title: ÐÐ¿ÑÐ¸Ð¼Ð¸Ð·Ð°ÑÐ¸Ñ Ð¼ÐµÑÐ¾Ð´Ð° Ð¸Ð·Ð¾ÑÐµÑÐ¼Ð¸ÑÐµÑÐºÐ¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸ Ð´Ð»Ñ Ð´Ð¸Ð°Ð³Ð½Ð¾ÑÑÐ¸ÐºÐ¸ Ð²Ð¸ÑÑÑÐ½ÑÑ Ð¸ Ð±Ð°ÐºÑÐµÑÐ¸Ð°Ð»ÑÐ½ÑÑ Ð¿Ð°ÑÐ¾Ð³ÐµÐ½Ð¾Ð²
title: Ð²ÑÐ¿ÑÑÐºÐ½Ð°Ñ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð±Ð°ÐºÐ°Ð»Ð°Ð²ÑÐ°
titleType: Subtitle
title: Optimization of the isothermal amplification method for viral and bacterial pathogens diagnostics
Creators: name: ÐÐ¾ÑÐ¾Ð±ÑÐµÐ²Ð°, ÐÐ°Ð»ÐµÑÐ¸Ñ
Publisher: name: Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¸Ð¹ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¸Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑ ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾
Year: 2024
Subjects: Ð´Ð¸Ð°Ð³Ð½Ð¾ÑÑÐ¸ÑÐµÑÐºÐ°Ñ Ð¿Ð»Ð°ÑÑÐ¾ÑÐ¼Ð°
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Ð¾Ð±ÑÐ°ÑÐ½Ð°Ñ ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ñ
diagnostic platform
recombinase polymerase amplification
crowding agent
plasmid vector
reverse transcription

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¿Ð¾Ð´Ð±Ð¾ÑÑ Ð¾Ð¿ÑÐ¸Ð¼Ð°Ð»ÑÐ½Ð¾Ð³Ð¾ ÑÐ¾ÑÑÐ°Ð²Ð° ÑÐµÐ°ÐºÑÐ¸Ð¾Ð½Ð½Ð¾Ð³Ð¾ Ð±ÑÑÐµÑÐ° Ð´Ð»Ñ Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ð¸Ñ ÑÐµÐºÐ¾Ð¼Ð±Ð¸Ð½Ð°Ð·Ð½Ð¾-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð½Ð¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸, Ð° ÑÐ°ÐºÐ¶Ðµ Ð¿Ð¾Ð´Ð±Ð¾ÑÑ Ð½Ð°Ð¸Ð±Ð¾Ð»ÐµÐµ Ð¿Ð¾Ð´ÑÐ¾Ð´ÑÑÐµÐ³Ð¾ ÑÐµÑÐ¼ÐµÐ½ÑÐ° Ð´Ð»Ñ Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ð¸Ñ Ð¾Ð±ÑÐ°ÑÐ½Ð¾Ð¹ ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ð¸ Ð² Ð¾Ð´Ð½Ð¾Ð¹ ÑÐµÐ°ÐºÑÐ¸Ð¸ Ñ RPA. ÐÐ°Ð´Ð°ÑÐ¸, ÐºÐ¾ÑÐ¾ÑÑÐµ ÑÐµÑÐ°Ð»Ð¸ÑÑ Ð² ÑÐ¾Ð´Ðµ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ: Ð¿Ð¾Ð´Ð±Ð¾Ñ ÑÐ¾ÑÑÐ°Ð²Ð° ÑÐµÐ°ÐºÑÐ¸Ð¾Ð½Ð½Ð¾Ð³Ð¾ Ð±ÑÑÐµÑÐ° Ð´Ð»Ñ ÑÐµÐºÐ¾Ð¼Ð±Ð¸Ð½Ð°Ð·Ð½Ð¾-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð½Ð¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸; Ð¾Ð¿ÑÐ¸Ð¼Ð¸Ð·Ð°ÑÐ¸Ñ Ð±ÑÑÐµÑÐ½Ð¾Ð¹ ÑÐ¸ÑÑÐµÐ¼Ñ Ð´Ð»Ñ Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ð¸Ñ ÑÐµÐ°ÐºÑÐ¸Ð¸ RPA; Ð¿Ð¾Ð´Ð±Ð¾Ñ ÑÐ¸Ð¿Ð° Ð¸ ÐºÐ¾Ð½ÑÐµÐ½ÑÑÐ°ÑÐ¸Ð¸ ÐºÑÐ°ÑÐ´Ð¸Ð½Ð³-Ð°Ð³ÐµÐ½ÑÐ° Ð² ÑÐµÐ°ÐºÑÐ¸Ð¸ RPA; Ð¿Ð¾Ð´Ð±Ð¾Ñ ÑÑÐ»Ð¾Ð²Ð¸Ð¹, Ð¿Ð¾Ð·Ð²Ð¾Ð»ÑÑÑÐ¸Ñ Ð¾Ð´Ð½Ð¾Ð²ÑÐµÐ¼ÐµÐ½Ð½Ð¾ Ð¿ÑÐ¾Ð²Ð¾Ð´Ð¸ÑÑ ÑÐµÐ°ÐºÑÐ¸Ñ Ð¾Ð±ÑÐ°ÑÐ½Ð¾Ð¹ ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ð¸ Ð¸ Ð¸Ð·Ð¾ÑÐµÑÐ¼Ð¸ÑÐµÑÐºÐ¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸. ÐÐ±ÑÐµÐºÑ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ â ÑÐ¸ÑÑÐµÐ¼Ð° Ð¸Ð·Ð¾ÑÐµÑÐ¼Ð¸ÑÐµÑÐºÐ¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸ RPA, Ð¸ÑÐ¿Ð¾Ð»ÑÐ·ÑÐµÐ¼Ð°Ñ Ð² ÑÐ°Ð·ÑÐ°Ð±Ð°ÑÑÐ²Ð°ÐµÐ¼Ð¾Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ°Ð»ÑÐ½Ð¾Ð¹ Ð´Ð¸Ð°Ð³Ð½Ð¾ÑÑÐ¸ÑÐµÑÐºÐ¾Ð¹ Ð¿Ð»Ð°ÑÑÐ¾ÑÐ¼Ðµ Ð´Ð»Ñ Ð²ÑÑÐ²Ð»ÐµÐ½Ð¸Ñ Ð¿Ð°ÑÐ¾Ð³ÐµÐ½Ð¾Ð² ÑÐ°Ð·Ð»Ð¸ÑÐ½Ð¾Ð¹ Ð¿ÑÐ¸ÑÐ¾Ð´Ñ. Ð Ð°Ð±Ð¾ÑÐ° Ð²ÑÐ¿Ð¾Ð»Ð½ÐµÐ½Ð° Ð½Ð° Ð±Ð°Ð·Ðµ ÐÐ°ÑÑÐ½Ð¾-Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÑÐºÐ¾Ð³Ð¾ ÐºÐ¾Ð¼Ð¿Ð»ÐµÐºÑÐ° Â«ÐÐ°Ð½Ð¾-Ð±Ð¸Ð¾ÑÐµÑÐ½Ð¾Ð»Ð¾Ð³Ð¸Ð¸Â». ÐÐ±Ð·Ð¾Ñ Ð»Ð¸ÑÐµÑÐ°ÑÑÑÑ Ð²ÐºÐ»ÑÑÐ°ÐµÑ Ð¾Ð¿Ð¸ÑÐ°Ð½Ð¸Ðµ Ð¼ÐµÑÐ¾Ð´Ð¾Ð² Ð¸Ð·Ð¾ÑÐµÑÐ¼Ð¸ÑÐµÑÐºÐ¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸, Ð° ÑÐ°ÐºÐ¶Ðµ Ð¼Ð¾Ð»ÐµÐºÑÐ»ÑÑÐ½ÑÐµ Ð¼ÐµÑÐ°Ð½Ð¸Ð·Ð¼Ñ ÑÐ¸ÑÑÐµÐ¼Ñ CRISPR-Cas. ÐÐ¾ ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÐ°Ð¼ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ð¹ Ð±ÑÐ»Ð¸ Ð²ÑÑÐ²Ð»ÐµÐ½Ñ Ð¾Ð¿ÑÐ¸Ð¼Ð°Ð»ÑÐ½ÑÐµ ÑÐ¾ÑÑÐ°Ð²Ñ Ð±Ñ-ÑÐµÑÐ½ÑÑ ÑÐ¸ÑÑÐµÐ¼, Ð¿Ð¾Ð´Ð¾Ð±ÑÐ°Ð½Ñ ÑÐ¸Ð¿ Ð¸ ÐºÐ¾Ð½ÑÐµÐ½ÑÑÐ°ÑÐ¸Ñ ÐºÑÐ°ÑÐ´Ð¸Ð½Ð³-Ð°Ð³ÐµÐ½ÑÐ°, Ð° ÑÐ°ÐºÐ¶Ðµ ÑÐµÑÐ¼ÐµÐ½Ñ Ð ÐÐÐÐ Ð¢Ð-L (ÐÐ¼Ð¿Ð»Ð¸Ð¡ÐµÐ½Ñ), Ð¿Ð¾Ð·Ð²Ð¾Ð»ÑÑÑÐ¸Ð¹ Ð¿ÑÐ¾Ð²Ð¾Ð´Ð¸ÑÑ ÑÐµÐ°ÐºÑÐ¸Ñ Ð¾Ð±ÑÐ°ÑÐ½Ð¾Ð¹ ÑÑÐ°Ð½ÑÐºÑÐ¸Ð¿ÑÐ¸Ð¸ Ð¸ ÑÐµÐºÐ¾Ð¼Ð±Ð¸Ð½Ð°Ð·Ð½Ð¾-Ð¿Ð¾Ð»Ð¸Ð¼ÐµÑÐ°Ð·Ð½Ð¾Ð¹ Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸ Ð² Ð¾Ð´Ð½Ð¾Ð¹ Ð¿ÑÐ¾Ð±Ð¸ÑÐºÐµ. Ð ÐµÐ·ÑÐ»ÑÑÐ°ÑÑ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ð¹ Ð±ÑÐ´ÑÑ Ð¿ÑÐ¸Ð¼ÐµÐ½ÐµÐ½Ñ Ð¿ÑÐ¸ ÑÐ°Ð·ÑÐ°Ð±Ð¾ÑÐºÐµ ÑÐ½Ð¸Ð²ÐµÑÑÐ°Ð»ÑÐ½Ð¾Ð¹ Ð´Ð¸Ð°Ð³Ð½Ð¾ÑÑÐ¸ÑÐµÑÐºÐ¾Ð¹ Ð¿Ð»Ð°ÑÑÐ¾ÑÐ¼Ñ Ð´Ð»Ñ Ð¾Ð±Ð½Ð°ÑÑÐ¶ÐµÐ½Ð¸Ñ Ð½ÑÐºÐ»ÐµÐ¸Ð½Ð¾Ð²ÑÑ ÐºÐ¸ÑÐ»Ð¾Ñ Ñ Ð²ÑÑÐ¾ÐºÐ¾Ð¹ ÑÑÐ²ÑÑÐ²Ð¸ÑÐµÐ»ÑÐ½Ð¾ÑÑÑÑ Ð¸ ÑÐ¿ÐµÑÐ¸ÑÐ¸ÑÐ½Ð¾ÑÑÑÑ Ð¸ Ð²ÑÑÐ¾ÐºÐ¾Ð¹ ÑÑÐµÐ¿ÐµÐ½ÑÑ Ð¿Ð¾ÑÑÐ°ÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸. ÐÑÐ¸ Ð½Ð°Ð¿Ð¸ÑÐ°Ð½Ð¸Ð¸ Ð»Ð¸ÑÐµÑÐ°ÑÑÑÐ½Ð¾Ð³Ð¾ Ð¾Ð±Ð·Ð¾ÑÐ° Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð»Ð¸ÑÑ ÑÐ»ÐµÐ´ÑÑÑÐ¸Ðµ Ð±Ð°Ð·Ñ Ð´Ð°Ð½Ð½ÑÑ: Scopus, PubMed, eLIBRARY.RU, cyberlenika.ru. ÐÐ±ÑÐ°Ð±Ð¾ÑÐºÐ° ÑÐºÑÐ¿ÐµÑÐ¸Ð¼ÐµÐ½ÑÐ°Ð»ÑÐ½ÑÑ Ð´Ð°Ð½Ð½ÑÑ Ð±ÑÐ»Ð° Ð²ÑÐ¿Ð¾Ð»Ð½ÐµÐ½Ð° Ñ Ð¿Ð¾Ð¼Ð¾ÑÑÑ Ð¿ÑÐ¾-Ð³ÑÐ°Ð¼Ð¼ Origin, ImageJ, ImageLab.
description: This work is devoted to the selection of the optimal reaction buffer composition for carrying out recombinase-polymerase amplification, as well as the selection of the most suitable enzyme for performing reverse transcription in one reaction with RPA. Problems that were solved during the study: selection of the recombinase-polymerase amplification reaction buffer compo-sition; optimization of the buffer system for the RPA reaction; selection of the type and concentration of crowding agent in the RPA reaction; selection of conditions that allow simultaneous implementation of reverse transcription and isothermal amplification reactions. The object of study is the RPA isothermal amplification system, used in the universal diagnostic platform being developed for identifying pathogens of various natures. The work was carried out based on the Nano-Biotechnologies Research Complex. The literature review includes a description of isothermal amplification methods, as well as the molecular mechanisms of the CRISPR-Cas system. Based on the research results, the optimal compositions of buffer systems were identified, the type and concentration of the crowding agent was selected, as well as the REVERTA-L enzyme (AmpliSens), which allows the reaction of reverse transcription and recombinase-polymerase amplification to be carried out in one test tube. The research results will be used in the development of a universal diagnostic platform for the detection of nucleic acids with high sensitivity and specificity and a high degree of portability. When writing the literature review, the following databases were used: Scopus, PubMed, eLIBRARY.RU, cyberlenika.ru. Processing of experimental data was carried out using Origin, ImageJ, ImageLab programs.