Development and study of the performance of a reagent kit for the identification of Vibrio cholerae and Vibrio parahaemolyticus by multilocus allele-specific polymerase chain reaction in clinical trials

BACKGROUND: Cholera belongs to the group of infections, and the fight against it is regulated by the WHO International Health Regulations (2005). To date, several test systems have been developed for the polymerase chain reaction (PCR) detection of Vibrio cholerae strains. However, no PCR test systems have been registered for detecting V. parahaemolyticus DNA in clinical material in Russia.

AIM: To conduct clinical trials and evaluate the performance of the “Reagent kit for identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR” (kit of reagents “Vibrio-screen FL”) for the state registration of the specified medical device.

MATERIALS AND METHODS: The study presents the results of the clinical trials for the reagent kit “Vibrio-screen FL” that has been developed to optimize the means and methods of identification and differentiation of pathogens of dangerous infections based on data on the diversity of genomes. Clinical trials were conducted at the Head Center of Hygiene and Epidemiology of the FMBA of Russia (license from 02.12.2013 No. ФС-99-01-008342; permission of the Ministry of Health from 16.05.2013 No. 300n) in accordance with the program of clinical trials No. 20.ПМКИ-042/10.20 from 23.09.2020 (Clinical Trial Certificate No. 01.AK-042/10.20 dated 12.10.2020). The clinical trials of the developed reagent kit “Vibrio-screen FL” used clinical strains of microorganisms of the genus Vibrio and other microorganism species as target analytes. Bacteriological method and multilocus PCR with hybridization-fluorescence detection were used.

RESULTS: The reagent kit “Vibrio-screen FL” is a medical device for professional use in clinical laboratory diagnostics. The analytical sensitivity of the reagent kit “Vibrio-screen FL” was at least 10⁶ copies of the SecY gene when determining belonging to the genus Vibrio; to the species V. cholerae, at least 10⁶ copies of the hlyA gene; and to the species V. parahaemolyticus, at least 10⁶ copies of the vppC gene. When determining the analytical specificity, no non-specific reactions with DNA samples of other microorganism species were found. A medical device for in vitro diagnostics “A reagent kit for the identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR (Vibrio-screen FL)” meets the requirements of the intended use, which allows it to be used for the differentiation of V. cholerae and V. parahaemolyticus strains isolated from a cultured clinical samples by multilocus allele-specific polymerase chain reaction with hybridization-fluorescent detection. The registration certificate No. РЗН 2021/13360 from 08.02.2021 was received. By the order of Roszdravnadzordated 08.02.2021 No. 1041, a reagent kit Vibrio-screen FL by the Rostov-on-Don Anti-Plague Institute of Rospotrebnadzor was admitted to distribution on the territory of the Russian Federation.

CONCLUSIONS: The reagent kit “Vibrio-screen FL” is a high-quality, effective, and safe diagnostic medical device for in vitro diagnostics with sufficient diagnostic sensitivity and specificity to identify specific fragments of Vibrio spp., V. cholerae, and V. parahaemolyticus nucleic acids. These allows it to be used in clinical laboratory diagnostics for epidemiological analysis and registration of diseases caused by pathogenic vibrions.