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Method for quickly detecting salmonella through loop-mediated isothermal amplification

A ring-mediated isothermal and Salmonella technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of short reaction time, high detection cost, and long reaction time, and achieve Strong primer specificity, fast completion of detection, and high sensitivity

Inactive Publication Date: 2018-06-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Salmonella can cause a variety of salmonellosis with different clinical manifestations in humans and animals. The study of Salmonella is still a serious issue. How to isolate Salmonella from the research object is a very important issue. Bacterial infection provides a scientific basis. At present, some can accurately detect Salmonella and identify its specificity, which requires expensive instruments and equipment, resulting in high detection costs and is not suitable for large sample screening. Traditional conventional culture and separation techniques are time consuming
[0004] At present, the general detection method of Salmonella adopts the method of PCR detection, but the reaction time of this method is long, and the sensitivity is average. Compared with the loop-mediated isothermal amplification method, the sensitivity is high, the reaction time is short, and the operation is simple, but the method requires primer design. relatively high

Method used

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Experimental program
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Effect test

Embodiment 1

[0018] A method for rapid detection of Salmonella by loop-mediated isothermal amplification, comprising the following steps:

[0019] S1. Extract the genomic DNA of the bacteria to be tested: Extract the DNA of the suspected Salmonella by boiling method, culture the suspected colony for 16 h, take 600 μL of the bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 10,000 r / min for 1 min, discard the supernatant, Add 300 μL of ultrapure water to resuspend, boil in boiling water for 10 minutes, immediately ice-bath for 10 minutes, and then centrifuge at 10,000 r / min for 5 minutes, take the supernatant as the template, and store it at -20°C for later use.

[0020] S2. Using the extracted genomic DNA as a template for LAMP amplification: the amplification primers are as follows:

[0021] F3_: 5'-gtctaacgagcttaccgaag-3' (SEQ ID NO: 1);

[0022] B3: 5'-gtttcaactctgccaagttc-3' (SEQ ID NO: 2);

[0023] FIP:: 5'-ggagttctgacacacgattttctg-3' (SEQ ID NO: 3);

[0024] BIP: 5'-ct...

Embodiment 3

[0036] A method for rapid detection of Salmonella by loop-mediated isothermal amplification, comprising the following steps:

[0037] S1. Extract the genomic DNA of the bacteria to be tested: Extract the DNA of suspected Salmonella by boiling method, culture the suspected colonies for 16.5 hours, take 650 μL of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 10,000 r / min for 1.5 minutes, and discard the supernatant , add 300 μL ultrapure water to resuspend, boil in boiling water for 10 min, immediately ice-bath for 10 min, and then centrifuge at 10,000 r / min for 5 min, take the supernatant as the template, and store it at -20°C for later use.

[0038] S2. Using the extracted genomic DNA as a template for LAMP amplification: the amplification primers are as follows:

[0039] F3_: 5'-gtctaacgagcttaccgaag-3' (SEQ ID NO: 1);

[0040] B3: 5'-gtttcaactctgccaagttc-3' (SEQ ID NO: 2);

[0041] FIP:: 5'-ggagttctgacacacgattttctg-3' (SEQ ID NO: 3);

[0042] BIP: 5'-ctcccg...

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Abstract

The invention discloses a method for quickly detecting salmonella through loop-mediated isothermal amplification. The method comprises the following steps: extracting the genome DNA (deoxyribonucleicacid) of bacteria to be detected: taking the extracted genome DNA as a template to carry out LAMP (Loop-mediated isothermal amplification), wherein the final concentration of magnesium sulfate in an amplification reaction system is 6.5-8mM / L, the final concentration of dNTPs (High quality deoxyribonucleotide triphosphates) is 4.2-5mM / L, the final concentration of F3 and B3 primers is 0.13-0.26mu m / L, the final concentration of FIP (Forward Inner Primer) and BIP (Backward Inner Primer) primers is 1.2-2 mu M / L, the final concentration of Bst DNA polymerase is 0.3-1.8U / mu L, the final concentration of Betaine is 1-2M / L, and amplification temperature is 60-62 DEG C for 1-1.5h and 85-87 DEG C for 1-3min. The method has the advantages of high primer specificity, high sensitivity and lower cost,whether a detected sample contains salmonella or not can be judged according to whether amplification is carried out or not, and detection can be quickly finished.

Description

technical field [0001] The invention relates to the detection of Salmonella, in particular to a method for rapid detection of Salmonella by ring-mediated isothermal amplification. Background technique [0002] Salmonella (Salmonella) belongs to the Enterobacteriaceae family and is a Gram-negative bacillus. Except for a few Salmonella (fowl typhoid and pullorum), all have flagellar motility. [0003] Salmonella can cause a variety of salmonellosis with different clinical manifestations in humans and animals. The study of Salmonella is still a serious issue. How to isolate Salmonella from the research object is a very important issue. Bacterial infection provides a scientific basis. At present, some can accurately detect Salmonella and identify its specificity, which requires expensive instruments and equipment, resulting in high detection costs and is not suitable for large sample screening. Traditional conventional culture and separation techniques are Time-consuming and la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12R1/42
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2565/125
Inventor 瞿孝云张建民廖明
Owner SOUTH CHINA AGRI UNIV
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