RPA-IAC primer for detecting vibrio mimicus and method

A technology of mimetic Vibrio and primer pairs, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high price of fluorescence detection equipment, not widely used, and limited popularization and application. , to save the primer design process, avoid false negatives, and have a wide range of applications.

Active Publication Date: 2017-10-03
INSPECTION & QUARANTINE TECH CENT SHANTOU CIQ +1
View PDF7 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the VIDAS-CHL method, EIA method, colloidal gold test paper method and API method all have defects such as poor specificity and low sensitivity, and have not been widely used; LAMP method, as a new detection method, has greatly improved the detection method. Efficiency, which reduces the cost of detection, but produces a higher false positive rate
As a highly sensitive, fast and simple detection method, PCR technology has been widely used in many fields, especially in the detection of pathogens, it has achieved revolutionary results and has become a gold standard for rapid detection of nucleic acids. On this basis, it has developed DNA probe technology, real-time fluorescent PCR technology and PCR combined with denaturing high-performance liquid chromatography (DHPLC) technology, etc., and the design of PCR primers has become a key factor restricting the success of this type of detection method. Conventional PCR primer design not only requires Repeatedly compare the specificity of the primers, and it is necessary to optimize the parameters and reaction conditions of the primers, especially the annealing temperature is repeatedly optimized to prevent non-specific amplification. Under the constraints of these conditions, the selected primers can easily affect the amplification. Increased validity and specificity
In addition, the price of fluorescence detection equipment is generally high, which also limits the popularization and application of this type of detection method to a certain extent.

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
  • RPA-IAC primer for detecting vibrio mimicus and method
  • RPA-IAC primer for detecting vibrio mimicus and method
  • RPA-IAC primer for detecting vibrio mimicus and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] This embodiment provides the RPA primer pair and the internal standard amplification sequence and its application.

[0068] The screening method for the RPA primer pair is as follows: Aiming at the Vibrio mimetic VMH gene (GenBank ID: CP016384.1), multiple RPA primers were designed and a large number of screenings were carried out, and the specificity, sensitivity, and the interaction between primers were integrated. As well as the adaptability of the primers used and the RPA amplification kit, the following RPA primer pairs with good specificity, good repeatability and high sensitivity that can detect Vibrio mimeticus were finally screened out:

[0069] VMH-F: 5'-GAGATATTGGCATCTTTACAAAATGGACGA-3' (SEQ ID NO: 1);

[0070] VMH-R: 5'-GATTGGCAATCAGAAGAAAGCCAATTACCCAG-3' (SEQ ID NO: 2);

[0071] Design and synthesis of internal standard amplified sequence: In order to avoid homologous interference of similar bacteria and individual nucleic acid contamination of testing per...

Embodiment 2

[0077] This embodiment provides a kit and application thereof, the kit comprising:

[0078] (1) SEQ ID NO: 1, SEQ ID NO: 2 (from Example 1), and a plasmid containing the internal standard amplification sequence shown in SEQ ID NO: 3;

[0079] (2) DNA extraction reagents;

[0080] (3) tube containing freeze-dried enzyme powder;

[0081] (4) rehydration buffer;

[0082] (5) Magnesium acetate solution.

[0083] The above tubes containing lyophilized enzyme powder, rehydration buffer (Rehydration Buffer) and magnesium acetate solution (280mmol / L) are reagents from the RPA amplification kit TwistAmp Basic kits (TwistDX company, product number TABAS03KIT).

[0084] The DNA extraction reagent is the reagent included in the animal genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP432 and the bacterial genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP302. .

[0085] The construction of...

Embodiment 3

[0091] This embodiment provides a method for detecting or assisting the detection of Vibrio mimicus, such as detecting or assisting in detecting whether a biological sample is infected with Vibrio mimicus, or detecting or assisting in detecting whether the pathogen is or is a candidate for Vibrio mimicus. The primer pair of Example 1 or the kit of Example 2.

[0092] Above-mentioned method comprises the steps:

[0093] Step 1: RPA-IAC amplification

[0094] Using the DNA of biological samples or pathogenic bacteria as a template, add the plasmid containing the internal standard amplification sequence shown in SEQ ID NO: 3 in Example 2, and use the primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 to perform RPA -IAC amplification to obtain the RPA-IAC amplification product, while setting a blank control (the template is ultrapure water).

[0095] The preparation method of the RPA-IAC amplification system is as follows: Add 29.5 μL of rehydration buffer (Rehydration Buf...

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 provides a primer pair and an internal amplification control sequence. The primer pair comprises a nucleotide sequence as shown in SEQ ID NO:1 and a nucleotide sequence as shown in SEQ ID NO:2, and the internal amplification control sequence comprises a nucleotide sequence as shown in SEQ ID NO:3. The invention further provides the application of the primer pair and the internal amplification control sequence to the preparation of products for the detection or aided detection of vibrio mimicus, and a method for detecting the vibrio mimicus, based on a RPA-IAC technology. Experiments prove that the RPA-IAC primer is good in specificity, high in sensitivity and short in detection time, does not need special instruments, and is wide in application scope, and the RPA-IAC detection method for vibrio mimicus, based on the primer matched with an internal amplification control, is sensitive, accurate, convenient and rapid, and has a guiding significance on quarantining imported and exported cargoes and products.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for detecting vibrio mimicus, a primer and a kit. Background technique [0002] Mimic Vibrio is a Gram-negative Vibrio with a single flagella and power. Like Vibrio parahaemolyticus and Vibrio cholerae, it belongs to the normal marine flora and widely exists in river water, seawater and seafood in nature. The strain grows well in nutrient broth containing no sodium or only 1% sodium chloride and can be isolated from aquatic animals. [0003] In the United States, North America, New Zealand, Bangladesh, and African countries, Vibrio mimicus has been reported to cause disease. It is also reported in Fujian, Beijing, Shanghai, Jiangsu, and Zhejiang in China. It is mostly sporadic or outbreaks. Many; the incubation period is 3 to 72 hours, with an average of 24 hours; the main symptoms are diarrhea, vomiting, abdominal pain, and watery stool or mucus and bloody stool; th...

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): C12Q1/68C12Q1/04C12N15/11C12M1/34C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2521/507C12Q2545/101C12Q2565/125
Inventor 周广彪凌莉易敏英陈冠武冼钰茵刘静宇袁慕云阳静李志勇魏霜
Owner INSPECTION & QUARANTINE TECH CENT SHANTOU CIQ
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products