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

A cryptic bacillus, real-time quantitative technology, applied in the field of microbial detection, can solve the problems of high cost, incorrect result judgment, low sensitivity, etc., and achieve the effect of a simple method of result judgment.

Inactive Publication Date: 2017-09-05
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 PCR detection method is lower than that of LAMP method, which may cause incorrect judgment of results. In addition, in terms of result judgment, PCR detection method requires Perform agarose gel electrophoresis on the PCR amplification products, and then use the ultraviolet transilluminator to judge, and the 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 arcanobacterium
  • Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting arcanobacterium
  • Real-time quantitative LAMP (Loop-Mediated Isothermal Amplification) primer, kit and method for detecting arcanobacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0068] The specificity result of embodiment 1 LAMP detection method

[0069] Perform LAMP amplification on 1 strain of Cryptobacterium, 6 strains of negative control bacteria and water control, the results are as follows figure 1 As shown, the cryptic bacilli reaction tube showed a rising curve of turbidity in about 30 minutes, which was a positive result, and the curves of the 6 negative control bacteria reaction tubes and the water control reaction tube showed no amplification, which was a negative result.

Embodiment 2

[0070] Example 2 Sensitivity results of LAMP detection method

[0071] Cryptobacteria recombinant plasmid pMD18-T- Plo The starting concentration is 1.05×10 1 ng / μL, LAMP and PCR amplification were performed after 10-fold dilution, the results were as follows figure 2 and image 3 As shown, the results show that the detection limit of the LAMP method is about 1.05×10 -4 ng / μL, while the detection limit of PCR method is 1.05×10 -3 ng / μL.

Embodiment 3

[0072] The drawing of embodiment 3 cryptobacteria quantitative standard curve

[0073] With the recombinant plasmid pMD18-T- Plo As a standard sample, its initial concentration was determined, and 8 dilutions were serially diluted 10 times with RNA-Free Water, and 2 μL of each dilution was used as a template for LAMP amplification.

[0074] Set the control: the concentration is 1.05×10 1 ng / μL, 1.05×10 0 ng / μL, 1.05×10 -1 ng / μL, 1.05×10 -2 ng / μL, 1.05×10 -3 ng / μL recombinant plasmid pMD18-T- Plo One standard sample, because the negative logarithmic value of the standard sample concentration is linearly related to the time value of the amplified turbidity value of 0.1, so the value captured by the turbidimeter and time (as shown in Table 1) can be made into a standard curve, Obtain the standard curve equation, y = 0.1912x - 5.8235, as Figure 4 As shown, the correlation coefficient R 2 = 0.9921, showing a good linear relationship. Taking time as the X value, you can...

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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 arcanobacterium. The primer is shown as SEQ ID NO: 1-4; the kit comprises the LAMP primer, 2X reaction buffer, Bst DNA polymerase, ultra-pure water and arcanobacterium DNA. The method comprises the steps: preparation of materials, design and synthesis of the LAMP primer, extraction of a reaction template, establishment of an LAMP reaction system, and specific detection, sensitivity detection and real-time quantitative detection by adopting the LAMP detection method. The specific detection and sensitivity detection prove that the LAMP detection method provided by the invention is capable of monitoring the reaction in real time and performing quantitative detection on a copy number of the arcanobacterium, the detection result can be rapidly and accurately obtained, and convenience is brought to simple and rapid detection of the arcanobacterium.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a loop-mediated isothermal amplification kit capable of rapid and real-time quantitative detection of cryptobacteria and its application. Background technique [0002] Arcanobacterium pyogenes (A.pyogenes), also known as Actinobacillus pyogenes, is a polymorphic, non-motile Gram-positive bacillus. Cryptobacterium is commonly found in the mucous membranes of cattle, sheep, pigs and other economically important animals, and is an opportunistic pathogen. Cryptobacterium can spread widely among livestock, causing a wide variety of non-specific purulent infections of the skin, internal organs and joints, such as acute suppurative mastitis, chronic abscessive mastitis, arthritis, endocarditis, liver abscess, Metritis, miscarriage and infertility, etc., have brought greater economic losses to the breeding industry. [0003] At present, the detection techniques of Cryptobac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2561/113C12Q2531/119C12Q2545/114
Inventor 李军吴翠兰彭昊冯世文潘艳陶立马春霞谢永平钟舒红胡帅杨威陈泽祥
Owner GUANGXI VETERINARY RES INST
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