RPA-LFD method and kit for detecting salmonella and application

A technology for Salmonella and Salmonella enteritidis, which is applied in the field of food safety testing to achieve accurate detection, strong specificity, and short time effects

Pending Publication Date: 2022-01-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current research mainly focuses on a single target, and the multiple detection challenge of cross-reactivity between several specific antibodies in the

Method used

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  • RPA-LFD method and kit for detecting salmonella and application
  • RPA-LFD method and kit for detecting salmonella and application
  • RPA-LFD method and kit for detecting salmonella and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) is used to extract the genomic DNA of the pathogenic bacteria to be tested, and the sample DNA to be tested is extracted as a template, and the primers designed by the present invention are used to carry out the RPA reaction. The specific steps are as follows:

[0049] Use the bacterial genomic DNA extraction kit to extract the genomic DNA of the pathogenic bacteria to be tested, and add 29.5 μL of RPA reaction buffer to the reaction tube containing lyophilized enzyme powder; forward primers InvA-F, Stm-F and Sen- F each 0.5 μL (concentration is 10 μM); Reverse primers InvA-R, Stm-R and Sen-R each 0.5 μL (concentration is 10 μM); sample DNA to be tested 2.0 μL; use deionized water to make up the volume to 47.5 μL, Then vortex and mix once; add 2.5 μL MgOAc to the inside of the PCR tube cap and centrifuge quickly for 10 s; fully vortex and mix once again and centrifuge quickly for 10 s; place ...

Embodiment 2

[0058] In order to verify the specificity of the RPA-LFD detection method, Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Escherichia coli O157:H7, Shigella flexneri, Staphylococcus aureus, Enterobacter cloacae and Listeria monocytogenes As the test material, the RPA reaction is carried out through the primers designed in the present invention, and the RPA amplification product is detected by target lateral flow chromatography test strips.

[0059] Detect according to the method of embodiment 1, detection result is as follows figure 2 shown. figure 2 It is the test result chart of RPA-LFD-specific test strips. In the figure, test strip number 1: Salmonella typhimurium and Salmonella enteritidis; number 2: Salmonella typhimurium; number 3: Salmonella enteritidis; number 4: Salmonella infantis; number 5: Escherichia coli ATCC 25922; Code 6: Enterobacter sakazakii ATCC 25914; Code 7: Staphylococcus aureus ATCC 29213; Code 8: Bacillus cereus ATCC 1220; Cod...

Embodiment 3

[0061] This embodiment mainly detects salmonella-contaminated chicken.

[0062] First, single colonies of Salmonella typhimurium, Salmonella enteritidis and Salmonella infantis were picked on the LB agar plate, inoculated in LB broth respectively, shaken at 200 rpm, and cultivated overnight at 37°C to obtain 10 5 CFU / mL bacterial solution; prepare five 10g chicken samples without Salmonella, numbered as samples No. 1-5; add 1 mL PBS to No. 1 sample; add 1 mL each of Typhimurium and Salmonella Enteritidis to No. 2 sample; add 1 mL to No. 3 sample Salmonella infantis; add 1mL Salmonella enteritidis to sample No. 4; add 1mL Salmonella typhimurium to sample No. 5; then add 9mL PBS to samples No. 1, 3-5, add 8mL PBS to sample No. 2, mix well, transfer the liquid to In a 15mL centrifuge tube, centrifuge at 1000rpm for 6min to remove larger impurities, transfer the supernatant to a new 15mL centrifuge tube, and centrifuge at 6000rpm for 6min to collect the precipitate. Using the met...

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Abstract

The invention relates to the field of food safety detection, and discloses primer pairs, a method and a kit for rapidly detecting salmonella through an RPA-LFD method and application. The primer pairs are InvA-F, InvA-R, Stm-F, Stm-R, Sen-F and Sen-R. The detection method comprises the steps of extracting total DNA of a sample, performing RPA reaction and parameter optimization by using the primer pairs, detecting by using a target lateral flow chromatography test strip, and judging whether the sample contains salmonella and serotypes such as mouse typhoid and enteritis or not according to the strip, wherein the whole detection process can be completed within 1 hour. The kit comprises sterile double distilled water, a buffer solution, standard positive DNA, a target lateral flow chromatography test strip and a product diluent. The primer pairs, the method and the kit for detecting the salmonella have the characteristics of being high in specificity, simple, convenient, rapid and the like, and can be used for rapid and accurate detection of the salmonella and the mouse typhoid and enteritis serotypes of the salmonella.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a primer pair, a method, a kit and an application for the rapid detection of Salmonella by the RPA-LFD method. [0002] technical background [0003] Salmonella (Salmonella) is a pathogenic bacterium that has received widespread attention. It can not only cause food poisoning by contaminating eggs, milk, meat and other foods, but also infect a variety of animals, and then spread to the food chain, bringing great harm to food safety. . Salmonella infection can cause clinical symptoms such as fever, gastroenteritis, diarrhea, vomiting, loss of appetite, headache, and lack of energy. In severe cases, it can even cause sepsis and bacteremia, leading to death of the patient. According to statistics, worldwide, Salmonella can cause 93.8 million infections and 155,000 deaths each year. In China, food poisoning caused by Salmonella accounts for 70%-80% of bacterial food poisoning. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/42
CPCC12Q1/689C12Q1/6844C12Q2600/16C12Q2521/507C12Q2522/101C12Q2537/143C12Q2563/131C12Q2565/625
Inventor 史贤明詹泽强周秀娟崔妍方正伟何守魁王旭
Owner SHANGHAI JIAO TONG UNIV
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