Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Kit and detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae

An absolute quantification technique for Vibrio cholerae, applied in the field of molecular biology, can solve the problems that the validity and practicability of the quantitative analysis of Vibrio cholerae are unknown, the quantitative detection of Vibrio cholerae has not been seen, and the composition of the background bacteria is complex, etc., to achieve The effect of shortening detection time, avoiding deviation, and reducing influence

Active Publication Date: 2018-05-11
中国海关科学技术研究中心
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the quantitative analysis of this technology for microorganisms is still in its infancy. Related studies such as human herpesvirus, methicillin-resistant Staphylococcus aureus, Chlamydia trachomatis, parasite Cryptosporidium in clinical samples, and Shigella in bovine feces samples Escherichia coli with toxins, and plant pathogenic bacteria that cause amylovora blight and potato brown rot, etc., have not yet seen relevant reports that can be used for the quantitative detection of Vibrio cholerae in food
Since the food matrix is ​​mostly rich in nucleic acid amplification inhibitors such as protein, fat, and pectin, and the background bacteria are complex and high in concentration, the effectiveness and practicability of ddPCR for the quantitative analysis of Vibrio cholerae in food is still unknown, and there is room for development. and broad application prospects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit and detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae
  • Kit and detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae
  • Kit and detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2d

[0046] The design of embodiment 2ddPCR primers, probes

[0047] In order to achieve the specific detection and absolute quantitative analysis of total Vibrio cholerae and pathogenic Vibrio cholerae, we selected a single copy of species-specific and highly conserved N-acetylglucosamine (GlcNAc) binding protein A The coding gene gbpA (GenBank no.EU072441.1) and the virulence co-regulatory pilus coding gene tcpA (GenBank no.KP187623.1) were used as target sequences, and the sequences were analyzed and compared through NCBI online tools, using Prime Express software V3.0 (ABI, Foster City, CA, USA) designed three pairs of primers and probes, and the sequences are shown in Table 1.

Embodiment 3d

[0048] Example 3 ddPCR detection

[0049] 1. Preparation of reaction premix

[0050] Prepare 20 μL ddPCR reaction master mix according to Table 2.

[0051] 2. Droplet generation

[0052] 20 μL of ddPCR reaction master mix and 70 μL of droplet generating oil were added to the 8-well droplet generating card, and placed in a droplet generator (QX100, Bio-Rad, Pleasanton, CA) to generate droplets.

[0053] 3. Amplification reaction

[0054] The generated water-in-oil droplets (40 μL) were slowly transferred to a 96-well plate, sealed and placed on a PCR machine (GeneAmp9700, Applied BioSystems, Foster City, CA) for amplification reaction. The amplification conditions are shown in Table 3 shown.

[0055] 4. Result judgment

[0056] The amplified 96-well plate was placed in a droplet reader (QX100, Bio-Rad, Pleasanton, CA), and the results were read and analyzed using QuantaSoft software. Positive microdroplets containing amplified products and negative microdroplets without a...

Embodiment 4

[0057] Embodiment 4qPCR detects

[0058] In order to facilitate comparison with the ddPCR method, the same set of primers and probes were selected for qPCR detection. The 25 μL double qPCR reaction system includes: 2×Master Mix 12.5 μL, 10 μM gbpA-F, gbpA-R, tcpA-F and tcpA-R 1 μL each, 10 μM gbpA-P and tcpA-P 0.5 μL each, to be tested DNA template 2 μL. The amplification was carried out on ABI7900 fluorescent PCR instrument (US, ABI company) or Light cycler 480II (Switzerland, Roche company), 50°C for 2min, 95°C for 10min, 95°C for 15s, 60°C for 1min, 45 cycles. After the reaction is completed, use SDS3.2 or SW1.5.1 software for analysis, and calculate the copy number of the target gene according to the standard curve.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a kit and a detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae. The present invention provides two sets of primers andprobes for the absolute quantitative detection of the total vibrio cholerae and the pathogenic vibrio cholerae. One set of the primers and probe is used for detection of the total vibrio cholera, thenucleotide sequences of the primers are as shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of the probe is as shown in SEQ ID No. 3. The other set of the primers and probe is usedfor detection of the pathogenic vibrio cholera, the nucleotide sequences of the primers are as shown in SEQ ID No. 4 and SEQ ID No. 5, and the nucleotide sequence of the probe is as shown in SEQ ID No. 6. The invention also provides a method for the absolute quantitative detection of the total vibrio cholerae and the pathogenic vibrio cholerae by use of a micro-droplet digital polymerase chain reaction. The method has strong specificity, high sensitivity, good amplification effect, strong operability and easy standardization.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer / probe and a kit for the absolute quantitative detection of total Vibrio cholerae and pathogenic Vibrio cholerae, more specifically, to an absolute quantitative detection of total Vibrio cholerae and pathogenic Vibrio cholerae A droplet digital polymerase chain reaction (ddPCR) kit and detection method for pathogenic Vibrio cholerae. Background technique [0002] Vibrio cholerae (V.cholera) is the pathogen of human cholera. Cholera is one of the ancient and widespread severe infectious diseases. It has caused many pandemics in the world, mainly manifested as severe vomiting, diarrhea and dehydration. , the death rate is very high. Vibrio cholerae mainly exists in coastal waters and can usually infect humans through contaminated water sources or food, especially aquatic products. O1 and O139 are the two main pathogenic serotypes of Vibrio cholerae, which are closely rela...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/63
CPCC12Q1/6851C12Q1/689C12Q2563/159C12Q2531/113Y02A50/30
Inventor 魏海燕曾静赵晓娟马丹魏咏新刘莉李丹周熙成汪琦徐蕾蕊张西萌付溥博
Owner 中国海关科学技术研究中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com