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LAMP primer for detecting Brucella and kit containing same

A Brucella and kit technology, applied in the field of microbial detection, can solve problems such as the inability to detect Brucella quickly and accurately, and achieve the effects of convenient product detection, high sensitivity, and avoiding time loss.

Active Publication Date: 2014-02-26
黄耀江
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the invention is to provide a fast, specific, simple and safe method for the deficiencies in the prior art that cannot detect Brucella quickly and accurately Methods for detecting Brucella;

Method used

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  • LAMP primer for detecting Brucella and kit containing same
  • LAMP primer for detecting Brucella and kit containing same
  • LAMP primer for detecting Brucella and kit containing same

Examples

Experimental program
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Effect test

Embodiment 1

[0064] Embodiment 1 is used to detect the design and synthesis of the LAMP primer of Brucella

[0065] Select the BCSP31 gene of Brucella melis as the target gene, and use the online tool http: / / primerexplorer.jp / e / to use Primer Explorer V4 to design, screen and synthesize a set of primers whose parameters meet the requirements of LAMP primer design. The primers include 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2 pairs of loop primers (LF1, LB1 and LF2, LB2), synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. . The sequences of the primers are shown in Table 1:

[0066] Table 1: LAMP primers for detection of Brucella

[0067]

Embodiment 2

[0068] Embodiment 2 utilizes LAMP primer to detect the specificity analysis of Brucella

[0069] The established LAMP method was used to amplify Escherichia coli, Staphylococcus aureus, Salmonella, Bacillus thuringiensis, Lactococcus lactis, Streptococcus thermophilus, yeast, Aspergillus flavus, etc. to verify the specificity of the method. Analyze the peak time and peak value of the LA-320 program, such as figure 2 and image 3 shown. From figure 2 It can be seen that none of CH1-8 reacted; image 3 Middle red represents Brucella positive, green represents Brucella negative; the turbidity changes during the 1-8 reaction process, all show green, so the amplification results are all negative. The result shows that the LAMP primer of the present invention has high specificity to BCSP31 gene.

Embodiment 3

[0070] Embodiment 3 utilizes LAMP primer to detect the sensitivity analysis of Brucella

[0071] After the DNA template purified from the blood of the confirmed diseased sheep (from the Animal Epidemic Prevention Station of Tuquan County, Inner Mongolia) was measured by the NANODROP2000 UV-Vis spectrophotometer, the concentration was measured by a 10-fold serial dilution, so that the concentration gradient was 3.5ng / μL, 350pg / μL, 35pg / μL, 3.5pg / μL, 350fg / μL, 35fg / μL, 3.5fg / μL, and 350ag / μL. Take 2.0 μL as a template for the LAMP reaction, and perform three replicates for each gradient. After the reaction, directly observe the color change in the reaction tube with the naked eye to judge the result. When LAMP amplification occurs, the result is called a white precipitate, while if no LAMP reaction occurs, the solution is clear. Through observation, it was found that when the DNA template concentration was as low as 35fg / μL, there was still white precipitate in the reaction tu...

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Abstract

The invention relates to an LAMP primer for detecting Brucella and a kit containing the same. The LAMP primer comprises inner primers, namely FIP and BIP, outer primers, namely F3 and B3, and two pairs of ring primers, namely LF1 and LB1 or LF2 and LB2; the kit comprises the LAMP primer, Bst DNA polymerase, and a reaction buffer solution containing dNTPs. The kit utilizes loop-mediated isothermal amplification reactions (LAMP), carries out a specific amplification on target gene BCSP31 by using Bst polymerase and the LAMP primer, and is capable of rapidly, accurately, and conveniently detecting the Brucella in a human blood sample or milk. The detection method has a high sensitivity, the lower limit of detection can reach 35 fg, the reaction time is short, expensive instruments are not needed, the operability is strong, moreover, the product detection is convenient, the product can be detected by naked eyes or through a dyeing method, so the kit has a very high practical value.

Description

technical field [0001] The invention relates to a method for rapidly detecting Brucella, LAMP primers and a kit containing the primers, belonging to the technical field of microbial detection. Background technique [0002] Brucella (Brucella spp.) is a Gram-negative bacterium with no flagella, no spores, smooth and capsuled. Often parasitic in cells. Endotoxin is its important pathogenic substance. Brucella can enter the host through intact skin and mucous membranes, and has strong invasiveness. According to the phenotype, antigenicity and host, Brucella is divided into 6 kinds, namely Brucella abortus (Brucella bovis), Brucella melitensis (Brucella ovis), Brucella ovis (sheep) Brucella epididymis), Brucella canis (Brucella canis), Brucella suis (Brucella suis) and Brucella neotomae (Brucella sahling); infects humans primarily of bovine species Brucella, Brucella melis and Brucella suis. Brucella has long been listed as a potential biological weapon. [0003] Brucellos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04C12R1/01
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 黄耀江蒋丹杨志达靳卫林闫强
Owner 黄耀江
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