▎ 摘　　要

NOVELTY - Kit comprises a real-time detection self-priming valve partition chip. The real-time detection self-sucking valve partition chip includes self-sucking valve partition chip, FAM (RTM: fluorescent dye) dye modified specific aptamer of Listeria monocytogenes, FAM dye modified specific aptamer of Vibrio parahaemolyticus ligand, FAM dye-modified specific aptamer of Salmonella typhimurium, blank control FAM dye-modified aptamer, hydroxy naphthol blue (HNB) dye and graphene oxide. USE - Kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium. ADVANTAGE - The kit has lowest detection concentration of 46.8, 22.9 and 32.3 CFU/mL for Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, standard addition recovery rate of 97.11-10.40%, and has high sensitivity and good stability. DETAILED DESCRIPTION - An INDEPENDENT CLAIM is included for a method for preparing a kit, which involves: (A) preparing self-priming valve partition chip and preparing mold by using CorelDRAW X8 to design channel and chamber pattern of self-priming valve partition chip, printing it on a transparent film as a mask, using spin coater, applying negative photoresist on the silicon wafer for a total of two layers, placing silicon wafer on single-sided lithography machine under mask, adjusting position of mask so that pattern is on wafer in middle of process, baking silicon wafer after exposure, rinsing processed silicon wafer with a developer until mold pattern appears completely, and baking it again to obtain a mold; (B) producing self-priming valve partition chip by using trimethylchlorosilane to process the mold, mixing polydimethylsiloxane component pre-curing agent A and cross-linking agent B to remove air bubbles, pouring it into mold, screwing one end of bolts with nut, baking and curing it on heating plate, removing polydimethylsiloxane from mold surface, using a punch to add samples, applying plasma treatment to channel surface of polydimethylsiloxane and surface of the glass sheet, binding treated surfaces to each other, and baking and solidifying it to form a chip; (C) preparing graphene oxide by adding graphite powder to mixture of sulfuric acid and sodium nitrate, stirring it in an ice bath, slowly adding potassium permanganate to the above mixture under vigorous stirring, heating it under stirring condition at 30-40 degrees C, and heating it to 140-150 degrees C, adding deionized water to terminate reaction system, neutralizing sulfuric acid with sodium hydroxide in an ice bath, adjusting pH to 5.5-6 to obtain light yellow solution, filtering yellow solution through ion filter membrane to remove large particles, and dialyzing it in a dialysis bag for 2-3 days to remove salt to obtain graphene oxide; (D) performing real-time detection of chip by closing valve, covering top of chip with scotch tape, placing it in a vacuum pump for degassing, separating graphene oxide from mononuclear list FAM dye-modified specific aptamer of bacteria, FAM dye-modified specific aptamer of Vibrio parahaemolyticus, FAM dye-modified specific aptamer of Salmonella typhimurium or blank control FAM dye-modified aptamer and HNB, mixing and distributing mixture uniformly into the sample holes under negative pressure through four substrate injection holes respectively, removing tape on top of chip, placing it on a 65 degrees C heating plate, drying and waiting it until the substrate is completely dried; and (E) removing chip, opening valve, reattaching chip with tape on top of chip, and placing it in a vacuum pump to vacuum to obtain final chip.