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Primer, kit and detection method for detecting salmonella at constant temperature on the basis of polymerase spiral reaction

A technology for detecting Salmonella and primers, applied in the biological field, can solve the problems of being unsuitable for rapid detection at the grassroots level, complex primer design, and high false positive rate, and achieve the effects of simple and fast operation, short time-consuming and good specificity

Pending Publication Date: 2018-11-06
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method requires an expensive cycler and is not suitable for rapid detection at the grassroots level
At present, the most widely used constant temperature amplification technology-LAMP also has its limitations, such as complex primer design, high false positive rate, high reagent price, and due to the protection of intellectual property rights in Japan, my country has strong limitations in transformation and application.

Method used

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  • Primer, kit and detection method for detecting salmonella at constant temperature on the basis of polymerase spiral reaction
  • Primer, kit and detection method for detecting salmonella at constant temperature on the basis of polymerase spiral reaction
  • Primer, kit and detection method for detecting salmonella at constant temperature on the basis of polymerase spiral reaction

Examples

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Effect test

Embodiment 1

[0038] Example 1 Microbiological method for detection of Salmonella based on polymerase helical reaction isothermal amplification technology

[0039] 1. A method for detecting pathogenic microorganisms based on polymerase helical isothermal amplification technology. In this embodiment, Salmonella is taken as an example. The reagents used are as follows:

[0040] a. Detection primers Ft aqueous solution and Bt aqueous solution, accelerated primer IF and IB aqueous solution with a concentration of 50 μM each, the primer sequences are as follows (5'-3'):

[0041] Detection primer Ft: cacaaagatgataatgatgccTACTGGAAAGGGAAAGCC (SEQ ID NO.1);

[0042] Detection primer Bt: ccgtagtaatagtagaaacacGACAGAGCGGAGGATAAA (SEQ ID NO.2);

[0043] Acceleration primer IF: TCATCGCACCGTCAAA (SEQ ID NO.3);

[0044] Acceleration primer IB: TGGCGGTATTTCGGTGGG (SEQ ID NO.4);

[0045] b.2×Reaction stock solution: Tris-HCl with a concentration of 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0m...

Embodiment 2

[0054] The specificity test of embodiment 2 polymerase helical reaction detection Salmonella

[0055] The genomic DNA of Salmonella and non-Salmonella was established according to the above-mentioned reaction system and conditions to establish a polymerase helical reaction detection method for specificity tests, wherein the non-Salmonella were: Salmonella ATCC14028, Salmonella ATCC29629, Escherichia coli ATCC43895, Escherichia coli ATCC438944, Pseudomonas aeruginosa bacteria ATCC27853, Listeria monocytogenes ATCC19116, Listeria monocytogenes ATCC19114, Staphylococcus aureus ATCC27664, Lactobacillus casei GBHM-21 (Bao Zhining. Lactobacillus casei GBHM-21 identification, pilot plant preparation and fermentation technology research [D ]. South China University of Technology, 2015.). Set the Salmonella genome as a positive control, ultrapure water as a negative control, the results are as follows figure 2 shown. The results showed that only the Salmonella genome showed a positi...

Embodiment 3

[0056] Embodiment 3PSR detects the sensitivity test of Salmonella

[0057] The genome of Salmonella was diluted 10 times in a concentration gradient, respectively 12.3ng / μL, 1.23ng / μL, 123pg / μL, 12.3pg / μL and 1.23pg / μL, and a negative control (deionized water) was set at the same time, according to the example The reaction system in 1 is used to construct the polymerase helical reaction amplification method to determine the sensitivity of the detection method, the results are as follows image 3 shown. The results showed that the established Salmonella polymerase helical reaction method could detect 123pg / μl of Salmonella DNA in the sample.

[0058] Conclusion: From the above experimental results, it can be seen that the amplification method based on polymerase helical reaction has the following advantages over conventional PCR and fluorescent PCR:

[0059] The operation and identification are simple and quick: the whole process of conventional PCR can produce results in 2-4...

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Abstract

The invention discloses a primer, a kit and a detection method for detecting salmonella at a constant temperature on the basis of polymerase spiral reaction. The invention designs a pair of detectionprimers Ft / Bt and one pair of acceleration primers IF / IB by aiming at the specific region of the specificity target sequence invA conserved region of the salmonella, the nucleotide sequence of the primer is disclosed in SEQ ID NO. 1-4, and the reliability of a detection result is guaranteed by the primer. The invention also provides a kit for detecting the salmonella on the basis of the polymerasespiral reaction. The kit has the characteristics of high sensitivity, good specificity, convenience and high speed in operation, accurate and reliable result and low cost and is suitable for small and medium size departments and field detection application. In addition, the invention also provides a detection method for detecting salmonella at the constant temperature on the basis of the polymerase spiral reaction. According to the method, under a constant temperature condition, short time is consumed, an amplified product does not need gel electrophoresis, and a result can be directly judgedwith eyes after fluorescent dye is directly used for color development.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer, a kit and a detection method for constant temperature detection of Salmonella based on a polymerase helical reaction. Background technique [0002] Salmonella is a large group of important food-borne pathogens in Enterobacteriaceae, and the poisoning cases caused by Salmonella rank first in food poisoning around the world. According to the WHO Foodborne Disease Surveillance Program, about 160,000 people in Europe are infected with Salmonella each year. The annual cost of prevention and treatment of Salmonella is as high as 2.8 billion euros. In some countries, 70% to 80% of bacterial food poisoning incidents are caused by Salmonella, and about 90% of the food that causes Salmonella poisoning is meat, eggs, milk and other livestock products. Salmonella can cause a series of diseases after infecting livestock and poultry. During animal slaughter, pathogenic bacteria in the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/42
CPCC12Q1/6844C12Q1/689
Inventor 徐振波徐行勇刘君彦苗健苏健裕刘丽艳李冰李琳
Owner SOUTH CHINA UNIV OF TECH
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