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Novel isothermal loop-mediated enterocolitis yersinia nucleic acid label detection reagent

A technology for Yersinia nucleic acid and enterocolitis, which is applied in the field of thermocycle-mediated Yersinia enterocolitica nucleic acid labeling detection reagents, and can solve problems affecting technology popularization and application, gel electrophoresis pollution, and magnesium pyrophosphate precipitation detection Poor sensitivity and other problems, to achieve the effect of improving detection sensitivity, fast detection results, and reducing detection costs

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 enterocolitis yersinia nucleic acid label detection reagent
  • Novel isothermal loop-mediated enterocolitis yersinia nucleic acid label detection reagent
  • Novel isothermal loop-mediated enterocolitis yersinia nucleic acid label detection reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Yersinia enterocolitica DNA genome gradient dilution marker amplification detection

[0039] Take the Yersinia enterocolitica DNA genome with known concentration and carry out 10-fold serial dilution on it, and dilute it to the final concentration of the target gene in the Yersinia enterocolitica DNA to be 10 0 copies / μL, take the positive reference material prepared from the Yersinia enterocolitica DNA template standard, the negative reference material, and the serial dilution of Yersinia enterocolitica DNA (Yersinia enterocolitica in a PCR tube The final concentration of the specific target gene was 10 5 copy, 10 4 copy, 10 3 copy, 10 2 copy, 10 1 copy and 10 0 copy) as a template for marker amplification detection.

[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 ) 2 SO 4 , 2 mM MgS...

Embodiment 2

[0046] Example 2: Detection of Yersinia enterocolitica

[0047] Sample: A DNA sample suspected to be Yersinia enterocolitica.

[0048] The sample was taken for isothermal marker amplification detection amplification, and at the same time, the Yersinia enterocolitica DNA template standard was used, which contained about 10 target genes. 3 Copy DNA sample; Yersinia enterocolitica DNA template standard containing about 10 genes of interest 2 Copy DNA sample; Yersinia enterocolitica DNA template standard containing about 10 genes of interest 1 Copy DNA sample; Yersinia enterocolitica DNA template standard containing about 10 genes of interest 0 Copy DNA sample; Yersinia pseudotuberculosis DNA template standard and negative control of 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-...

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Abstract

The invention relates to a novel isothermal loop-mediated enterocolitis yersinia nucleic acid label detection reagent, which is characterized in that a front inner primer used in isothermal loop-mediated amplification is labeled with an antigen, meanwhile, a back inner primer used in isothermal loop-mediated amplification is labeled with another antigen, and a gene can be labeled during the amplification of specific target genes of enterocolitis yersinia. After the labeling, a matched colloidal gold strip can be used for fast detecting amplification products of target genes, so the enterocolitis yersinia is detected. The detection reagent provided by the invention has the advantages that the operation is simple and fast, the specificity is high, and the sensitivity is high.

Description

technical field [0001] The invention relates to a reagent for nucleic acid detection, in particular to a novel isothermal ring-mediated Yersinia enterocolitica nucleic acid labeling detection reagent. [0002] Background technique [0003] Yersinia enterocolitica is widely distributed in nature and is one of the few enteric pathogens that can grow at refrigerated temperatures. This bacterium naturally lives in a variety of animals, such as pigs, rats, livestock, etc., and is infected by contaminated food (milk, pork, etc.) and water through the fecal-oral route or by contact with infected animals. In addition to causing gastrointestinal symptoms, it can also cause diseases such as the respiratory system, cardiovascular system, bone connective tissue, and even sepsis, resulting in death. This bacterium is also an important food-borne pathogen, and many countries have listed this bacterium as a routine inspection item for imported and exported food. Clinicians rarely consid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 祁军左锋王馨刘寅韩静娜姜智贤叶正
Owner 天津国际旅行卫生保健中心
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