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

An isothermal ring-mediated, Vibrio nucleic acid technology, which is applied in the determination/testing of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of poor detection sensitivity of magnesium pyrophosphate precipitation, gel electrophoresis pollution, Affecting technology popularization and application and other issues, to achieve rapid detection results, reduce detection costs, and improve detection sensitivity

Inactive Publication Date: 2014-03-26
天津国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

[0040] The composition ratio of each component in isothermal amplification is as follows:

[0041]

[0042] The composition of each substance in 1X buffer is as follows: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4...

Embodiment 2

[0046] Example 2: Detection of Vibrio parahaemolyticus

[0047] Sample: A DNA sample suspected to be Vibrio parahaemolyticus.

[0048] 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.

[0049] The composition ratio of each component in isothermal amplification is as follows:

[0050]

[0051] The composition of each substance in 1X buffer is as follows: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 And the mass concentration is 0.1% Triton X-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.

[0052] Add the above mixture into a PCR tube, and carry out isothermal amplification in a...

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Abstract

The invention relates to a novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid labeling detection reagent. By labeling a front inner primer in a primer group used for isothermal loop-mediated amplification with one antigen and meanwhile, labeling a rear inner primer with another antigen; a vibrio parahaemolyticus specific target gene can be labeled while the vibrio parahaemolyticus specific target gene is amplified. After the vibrio parahaemolyticus specific target gene is labeled, an amplification product of the target gene can be rapidly detected by using matched colloidal gold test paper, so that vibrio parahaemolyticus is detected. The detection reagent disclosed by the invention is simple and rapid in operation and has strong specificity and high 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. [0002] Background technique [0003] 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. [0004] Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification) is a new type of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/6844C12Q2531/119C12Q2563/137Y02A50/30
Inventor 关淳王馨祁军左锋刘寅
Owner 天津国际旅行卫生保健中心
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