Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent

An isothermal loop-mediated, labeling detection technology, applied in measurement devices, microbial determination/inspection, instruments, etc., can solve the problems of gel electrophoresis pollution, poor detection sensitivity of magnesium pyrophosphate precipitation, affecting technology popularization and application, etc. The effect of fast detection results, improved detection sensitivity, and reduced detection costs

Inactive Publication Date: 2014-04-02
天津国际旅行卫生保健中心
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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 yersinia pestis nucleic acid mark detection reagent
  • Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent
  • Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Detection of Yersinia pestis DNA genome gradient dilution marker amplification

[0037] Take the DNA genome of Yersinia pestis with known concentration and carry out 10-fold gradient dilution, and dilute to the final concentration of the target gene in Yersinia pestis DNA is 10 0 Copies / μL, take the positive reference material, negative reference material prepared by Yersinia pestis DNA template standard, and Yersinia pestis DNA serial dilution (the final target gene of Yersinia pestis-specific target gene in PCR tube). The concentration is 10 4 copy, 10 3 copy, 10 2 copy, 10 1 copy and 10 0 copy), as a template for marker amplification detection.

[0038]

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

[0040]

[0041] The composition of each substance in the 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 p...

Embodiment 2

[0045] Example 2: Detection of Yersinia pestis

[0046] Sample: A DNA sample suspected of Yersinia pestis.

[0047] Take the sample for isothermal labeling amplification detection amplification

[0048] The composition ratio of each component in isothermal amplification is as follows:

[0049]

[0050] The composition of each substance in the 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.

[0051] The above mixture was added to a PCR tube, and isothermal amplification was performed in a water bath. The amplification steps were: 60°C, 30 minutes; 80°C, 2 minutes.

[0052] Add the amplified product to the colloidal gold test strip, and read the result within 15 minutes. When the test line and the quality control line...

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Abstract

The invention relates to a novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent. A front inner primer in a primer group for isothermal loop-mediated amplification is used for marking an antigen, and meanwhile, a rear inner primer in the primer group is used for marking another antigen, so that a yersinia pestis specific target gene can be simultaneously amplified and marked. A matched colloidal gold strip can be used for rapidly detecting an amplification product of the target gene after marking, thereby detecting yersinia pestis. The detection reagent can be simply and rapidly operated, and is high 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 Yersinia pestis nucleic acid labeling detection reagent. Background technique [0002] Yersinia pestis, commonly known as Yersinia pestis, is the pathogenic bacteria of plague. Plague is a zoonotic natural foci of severe infectious diseases. Human plague is mostly infected by the flea bites of plague rats. It is a statutory Class A infectious disease in my country. It was not until the end of the nineteenth century that Yersinia pestis was isolated and named. This bacterium is a natural foci disease in rodents. It is highly contagious and has a high fatality rate, and it is easy to cause a pandemic. There have been three pandemics from the 6th to the 19th century AD. This bacterium mainly involves the skin and lymph nodes, followed by sepsis, pneumonia, and meningitis. During the Second World War, the Japanese invading army used Yersinia ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/533
CPCC12Q1/6804C12Q2531/119C12Q2563/131C12Q2565/625
Inventor 祁军刘寅王馨关淳
Owner 天津国际旅行卫生保健中心
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