METHOD FOR TESTING TARGET NUCLEIC ACID IN SAMPLE

• Main Authors: ZHANG, Zhuo, SUN, Yang, CHENG, Xuejia, PENG, Qiongfang, PAN, Weiye, CHEN, Chongjian
• Format: Patent
• Language: English
• Published: 04.01.2024
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; VINEGAR; WINE

Provided is a method for detecting a target nucleic acid in a sample, comprising the following steps: reacting the sample with a mixed reaction system consisting of a sgRNA-Cas system and a recombinase-aid isothermal amplification system; and detecting a detectable signal generated by the reaction after the reaction is completed. The sgRNA-Cas system comprises a Cas12b protein and a sgRNA targeting the target nucleic acid; the recombinase-aid isothermal amplification system comprises a primer, a single-stranded DNA reporter molecule that generates the detectable signal after being cleaved. When a reagent user operates, a sample adding and mixing operation of stopping the reaction midway to add reaction component of the second step is leaved out, so that uniformity and coherence of the operation are better, facilitating the improvement of reaction precision. The operation of opening a reaction tube after the amplification reaction of recombinant polymerase is omitted. In addition, when these two reactions are mixed, the cis-cleavage activity of Cas protease will digest the amplification product, so that it will not be released into the environment to become a pollution source, which is especially important for high-throughput and high sensitivity detection; and the reaction sensitivity is improved.