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LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit

A technology for Staphylococcus and Salmonella, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems that the freshest products cannot be put on the market and delivered to consumers, so as to prevent proliferation and Contamination, high sensitivity, and easy operation

Pending Publication Date: 2020-03-31
JILIN BUSINESS & TECH COLLEGE
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Staphylococcus aureus and Salmonella are two food-poisoning bacteria that are easily contaminated by liquid milk. The national standard stipulates that they must not be detected in liquid milk, but the existing national standard detection method for these two pathogenic bacteria takes 3 days, resulting in It solves the problem that enterprises cannot put the freshest products on the market in time and deliver them to consumers

Method used

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  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit
  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit
  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 liquid milk sample collection

[0037] Use cleaned sampling tools and sample bottles, and the sample bottles must be free from residual disinfectants, water and detergents. Sampling was carried out in the milking hall of the milk station, milk storage tanks in the milk station, milk tanks in milk trucks, liquid milk production lines and supermarkets, and detailed records were made. Fresh samples collected were tested immediately.

Embodiment 2

[0039] A method for loop-mediated isothermal amplification technology detection of Staphylococcus aureus and Salmonella in liquid milk, comprising the following steps:

[0040] S1. Genome extraction

[0041] (1) Sample preparation: Take 1ml of liquid milk and put it into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 5min, discard the supernatant; add 1ml of normal saline, blow and mix, centrifuge at 12000rpm for 1min, discard the supernatant, add 1ml of normal saline to the precipitate Mix by pipetting.

[0042] (2) Bacterial lysis: add lysozyme to the precipitate obtained in step (1), 37 ° C for 10 min, then add lysate and mix, 37 ° C for 5 min, lyse, then add proteinase K, 70 ° C for 10 min, Obtain the lysate.

[0043] (3) DNA extraction: use phenol chloroform to extract the lysate obtained in step (2) to obtain a white suspension, carefully collect the supernatant, add isopropanol and sodium acetate, mix well, and collect the precipitate after centrifugation. Wash ...

Embodiment 3

[0067] A kind of loop-mediated isothermal amplification technique detects the method for Staphylococcus aureus and Salmonella in liquid milk, and its difference with embodiment 2 is:

[0068] S3. LAMP detection

[0069] The isothermal amplification temperature is 60°C constant temperature water bath for 1 hour, and then 80°C to inactivate the polymerase used. In a 20 μL reaction system, the concentrations of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LB were all 10 μM, and the amounts added were 0.5 μL, 0.5 μL, 2 μL, 2 μL, 1 μL, 1 μL, the molar ratio in the reaction system is 1:1:4:4:2:2; the amount of 0.16U / μL Bst DNA polymerase added is 0.4 μL.

[0070] Visual analysis of S4.LAMP detection results

[0071] In this experiment, hydroxynaphthol blue (HNB) was used as the chromogenic agent of the LAMP visualization method, and the reaction was judged according to the change of hydroxynaphthol blue (100 μmol / L) before a...

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Abstract

The invention relates to a LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and a kit, which belong to the technical field of food safety rapid detection. Themethod comprises the following steps: extracting genomic DNA of the liquid milk on site, designing a primer group on the basis of specific molecular marker characteristics of genomes of staphylococcusaureus and salmonella, and synchronously detecting polluted staphylococcus aureus and salmonella in the liquid milk by adopting a loop-mediated isothermal amplification technology. The whole detection process has low requirements on environment and equipment, and whether a to-be-detected sample contains staphylococcus aureus or salmonella or not is determined by adding a color developing agent toobserve the color change in the tube. The method has the advantages of high sensitivity, good specificity, safety, harmlessness, no organic reagent and no nucleic acid binding dye, all reagents can be directly released into the environment, and the method has important significance for improving the quality safety of dairy foods and improving the food-borne pathogenic bacteria analysis and detection technology.

Description

technical field [0001] The invention relates to the technical field of rapid detection of food safety, in particular to the rapid detection of two important food-borne pathogens, Staphylococcus aureus and Salmonella, specifically a detection sample for a LAMP detection method for detecting Staphylococcus aureus and Salmonella in dairy products Preparation method, detection primer set, detection kit and specific detection method. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a widespread Gram-positive bacterium that can be widely distributed in soil, air, water, and human skin. As a common pathogen, Staphylococcus aureus can produce various virulence factors such as enterotoxin. These virulence factors are highly virulent, low poisoning dose, good stability, difficult to remove during food processing, and have serious hazards. Pork, chicken, milk, milk powder, eggs, bread, ice cream and other foods have been reported to be contaminated by St...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2600/166C12Q2531/119C12Q2537/143C12Q2537/1376C12Q2563/103C12Q2527/125C12Q2545/113Y02A50/30
Inventor 孟宪梅李岩松刘熙孙宇宿甲子刘东朱媛媛齐雪峰关玉婷常江马昀钊杨柳
Owner JILIN BUSINESS & TECH COLLEGE
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