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Novel isothermal loop-mediated legionella pneumophila nucleic acid labeling detection reagent

A technology mediated by Legionella pneumophila and isothermal rings, applied in the field of reagents for nucleic acid detection, can solve problems affecting the popularization and application of technology, poor detection sensitivity of magnesium pyrophosphate precipitation, gel electrophoresis pollution, etc., to improve detection sensitivity, detect The effect of rapid results and reduced testing 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 legionella pneumophila nucleic acid labeling detection reagent
  • Novel isothermal loop-mediated legionella pneumophila nucleic acid labeling detection reagent
  • Novel isothermal loop-mediated legionella pneumophila nucleic acid labeling detection reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: DNA genome gradient dilution marker amplification detection of Legionella pneumophila

[0037] Take the Legionella pneumophila DNA genome with known concentration and carry out 10-fold serial dilution to the final concentration of the target gene in the Legionella pneumophila DNA to 10 0 copy / μL, take the positive reference substance prepared from the Legionella pneumophila DNA template standard, the negative reference substance, and the Legionella pneumophila DNA gradient dilution solution (the final concentrations of the specific target gene of Legionella pneumophila in the PCR tube are respectively 10 3 copy, 10 2 copy, 10 1 copy and 10 0 copy) as a template for marker amplification detection.

[0038] The composition ratio of each component in isothermal amplification is as follows:

[0039]

[0040] 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 concentr...

Embodiment 2

[0044] Embodiment 2: Detection of Legionella pneumophila

[0045] Sample: A DNA sample suspected to be Legionella pneumophila.

[0046] The sample was taken for isothermal marker amplification detection amplification, and at the same time, the Legionella pneumophila DNA template standard was used, which contained about 10 target genes. 5 Copy DNA sample; Legionella pneumophila DNA template standard containing about 10 genes of interest 0 The copied DNA sample; the Bacillus subtilis DNA template standard; the Bacillus licheniformis DNA template standard; the Bacillus subtilis DNA template standard and the negative control of sterile double distilled water were tested simultaneously.

[0047] The composition ratio of each component in isothermal amplification is as follows:

[0048]

[0049] 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 v...

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Abstract

The invention relates to a novel isothermal loop-mediated legionella pneumophila 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 legionella pneumophila specific target gene can be labeled, while the legionella pneumophila specific target gene is amplified. After the legionella pneumophila 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 legionella pneumophila 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 legionella pneumophila nucleic acid labeling. Background technique [0002] In 1976, acute febrile respiratory disease broke out among members of the Philadelphia Legion in the United States. More than 200 participants developed pneumonia, and 34 of them were killed. After research, a bacterium named Legionella pneumophila was found. Subsequently, many related bacteria were temporarily included in this genus, and retrospective research found that there were cases of Legionnaires' disease as early as 1943. More than 30 species of Legionella have been proposed, at least 19 of which are pathogens of human pneumonia. Among them, the most common pathogen is Legionella pneumophila (accounting for 85%~90% of cases). This incident reflects that Legionella pneumophila is not a small-scale human-to-human infection. Strengtheni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N33/569G01N33/558C12R1/01
CPCC12Q1/04C12Q1/6844C12Q2531/119C12Q2563/137
Inventor 关淳王馨祁军左锋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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