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A method for simultaneous detection of Salmonella and Staphylococcus aureus in food

A technology for Salmonella and Staphylococcus, which is applied in the field of microbial detection, can solve the problems of simultaneous detection of Salmonella and Staphylococcus aureus, long detection time, low sensitivity, etc., to improve the coupling efficiency of magnetic beads, easy to operate, and reduce interference Effect

Active Publication Date: 2018-01-05
HENAN BUSINESS SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After years of research by scholars from various countries, the detection method of Salmonella and Staphylococcus aureus in food is becoming more and more mature, but its shortcomings are: long detection time, low sensitivity, high cost, and cannot detect Salmonella and Staphylococcus aureus simultaneously

Method used

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  • A method for simultaneous detection of Salmonella and Staphylococcus aureus in food
  • A method for simultaneous detection of Salmonella and Staphylococcus aureus in food
  • A method for simultaneous detection of Salmonella and Staphylococcus aureus in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Effect test of aminotriacetic acid solution

[0028] After the standard strain of Salmonella (CMCC 50115) and the standard strain of Staphylococcus aureus (ATCC6538) were cultured overnight, they were diluted by a 10-fold gradient, and 10 -4 —10 -9 And 10 -4 —10 -10 Dilution test for sensitivity of immunomagnetic beads. Dilute the dilutions of each gradient of the two bacterial cultures into two groups: the test group and the control group. The control group was added with 65% EDTA solution with a mass concentration of 6:1. The volume ratio of the diluent to the EDTA solution was 6:1. The test group was added For the aminotriacetic acid solution with a mass concentration of 50%, the volume ratio of the diluent to the aminotriacetic acid solution is 6:1. The test steps are as follows:

[0029] 1) Take 100μL of each gradient dilution solution and add 200μL of immunomagnetic beads to mix thoroughly, incubate with slow shaking at 37°C for 20min, and let stand on a magn...

Embodiment 2

[0037] A method for simultaneously detecting Salmonella and Staphylococcus aureus in milk includes the following steps:

[0038] 1) Take 25mL of milk sample to be tested, add 225mL PBS buffer, shake for 30s, adjust pH to 7 with 1mol / mL sterile NaOH solution to obtain sample homogenate; add 50.0ml 50 wt% aminotriacetic acid to sample homogenate The solution was oscillated by ultrasonic at 37°C for 5 minutes (ultrasonic frequency 20KHz), washed with 0.01 mol / L PBS buffer, centrifuged at 8000r / min for 5 minutes, layered, and the upper layer was discarded;

[0039] 2) Take 100 μL of the sample processed in step 1) and add 200 μL of immunomagnetic beads (magnetic beads made of streptavidin-modified magnetic beads coupled with biotin-labeled Salmonella and Staphylococcus aureus antibodies), mix at 37°C, and shake After 20 minutes, let stand on the magnetic stand for 2 minutes to layer, discard the upper layer, wash the lower layer sample with 200μL 0.01mol / L PBS buffer, mix well, and let...

Embodiment 3

[0042] A method for simultaneously detecting Salmonella and Staphylococcus aureus in eggs includes the following steps:

[0043] 1) Rinse the egg shell to be tested with running water, then wipe and disinfect with 75% alcohol cotton ball, take 25mL egg liquid, add 225mL PBS buffer, beat with a slap homogenizer for 60s, adjust with 1mol / mL sterile NaOH solution pH to 7 to obtain a sample homogenate; add 45.5mL of 50wt% aminotriacetic acid solution to the sample homogenate, oscillate at 37°C for 5 minutes with ultrasound (ultrasonic frequency 20KHz), and wash twice with 0.01mol / L PBST buffer. Centrifuge at 8000r / min for 5min, stratify, discard the upper layer;

[0044] 2) Take 100μL of the sample processed in step 1) and add 200μL of immunomagnetic beads (magnetic beads made of streptavidin-modified magnetic beads coupled with biotin-labeled Salmonella and Staphylococcus aureus antibodies), mix and shake at 37°C After 20 minutes, let stand on the magnetic stand for 2 minutes, layer ...

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Abstract

A method for simultaneously detecting Salmonella and Staphylococcus aureus in food, comprising the following steps: 1) diluting a food sample to be tested with reference to GB 4789 to obtain a 1:10 sample homogenate; adding 50-65 wt wt to the sample homogenate % aminotriacetic acid solution, the volume ratio of sample homogenate to aminotriacetic acid solution is 5-7:1, shake, add phosphate buffer or phosphate buffer containing 0.05% Tween-20, centrifuge for layers, discard the upper layer ; 2) Add immunomagnetic beads to the sample treated in step 1), mix and shake, then place it on a magnetic stand for stratification, discard the upper layer, and wash the lower sample; the immunomagnetic beads are made of streptavidin-modified magnetic Bead-conjugated biotin-labeled Salmonella and Staphylococcus aureus antibodies were prepared; 3) phosphate buffer was added to the washed samples, shaken, coated on LB plates, and cultured for observation. The detection method of the invention is easy to operate, accurate and efficient, and has low cost, and can enrich two target bacteria at the same time, and can shorten the detection time from 72 hours to 25 hours.

Description

Technical field [0001] The invention belongs to the technical field of microorganism detection, and specifically relates to a method for simultaneously detecting Salmonella and Staphylococcus aureus in food. Background technique [0002] Salmonella is one of the main pathogenic bacteria in Enterobacteriaceae. Livestock and poultry infected with Salmonella can cause a series of diseases. Eating infected food can cause food poisoning. In my country, 70% to 80% of bacterial food poisoning incidents are caused by Salmonella. Approximately 90% of the food poisoned by Salmonella infection are animal products such as meat, eggs, and milk. [0003] Staphylococcus aureus can cause multi-tissue infections, can spread in a wide range of people and cause a variety of diseases, and sometimes even life-threatening, such as bacteremia, endocarditis and pneumonia. In addition, Staphylococcus aureus is highly prone to drug resistance, especially the large number of methicillin-resistant strains ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/10C12Q1/14G01N33/569
CPCY02A50/30
Inventor 周莉张立攀王法云朱海华徐钟关炳峰平洋张亚勋任钊洪慧杰谭静李栋
Owner HENAN BUSINESS SCI RES INST
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