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Method for label-free visual detection of vibrio parahaemolyticus genes based on CRISPR(clustered regularly interspaced short palindromic repeats)/Cas12a

A hemolytic vibrio, label-free technology, applied in the field of microbial detection

Active Publication Date: 2021-09-03
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem that nucleic acid detection based on CRISPR / Cas12a relies on nanomaterials or fluorescent labels, the present invention provides a method that uses CRISPR / Cas12a to cut the G-quadruplex so that it cannot bind to ThT and cannot enhance the fluorescence emission of ThT. Method for genetic detection of vibrio hemolyticus

Method used

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  • Method for label-free visual detection of vibrio parahaemolyticus genes based on CRISPR(clustered regularly interspaced short palindromic repeats)/Cas12a
  • Method for label-free visual detection of vibrio parahaemolyticus genes based on CRISPR(clustered regularly interspaced short palindromic repeats)/Cas12a
  • Method for label-free visual detection of vibrio parahaemolyticus genes based on CRISPR(clustered regularly interspaced short palindromic repeats)/Cas12a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: sensitivity test

[0063] Genomic DNA of Vibrio parahaemolyticus ATCC 17802 was extracted using a commercially available bacterial genomic DNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.), its concentration was measured by a micro-volume ultraviolet spectrophotometer (Thermo Fisher, USA), and the copy number was calculated.

[0064] The extracted Vibrio parahaemolyticus genomic DNA was diluted tenfold with sterile water so that its concentration was 10 4 , 10 3 , 10 2 , 10 1 Copies / μL (copies / μL).

[0065] Using the gradient diluted Vibrio parahaemolyticus genomic DNA as a template, use LAMP to amplify: 12.5 μL amplification system includes 2 μL of Vibrio parahaemolyticus DNA template, 1.6 μM internal primer 1 and internal primer 2, and 0.2 μM external primer 1 and outer primer 2, 0.4μM loop primer 1 and loop primer 2, 1.4mM dNTPs, 8U Bst DNA Polymerase Large Fragment, 1×NEBuffer2.1, and incubated at 65°C for 60min to obtain the LAMP produc...

Embodiment 2

[0067] Embodiment 2: specificity experiment

[0068]Using a commercially available bacterial genomic DNA extraction kit (Beijing Baitaike Biotechnology Co., Ltd.) from Vibrio parahaemolyticus ATCC 17802, Salmonella typhimurium CMCC (B) 50115 (Salmonella 50115), Acinetobacter DSM 25388 (Acinetobacter 25388), Genomic DNA was extracted from Escherichia ferguson 056 (Escherichia ferguson 056) and Escherichia coli ATCC25922 (Escherichia coli 25922), and its concentration was measured by a micro-ultraviolet spectrophotometer (ThermoFisher, USA) to calculate the copy number.

[0069] Using the DNA of the above five kinds of bacteria as templates, amplified with Tlh primers respectively to obtain five kinds of amplification products: 12.5 μL amplification system including 2 μL DNA templates corresponding to bacteria, 1.6 μM inner primer 1 and inner primer 2, 0.2 μM outer primer Primer 1 and outer primer 2, 0.4 μM loop primer 1 and loop primer 2, 1.4 mM dNTPs, 8U Bst DNA Polymerase Lar...

Embodiment 3

[0072] Example 3: Detection of Vibrio parahaemolyticus in fresh fresh shrimp

[0073] The preserved strain of Vibrio parahaemolyticus was separated by streaking on the plate medium, and then a single colony was picked and transferred to LB broth liquid medium, cultured at 37°C for 6 hours, diluted 10 times with normal saline, and calculated according to the plate count Contains 6.1×10 5 CFU / mL to 6.1×10 0 CFU / mL of Vibrio parahaemolyticus. Dip the aseptic prawns into the diluent to infect the bacteria, and then let them stand at room temperature for 30 minutes in a sterile environment to make Vibrio parahaemolyticus tightly adhere to the surface of the prawns.

[0074] The prawns contaminated by the above-mentioned Vibrio parahaemolyticus were used as samples, and DNA was extracted from them using a commercially available bacterial genomic DNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.).

[0075] The DNA extracted from the polluted prawns was used as a template...

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Abstract

The invention discloses a method for label-free visual detection of vibrio parahaemolyticus genes based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a. A characteristic gene of vibrio parahaemolyticus is amplified through LAMP, a compound assembled by Cas12a and crRNA is utilized to recognize and is combined with a characteristic sequence on a specific product amplified by LAMP to activate the affiliated cleavage activity of Cas12a, so that a G quadruplex is cleaved, the G quadruplex cannot be combined with thioflavin T (ThT) and loses the fluorescence enhancement capability, and finally, gene information of the vibrio parahaemolyticus is converted into visible fluorescence change. According to the invention, specific, label-free and visual detection of the vibrio parahaemolyticus genes is realized.

Description

technical field [0001] The invention belongs to the field of microbial detection, and in particular relates to a method for realizing specific, label-free and visual detection of Vibrio parahaemolyticus by using CRISPR / Cas12a to cut G-quadruplex and regulate the fluorescence emission of Thioflavin T (ThT). Background technique [0002] Vibrio parahaemolyticus is a major foodborne pathogen. After people eat food contaminated by Vibrio parahaemolyticus, it is very likely to cause acute gastroenteritis with abdominal pain, diarrhea, nausea, vomiting, and fever as the main symptoms. Vibrio parahaemolyticus widely exists in seawater, seabed sediments and seafood such as fish, shellfish, shrimp and crab. At present, Vibrio parahaemolyticus has become the main food-borne pathogen in my country, and food poisoning incidents caused by Vibrio parahaemolyticus contamination have surpassed Salmonella in number. Especially in some coastal cities, food poisoning incidents caused by this...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12R1/63
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2521/327C12Q2525/161C12Q2563/107
Inventor 王柳陈雪雲何开雨徐霞红
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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