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Novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent

An isothermal loop-mediated, Vibrio nucleic acid technology, used in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problem of poor detection sensitivity of magnesium pyrophosphate precipitation, gel electrophoresis pollution, Affect the promotion and application of technology and other issues, and achieve the effects of rapid detection results, lower detection costs, and improved detection sensitivity

Inactive Publication Date: 2016-07-27
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has certain defects, mainly in the detection of amplification results. Gel electrophoresis detection is easy to cause pollution, magnesium pyrophosphate precipitation detection sensitivity is poor, and the method of fluorescent substance color change is limited by the cost of fluorescent substances or the need to use detection instruments.
Therefore, the restrictions in the product detection link have affected the popularization and application of this technology.

Method used

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  • Novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent
  • Novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent
  • Novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: DNA genome gradient dilution marker amplification detection of Vibrio parahaemolyticus

[0038] Take the Vibrio parahaemolyticus DNA genome with known concentration and carry out 10-fold serial dilution to it, and dilute it to the final concentration of the target gene in the Vibrio parahaemolyticus DNA to be 10 0 copies / μL, take the positive reference material prepared from the standard Vibrio parahaemolyticus DNA template, the negative reference material, and the gradient dilution of Vibrio parahaemolyticus DNA (the final concentrations of the specific target gene of Vibrio parahaemolyticus in the PCR tube are respectively 10 4 copy, 10 3 copy, 10 2 copy, 10 1 copy and 10 0 copy) as a template for marker amplification detection.

[0039] The composition ratio of each component in isothermal amplification is as follows:

[0040]

[0041] The composition of each substance in 1 times buffer is as follows: 20mM Tris-HCl, 10mMKCl, 10mM (NH 4 ) 2 SO 4...

Embodiment 2

[0045] Example 2: Detection of Vibrio parahaemolyticus

[0046] Sample: A DNA sample suspected to be Vibrio parahaemolyticus.

[0047] Take the sample for isothermal label amplification detection amplification, and use Vibrio parahaemolyticus DNA template standard at the same time, which contains about 10 target genes. 4 copy ~10 0 Copied DNA samples and a negative control in sterile double distilled water were tested simultaneously.

[0048]

[0049] The composition of each substance in 1 times buffer is as follows: 20mM Tris-HCl, 10mMKCl, 10mM (NH 4 ) 2 SO 4 , 2mMMgSO 4 And the mass concentration is 0.1% TritonX-100, the pH value of the buffer is 8.8, and the concentration of each component in the 10-fold buffer is 10 times the concentration of each component in the 1-fold buffer.

[0050] Add the above mixture into a PCR tube, and carry out isothermal amplification in a water bath. The amplification steps are: 60°C, 30 minutes; 80°C, 2 minutes.

[0051] Add the am...

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Abstract

The invention relates to a novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent. According to the reagent, an antigen is marked for a front inner primer in a primer group used in isothermal loop-mediated amplification, meanwhile another antigen is marked for a rear inner primer, the operation can be performed when a vibrio parahaemolyticus specific 16S-23S rRNA intergenic region sequence is amplified, subsequently a piece of colloidal gold test paper in match is adopted to detect an amplification product, and then vibrio parahaemolyticus can be detected. As the primer for the 16S-23S rRNA intergenic region sequence is designed for RNA secondary structure prediction, the situation that the detection sensitivity and the specificity can be influenced by mutation caused if other protein genes are used or the rRNA gene is blindly used can be avoided as much as possible, and the detection reagent provided by the invention is good in specificity and sensitivity.

Description

technical field [0001] The invention relates to a reagent for nucleic acid detection, in particular to a novel isothermal ring-mediated detection reagent for nucleic acid labeling of Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus is a Gram-negative polymorphic bacillus or Vibrio slightly curved. Vibrio parahaemolyticus food poisoning, also known as halophilic bacteria food poisoning, is caused by eating food containing this bacteria, mainly from seafood or salt pickled products, common ones are crabs, squid, jellyfish, fish, yellow mud snails, etc. , followed by eggs, meat or vegetables. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stool. The rapid detection of Vibrio parahaemolyticus is of great significance for the prevention and control of the diseases caused by it. [0003] Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification) is a new type of nucleic a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6804C12Q1/6844C12Q2531/119C12Q2521/101C12Q2563/107Y02A50/30
Inventor 于佳魏玉西刘寅何雨蓉陈双张文嘉
Owner QINGDAO UNIV
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