A method for label-free visualization of Vibrio parahaemolyticus genes based on CRISPR/Cas12a

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

Active Publication Date: 2022-08-05
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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  • A method for label-free visualization of Vibrio parahaemolyticus genes based on CRISPR/Cas12a
  • A method for label-free visualization of Vibrio parahaemolyticus genes based on CRISPR/Cas12a
  • A method for label-free visualization of Vibrio parahaemolyticus genes based on CRISPR/Cas12a

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1: Sensitivity test

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

[0064] The extracted genomic DNA of Vibrio parahaemolyticus was diluted ten-fold with sterile water to make its concentration 10 4 , 10 3 , 10 2 , 10 1 copies / μL (copies / μL).

[0065] Using the serially diluted Vibrio parahaemolyticus genomic DNA as a template, using LAMP to amplify: a 12.5 μL amplification system includes 2 μL of Vibrio parahaemolyticus DNA template, 1.6 μM inner primer 1 and inner primer 2, and 0.2 μM outer 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 incubate at 65°C for 60min to obtain the LAMP product.

[0066] 1 μL...

Embodiment 2

[0067] Example 2: Specificity Experiment

[0068]Using commercially available bacterial genomic DNA extraction kits (Beijing Biotech 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 25922 (Escherichia coli 25922), the concentration was measured by a micro UV spectrophotometer (ThermoFisher, USA), and the copy number was calculated.

[0069] Using the DNA of the above five bacteria as the template, the Tlh ​​primers were used for amplification to obtain five amplification products: 12.5 μL of the amplification system included 2 μL of the DNA template of the corresponding bacteria, 1.6 μM inner primer 1 and inner primer 2, and 0.2 μM outer primer. Primer 1 and outer primer 2, 0.4μM loop primer 1 and loop primer 2, 1.4mM dNTPs, 8U Bst DNA Polymerase Large Fr...

Embodiment 3

[0072] Example 3: Detection of Vibrio parahaemolyticus in New Bright Shrimp

[0073] The preserved strains of Vibrio parahaemolyticus were streaked and separated on the plate medium, and then a single colony was picked and transferred to LB broth liquid medium, cultivated at 37 °C for 6 hours, and diluted 10 times with normal saline, and the dilution was calculated according to the plate count. containing 6.1×10 5 CFU / mL to 6.1×10 0 CFU / mL of Vibrio parahaemolyticus. The sterile prawns were immersed in the diluent for bacterial infection, and then allowed to stand at room temperature for 30 minutes in a sterile environment to make Vibrio parahaemolyticus closely attached to the surface of the shrimp.

[0074] Taking the prawn contaminated by Vibrio parahaemolyticus as a sample, DNA was extracted from it using a commercially available bacterial genomic DNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.).

[0075] The DNA extracted from the contaminated prawns was us...

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Abstract

The invention discloses a method for label-free visual detection of Vibrio parahaemolyticus genes based on CRISPR / Cas12a. In the present invention, the characteristic gene of Vibrio parahaemolyticus is amplified by LAMP, and the complex assembled by Cas12a and crRNA is used to recognize and combine the characteristic sequence on the specific product amplified by LAMP to activate the accessory cutting activity of Cas12a, thereby to G-quadruplex The body is cleaved, making it unable to bind to Thioflavin T (ThT) and losing the ability to enhance fluorescence, finally converting the genetic information of Vibrio parahaemolyticus into visible fluorescence changes. The invention realizes the specific, label-free and visual detection of Vibrio parahaemolyticus gene.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and in particular relates to a method for realizing specific, label-free and visual detection of Vibrio parahaemolyticus by utilizing 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. People who eat food contaminated with Vibrio parahaemolyticus are likely to cause acute gastroenteritis with abdominal pain, diarrhea, nausea, vomiting, and fever as the main symptoms. Vibrio parahaemolyticus is widely found in seawater, seabed sediments and seafood such as fish, shells, shrimps and crabs. At present, Vibrio parahaemolyticus has become the main food-borne pathogen in my country, and the number of food poisoning incidents caused by Vibrio parahaemolyticus contamination has exceeded that of Salmonella. Especially in some coastal cities, food poisoning incidents cau...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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