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

LAMP represents an attractive approach for rapid and deployable pathogen detection. Since its development by Notomi et al. [8], LAMP has been widely used as a molecular detection tool. One of the most outstanding advantages of LAMP is its speed. For instance, LAMP results can be obtained within 30 min, whereas PCR, which is one of the most widely used molecular detection tools, would require at least 90 min to obtain a result. However, the most critical advantage of LAMP is its simplicity whereby LAMP runs under isothermal conditions, and the method requires only a water bath or heating block [1].

To date, the usage of LAMP has been widely reported and most studies have highlighted its advantage of being rapid, while capable of detecting pathogens with high sensitivity and specificity [7, 8]. Due to its advantages, LAMP is particularly useful in epidemics as a rapid diagnostic tool to play a vital role in outbreak management. For example, during the current coronavirus disease 2019 (COVID-19) pandemic, LAMP has been deployed as molecular tool to detect and contain the spread of COVID-19 [16]. Similarly, LAMP may be used as a method to detect and control the spread of leptospirosis. Unfortunately, the requirement of cold storage of LAMP reagents limits the application of the method, especially in resource-limited settings where cold storage facilities are limited [7].

Alternatively, researchers have opted for lyophilized LAMP to eliminate the use of cold storage. Engku Nur Syafiraha et al. indicated that lyophilization of LAMP reagents with addition of sugar in LAMP enabled the reagent to maintain functionality and stability at room temperature [7]. However, a drawback of lyophilization lies in the requirement for sophisticated equipment such as a rotary or manifold freeze-dryer. The use of a freeze-dryer may alter material structure, which could affect the sensitivity and specificity of the test. Moreover, freeze-dryers are expensive and not readily available in many laboratories or in resource-limited settings [13].

Othman et al. developed and optimized a LAMP protocol using clinical and animal samples targeting the same secY gene region. The sensitivity of the developed LAMP system was found to be as low as 2 × 10⁴ copies of genomic DNA per reaction [10]. Despite the high sensitivity reported in the previous study, the method is not appropriate if the test was carried out in less-equipped laboratory settings due to the restriction of cold storage for LAMP reagents. Therefore, the present study modified the developed LAMP system with addition of sucrose to eliminate the need of cold storage for LAMP reagents. The 20 rat kidney samples used in the present study were the same as those tested with the LAMP method developed in previous studies [10, 15].

The LAMP reaction using the sucrose-free premixed reagents stored at room temperature showed no amplification in all rat kidney samples on day 1 (Figure 1A). The test was conducted only for up to 14 days because it consistently showed no amplification, which presumably was because the activity of the Bst DNA polymerase had been lost in the sugar-free premixed reagents. Conversely, amplification was observed in the rat kidney samples that contain leptospiral DNA using the premixed LAMP reagents with sucrose stored at room temperature on day 1 (Figure 1B). Moreover, we found that the premixed LAMP reagents with sucrose remained stable for 45 days at room temperature (Figure 2). The positive samples showed changes in the calcein dye color from orange to yellow–green under visible light and emitted a fluorescent signal under UV light. For the sample known to be negative, the calcein dye remained orange, and no emission of fluorescence was observed. LAMP was also analyzed by agarose gel electrophoresis to confirm the positive samples showed ladder-like bands on the gel.

Figure 1

Figure 2

To date, studies on the use of sugar in LAMP reagents remain limited. Most of the studies reported have used treha-lose as stabilizer for the LAMP reagents because trehalose has been reported to provide a good stability for proteins in room temperature storage [7, 9]. However, others indicated that sucrose tends to provide better stability to maintain the enzymatic activity than trehalose [17]. Foo et al. adapted the preparation of premixed LAMP reagents with minor modifications of the use of a freeze-drying process and the type of sugar [11]. Meanwhile, the selection of sucrose was inspired by the study by Chen et al. to test for its applicability in the LAMP method [9]. Sucrose has been used widely to preserve enzymatic activity because it provides good stabilizing effects and the recovery of glucose-6-phosphate dehydrogenase activity in its presence is 94%–98% [18]. Sucrose can tolerate various temperature conditions and resist dehydration, while allowing reagents to be stored at room temperature [19]. Louwrier and Valk described that sucrose can maintain the enzymatic activity of Taq DNA polymerase for longer than reducing sugars such as glucose, raffinose, or maltose [12].

Although the exact mechanism of thermal protection by sucrose remains unclear, it is presumed that the possible mechanism of sugar in preserving the protein is due to a “glassy dynamics” hypothesis in which the “sugar forms a rigid, inert matrix in which the protein is molecularly dispersed, and the limited mobility in the glassy matrix dampens the protein mobility necessary for movement along the degradation pathway” [17]. As a result, degradation of protein is reduced by its kinetic control. In the present study, we found that sucrose maintained the enzymatic activity of the Bst DNA polymerase during storage at room temperature for at least 45 days.

Detection of leptospiral DNA in rat kidney samples using the room-temperature stable premixed LAMP reagents suggests that the method may also be applied to detect leptospiral DNA in clinical samples. The modification introduced in the present study would enhance the original LAMP method [10] attributes of ease-of-operation, minimal training requirements, and cost-effectiveness, thereby enhancing its potential as a point-of-care-testing method. The elimination of cold storage and machine dependence would allow the modified LAMP to be used on site to detect Leptospira spp. in potential outbreak areas or field surveillance where resources are limited. This would facilitate the epidemiology of leptospirosis and implementation of prevention and control strategies, particularly in the control of the reservoir animal.

There are some limitations to the present study. Various concentrations of sucrose were not tested. Thus, we are uncertain whether another concentration would provide better stability of LAMP reagents. The present study only tested sucrose. Further studies that test other concentrations of sucrose and types of sugar to optimize conditions to preserve activity of Bst DNA polymerase may be warranted.

This improved method addresses the limitations of cold storage requirements for LAMP reagents. The premixed LAMP reagents with sucrose as stabilizer could maintain the Bst DNA polymerase activity at room temperature for at least 45 days. The formulation of room-temperature stable premixed LAMP reagents for the rapid detection of leptospiral bacteria may facilitate the early detection and control of leptospirosis, and enable preventive actions to be undertaken when a leptospirosis outbreak is anticipated, particularly in resource-limited settings and under field conditions.