Testing of a proposed reagent kit for detecting enteric pathogens by isothermal DNA amplification

BACKGROUND: Loop-Mediated Isothermal Amplification (LAMP) is a promising diagnostic tool for infectious diseases with high potential for transitioning to a point-of-care test format. It is used to detect a wide range of infectious pathogens, but there are virtually no similar tests for enteric pathogens in Russia.

AIM: The aim of the study was to evaluate a diagnostic reagent kit for the detection of DNA of enteric pathogens such as Shigella spp., enteroinvasive E. coli (EIEC), Salmonella spp., thermophilic Campylobacter, and group F enteric adenoviruses using LAMP.

MATERIALS AND METHODS: Fragments of ipaH Shigella, invA Salmonella, CJE0832 Campylobacter jejuni and Campylobacter coli genes and the adenovirus F 40 hexon gene were used as targets for amplification. A total of 254 clinical specimens were collected from patients with typical symptoms of diarrhea and asymptomatic patients. A 10% suspension in phosphate-buffered saline was prepared and solid particles were precipitated to obtain the fecal extract. DNA was isolated from the extracts using AmpliTest®Ribo-Prep (MA No. РЗН 2020-12985 (RZN 2020-12985), 22 December 2020) and AmpliTest® Magno-Sorb-Combo (MA No. РЗН 2022_19200 (RZN 2022_19200), 21 December 2022) reagent kits manufactured by the Centre for Strategic Planning of the Federal Medical and Biological Agency of Russia. The amplification results were detected using specific fluorescent probes. Amplification was performed in a multiplex format using two mixtures, the first for the detection of Shigella , EIEC, Campylobacter, and internal control sample DNA, and the second for the detection of Group F Adenovirus and Salmonella DNA.

RESULTS: Analytical sensitivity was evaluated using model biomaterial samples containing target DNA at known concentrations. Shigella, EIEC, Salmonella, Campylobacter and Group F Adenovirus DNA was reproducibly detected at a level of at least 5×10³ copies/mL (total number of repeats n =60). Specificity was confirmed on a panel of DNA from different strains of adenoviruses and bacteria. A study of 254 clinical samples showed concordance between results obtained using the proposed LAMP protocol and a PCR (polymerase chain reaction) technique (a reference AmpliSens® OKI-screen-FL kit). The diagnostic sensitivity of the LAMP protocol was at least 92.6% and the specificity was at least 98.2% at the 95% confidence level (p =0.95). The high analytical and diagnostic characteristics of the proposed AmpliTest® OKI LAMP reagent kit were confirmed by clinical laboratory tests. The kit was registered as a medical device for in vitro diagnostics (MA No. РЗН 2024/23503 (RZN 2024/23503) dated September 4, 2024).

CONCLUSION: The proposed LAMP reagent kit is designed to detect enteric pathogens in less than one hour with analytical and diagnostic characteristics comparable to PCR.