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Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting erysipelothrix rhuriopathiae

A real-time quantitative technology for Erysipelas suis, applied in the field of microbial detection, can solve the problems of high cost, laboratory pollution, complex equipment and other problems, and achieve the effect of simple result judgment method

Inactive Publication Date: 2017-09-01
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, complex instruments and equipment are required, the cost is high, and it is not suitable for grassroots and on-site detection. The sensitivity of the PCR detection method is lower than that of the LAMP method, which may cause incorrect judgment of the results. Amplified products are subjected to agarose gel electrophoresis, and then judged by ultraviolet transilluminator irradiation, and judgment by naked eyes has human subjective factors, and it is easy to cause laboratory pollution

Method used

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  • Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting erysipelothrix rhuriopathiae
  • Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting erysipelothrix rhuriopathiae
  • Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting erysipelothrix rhuriopathiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Specific results of LAMP detection method

[0068] LAMP amplification of 1 strain of Erysipelas suis, 5 strains of negative control bacteria and water control, the results are as follows figure 1 As shown, the Erysipelas suis reaction tube showed a turbidity rising curve in about 40 minutes, which was a positive result. The 5 strains of negative control bacteria reaction tube and the water control reaction tube curve showed no amplification, which was a negative result.

Embodiment 2

[0069] Example 2 Sensitivity results of LAMP detection method

[0070] The initial concentration of Erysipelas DNA is 1.68×10 2 ng / μL, LAMP and PCR amplification after 10-fold dilution, the results are as follows figure 2 with image 3 As shown, the result shows that the detection limit of the LAMP method is about 1.68×10 -3 ng / μL, and the detection limit of PCR method is 1.68×10 -2 ng / μL.

Embodiment 3

[0071] Example 3 Drawing of the quantitative standard curve of Erysipelas suis

[0072] Determination of recombinant plasmid pMD18-T- Spa As a standard sample, use RNA-Free Water to carry out a 10-fold dilution of 8 dilutions, and take 2 μL of each dilution as a template for LAMP amplification.

[0073] Set the control: the concentration is 1.68×10 1 ng / μL, 1.68×10 0 ng / μL, 1.68×10 -1 ng / μL, 1.68×10 -2 ng / μL, 1.68×10 -3 ng / μL recombinant plasmid pMD18-T- Spa There is one standard sample, because the negative logarithm of the standard sample concentration has a linear relationship with the time value of the amplified turbidity value of 0.1, so the negative logarithm of the sample concentration and the time value captured by the turbidimeter can be combined (see Table 1) Make a standard curve and obtain the standard curve equation, y = 0.1766x-5.3619, such as Figure 4 As shown, the correlation coefficient R 2 = 0.9961, showing a good linear relationship. Taking time as the X valu...

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Abstract

The invention discloses a real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, a kit and a method for detecting erysipelothrix rhuriopathiae. The primer is as shown in SEQ ID NO: 1 to SEQ ID NO: 4. The kit comprises the LAMP primer, a 2x reaction buffer solution, Bst DNA polymerase, superpure water and a erysipelothrix rhuriopathiae DNA. The method comprises the following steps: preparation of materials, design and synthesis of the LAMP primer, extraction of a reaction template, construction of a LAMP reaction system and specificity detection, sensitivity detection and real-time quantitative detection which are carried through a LAMP detection method. The specificity detection and the sensitivity detection show that the LAMP detection method provided by the invention can monitor reaction in real time and quantitatively detect the copy number of the erysipelothrix rhuriopathiae, thus quickly and accurately obtaining a detection result and bringing convenience to simple and quick detection of the erysipelothrix rhuriopathiae.

Description

Technical field [0001] The invention relates to the technical field of microbial detection, in particular to a ring-mediated isothermal amplification kit for rapid, real-time quantitative detection of Erysipelas and its application. Background technique [0002] Erysipelothrix rhusiopathiae, commonly known as Erysipelothrix rhusiopathiae or Erysipelothrix, belongs to the genus Erysipelothrix. It is a slender small bacillus with positive Gram stain and is a zoonotic pathogen. The bacterium was first isolated by Pasteur in 1882, and in 1886 Loffler described the pathogen causing swine erysipelas for the first time. The serotype of this bacteria is complex, and 26 species such as 1, 1a, 2, 2a, 2b, 3-24 and N types have been found. Swine erysipelas is an acute and febrile infectious disease caused by Erysipelas swine, which is clinically manifested as acute septicemia, subacute rash, chronic arthritis and endocarditis. Erysipelas can also infect more than 70 animals, including mamm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2561/113C12Q2545/114
Inventor 李军禤雄标冯世文彭昊潘艳胡帅钟舒红陶立马春霞谢永平杨威陈泽祥
Owner GUANGXI VETERINARY RES INST
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