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LAMP (Loop-Mediated Isothermal Amplification) detection method for calcein fluorescence visualization salmonella

A technology of salmonella and calcein, which is applied in the field of detection of salmonella with warm amplification technology, can solve the problems of false positive interference, aerosol pollution, high price, etc., and achieve the effect of simple operation, pollution avoidance and accurate judgment of results

Inactive Publication Date: 2013-11-27
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the second and third methods, due to the high-efficiency amplification of LAMP, it is easy to cause aerosol pollution after opening the lid after the reaction, causing false positive interference.
The fourth is to use a turbidity meter to detect the turbidity in real time and judge the reaction result. The use of a turbidity meter is expensive and the operation is relatively complicated

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) detection method for calcein fluorescence visualization salmonella
  • LAMP (Loop-Mediated Isothermal Amplification) detection method for calcein fluorescence visualization salmonella
  • LAMP (Loop-Mediated Isothermal Amplification) detection method for calcein fluorescence visualization salmonella

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Experimental program
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Effect test

Embodiment 1

[0031] Implementation 1. Design of specific primers for Salmonella LAMP detection method

[0032]Search and download all Salmonella fimY genes from the American gene sequence database Genbank, and perform homology analysis with Blast software to obtain a 381bp Salmonella-specific conserved fimY sequence (Genbank number: AM933172.1, see the nucleotide sequence list for detailed sequences 1) As the target sequence, use PrimerExplorer V4 software (Eiken Co., Ltd., Japan) to design primers yourself. Among the 4 primers, 2 are internal primers (FIP and BIP), and 2 are external primers (F3 and B3). The inner primer FIP includes the complementary sequence of the F1 region and the F2 sequence, that is, F1c-F2; the inner primer BIP includes the B1c sequence and the complementary sequence of the B2 region, that is, B1c-B2. Outer primers F3 and B3 are located outside the regions of F2 and B2, respectively. See the nucleic acid sequence Table 2-Table 5 for the specific sequence.

Embodiment 2

[0033] Implementation 2, the preparation of Salmonella genomic DNA template

[0034] Genomic DNA was extracted by boiling method, the specific method is as follows:

[0035] (1) Take 1ml of the bacterial solution cultured at 37°C for 10-14 hours in a 1.5ml Eppendorf tube, centrifuge at 5000rpm for 5min, discard the supernatant, wash twice with double distilled water,

[0036] (2) Suspend the precipitate in 50ul double-distilled water, pipette the tip of the pipette to mix evenly, boil in boiling water for 10 minutes,

[0037] (3) Immediately take out the Eppendorf tube and place it in the freezer at -80°C for 15 min.

[0038] (4) Immediately take out the Eppendorf tube, boil in boiling water for 1 min, centrifuge at 10,000 rpm for 3 min, take the supernatant, and store it at -20°C for later use.

Embodiment 3

[0039] Implementation 3. Establishment of calcein fluorescence visualized Salmonella LAMP detection method

[0040] 1. Preliminary establishment of calcein fluorescence visualized detection method for Salmonella LAMP

[0041] The initial reaction system is: 2 pairs of primers FIP and BIP 0.8uM, F3 and B3 0.1uM for the specific conserved sequence fimY gene of Salmonella, 2ul of the genomic DNA template of the sample to be tested, MgCl 2 3mM, betaine Betaine 0.4M, 10×Thermpol reaction buffer 2.5ul, 10×SYBRGreenⅠ2.5ul, dNTP 0.6mM, ddH 2 O 5.5ul, Bst DNA polymerase 320U / ml. The initial reaction conditions are: constant temperature reaction at 63° C. for 1 hour. Since the optimization of the LAMP reaction system and reaction conditions uses a fluorescent quantitative PCR instrument to detect fluorescence in real time for analysis, the fluorescent dyes in the initial reaction system can be used for IQ TM 5 The optical system of the fluorescent quantitative PCR instrument detects...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) detection method for calcein fluorescence visualization salmonella. According to the method, primers special for LAMP reaction and optimization, expected reagents and reaction conditions are adopted, two pairs of primers special for the LAMP reaction and the optimization are designed according to conserved sequence fimY genes of the salmonella. According to the method, an optimized prepared calcein MnCl2 mixed solution is taken as a fluorescence indicator, an LAMP reaction system is added before the reaction, and the result can be accurately judged with naked eyes without opening a cover after the one-step reaction. The method is efficient and rapid in reaction, has the characteristics that the operation is easy and convenient, the cost is low, the specificity is good, the university is wide, and the sensitivity is high, can be popularized in cultivation plants, local medical organizations, and laboratories for daily detection, and has wide application prospects and good economic benefits.

Description

Technical field [0001] The present invention is a molecular biological test method for bacteria in the field of biotechnology, which involves a Salmonella detection method that uses temperature amplification technologies such as circular guidance. Background technique [0002] Salmonella (Salmonella) is an important human and animal in the Enterobacteriacidae, and the Gram -negative pathogenic bacteria.Animal derived Salmonella is likely to cause acute diarrhea, and even outbreak of food poisoning, leading to gastrointestinalitis, typhoid fever, and parathylean.In recent years, the harm caused by Salmonella has been increasing globally, and it is of great significance in public health. [0003] Due to the large number of Salmonella blood, the various biochemical reactions are complicated. At present, a variety of Salmonis detection methods have been established, such as traditional culture method, enzyme -linked immune method, biosensor technology, and nucleic acid amplification ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCY02A50/30
Inventor 王红宁程菡张安云雷昌伟刘必慧杨帆夏青青管中斌
Owner SICHUAN UNIV
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