works. [48][49][50] The activities of the CQMED enzymes, Bst-LF and HIV-RT, were evaluated after several weeks of storage in non-ideal temperatures, compared to the enzymes kept at the recommended storage condition of −20 °C. Figure 3(A) demonstrates that average LAMP Ct (Cycle threshold) values ranging from 16 to 20 were observed for CQMED Bst-LF across the tested time points.…”

RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection

“…Therefore, employing this detection kit would be difficult at a point of care, especially in rural areas where the cold chain transportation and electricity could not be accessed. Several studies have attempted to prolong the stability of the reaction components at ambient temperature 48 – 50 . In this study, we have overcome this issue by adding specific stabilizers to preserve the structure of the biological molecules.…”

A versatile isothermal amplification assay for the detection of leptospires from various sample types

“…To design the LAMP primers, OptiGene LAMP Designer software ( http://www.optigene.co.uk/lamp-designer/ ) was used. The LAMP primer set consists of two outermost primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB) ( Lee et al, 2021 ). The primer sequences are listed in Table 1 and their respective mapped regions are shown in Appendix B.…”

“…The majority of human infections have been attributed to rats and other rodent species, which are known to be a permanent carrier of Leptospira spp. Previous outbreaks of leptospirosis have been linked with intense periods of high rainfall, which facilitates the spreading of leptospires shedding in the urban environment ( Lee et al, 2021 ). In humans, a wide range of non-specific symptoms such as high fever, headache, muscle ache, and vomiting may appear, and these are often mistaken for other diseases.…”