LAMP primer set for quickly detecting salmonella and application thereof

A Salmonella and primer set technology, applied in the biological field, can solve the problems of insufficient versatility and specificity of primers, inability to amplify nucleic acids, primer mismatches, etc., and achieve high repeatability, efficient amplification, and primer specificity. Effect

Inactive Publication Date: 2017-08-08
温和心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third is high specificity, requiring 4 specific primers, and nucleic acid amplification cannot be performed if the primers do not match
[0004] In the existing LAMP technology, the primer design has the disadvantages of p

Method used

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  • LAMP primer set for quickly detecting salmonella and application thereof
  • LAMP primer set for quickly detecting salmonella and application thereof
  • LAMP primer set for quickly detecting salmonella and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This example is used to illustrate the versatility of the primer set of the present invention for the detection of different forms of LAMP. The different forms of LAMP detection include electrophoresis LAMP, precipitation LAMP and chromogenic LAMP. The steps are as follows:

[0047] (1) Concentration gradient dilution of Salmonella

[0048] The Salmonella strain used for detection comes from the China Industrial Microbiology Culture Collection and Management Center, and the number is CICC10420 Salmonella typhimurium. In a liquid medium, culture overnight at 36°C, centrifuge the pure culture of Salmonella at 12000rpm for 10min, discard the supernatant, wash the precipitate with 0.9% normal saline and centrifuge once, and wash the precipitate with ddH 2 O resuspended, and the concentration of bacteria was calibrated by McFarland turbidity method, that is, 1MCF≈3×10 8 cfu / mL. were diluted to 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10 1 cfu / ...

Embodiment 2-5

[0060] Embodiment 2-5 Salmonella reaction system and detection method

[0061] The concentration gradient dilution of Salmonella and the extraction steps of genomic DNA are as steps (1) and (2) in Example 1, and the reaction system is shown in Table 3, wherein the primers of Examples 2 and 3 adopt primer set A, and the primers of Examples 4 and 3 Primer set B was used for primer 5, and the amplification results were confirmed by turbidity detection. It can be seen from Table 3 that the primer set, detection method and reaction system of the present invention can well amplify the Salmonella specific fragment and obtain detection results.

[0062] Each reaction system, reaction condition and reaction result of table 3 embodiment 2~5

[0063]

[0064]

Embodiment 6

[0066] This example is used to illustrate the versatility of the primer set of the present invention to Salmonella.

[0067] According to the method in Example 1, DNA was extracted from 5 kinds of Salmonella (Table 4), amplified by LAMP, and detected by chromogenic method LAMP.

[0068] The results of the experimental group are as Figure 4 As shown, the amplification reactions of the Salmonella strains of the A and B primer groups were all positive, showing blue-green, and the reaction tube of the negative control (NTC) was blue-purple without change. It shows that the primer set of the present invention has better versatility to the above five kinds of Salmonella.

[0069] Table 4 primer group of the present invention is used bacterial strain and detection result to the universality test of Salmonella

[0070]

[0071] Note: 1. CMCC: China Medical Bacteria Culture Collection Management Center; CICC: China Industrial Microbiology Culture Collection Management Center. 2....

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Abstract

The invention relates to the technical field of biology, in particular to LAMP primer set for quickly detecting salmonella, a detection method and a kit. The primer set includes inner primer FIP/BIP and outer primer F3/B3. The detection method comprises the following steps: firstly extracting genome DNA of bacterium to be detected, performing LAMP amplification with the extracted genome DNA as a formwork, then detecting an amplification product and determining whether a sample to be detected contains salmonella or not by judging whether a reaction result is positive or not. The kit comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTPs, MgSO4 and Betaine. The LAMP primer set for quickly detecting the salmonella has advantages of strong specificity, high flexibility, convenience to operate, strong repeatability, good practicability and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set for rapid detection of Salmonella. Background technique [0002] Salmonella can cause gastroenteritis in humans and many other animals, and is one of the most common pathogens of diarrheal diseases. The pathogenic bacteria are widely distributed and can be isolated from the human population, animals, environment and food. Salmonella is one of the routine inspection items for food safety, disease diagnosis, and animal quarantine. [0003] The traditional isolation and identification method for the detection of Salmonella takes 4 days from enrichment to isolation and cultivation to identification, which is inefficient and labor-intensive. During the cultivation process, a large number of miscellaneous bacteria will interfere with the selection of the target colony, and even the miscellaneous colony will cover the target colony, resulting in a low detection rate. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/42
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 温和心龙英全蒋荣华
Owner 温和心
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