Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

Abstract

Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts.The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

Naked‐eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens

“…Different chemicals were reported to improve LAMP assays. The most common ones are betaine (0.2−1.2 M), Dimethyl sulfoxide (DMSO, 5%–10%), and guanidine chloride (20–100 mM), but others such as tetramethylammonium chloride, tetramethylene sulfoxide, and formamide have also been tested (Sun et al., 2020; Wang, Brewster, et al., 2015; Zasada et al., 2020; Zhang et al., 2020).…”

“…This led researchers to consider it as a way to assess results. These studies evaluated different pure pH‐sensitive dyes, combination of dyes (i.e., bromothymol blue with phenol red [PR] and cresol red [CR] with thymol blue), or even pH‐sensitive and independent dyes (Scott et al., 2020; Tanner et al., 2015; Xiong, Huang, et al., 2020; Zasada et al., 2020). The type of change is dependent on the type of dye selected.…”

“…The type of change is dependent on the type of dye selected. It is worth mentioning that even though these dyes represent a straight forward approach to assess whether a LAMP reaction is positive or negative, the performance is limited by the capacity of the assay to generate sufficient pH variation, which has been reported not always possible, depending on the microbiological target and the type of sample under analysis (Zasada et al., 2020). Diagrams of the different molecules and typical color changes can be observed in Figure 8a–e…”

“…Due to their simplicity of use and low cost, these type of assays have been combined with different types of isothermal methods such as LAMP, RPA, or SEA for naked‐eye detection of different microorganisms in foods, surfaces, and even clinical samples (Azinheiro et al., 2020; Du et al., 2018; Wang et al., 2020; Zasada et al., 2020), with particular interest in low‐resource settings due to the need of very little instrumentation (Mei et al., 2019; Poulton & Webster, 2018; Srisrattakarn et al., 2020; van Dam et al., 2013).…”