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Method for detecting salmonella and staphylococcus aureus in food simultaneously

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: 2015-09-09
HENAN BUSINESS SCI RES INST
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  • 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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  • Method for detecting salmonella and staphylococcus aureus in food simultaneously
  • Method for detecting salmonella and staphylococcus aureus in food simultaneously
  • Method for detecting salmonella and staphylococcus aureus in food simultaneously

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 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 in a 10-fold gradient, and 10 -4 —10 -9 and 10 -4 —10 -10 Dilution for immunomagnetic bead sensitivity test. Divide the dilutions of each gradient of the two strain culture solutions into the test group and the control group. The control group added EDTA solution with a mass concentration of 65%. The volume ratio of the dilution solution to the EDTA solution was 6:1, and the test group added The mass concentration is 50% aminotriacetic acid solution, the volume ratio of diluent and aminotriacetic acid solution is 6:1, and the test steps are as follows:

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

Embodiment 2

[0037] A method for simultaneously detecting salmonella and staphylococcus aureus in milk, comprising the following steps:

[0038] 1) Take 25mL of the milk sample to be tested, add 225mL of PBS buffer, shake for 30s, adjust the pH to 7 with 1mol / mL sterile NaOH solution to obtain a homogeneous sample; add 50.0ml of 50 wt % aminotriacetic acid to the homogenous sample Solution, after oscillating by ultrasonic wave at 37°C for 5 minutes (ultrasonic frequency 20KHz), washed with 0.01 mol / LPBS buffer solution, centrifuged at 8000r / min for 5 minutes, separated into layers, and discarded the upper layer;

[0039] 2) Take 100 μL of the sample treated in step 1) and add 200 μL of immunomagnetic beads (magnetic beads obtained by coupling streptavidin-modified magnetic beads with biotin-labeled Salmonella and Staphylococcus aureus antibodies) to mix and shake at 37°C After 20 minutes, let stand on the magnetic stand for 2 minutes to separate layers, discard the upper layer, wash the lo...

Embodiment 3

[0042] A method for simultaneously detecting salmonella and staphylococcus aureus in eggs, comprising the following steps:

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

[0044] 2) Take 100 μL of the sample treated in step 1) and add 200 μL of immunomagnetic beads (magnetic beads obtained by coupling streptavidin-modified magnetic beads with biotin-labeled Salmonella and Staphylococcus aureus antibodies), mix and ...

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Abstract

The invention discloses a method for detecting salmonella and staphylococcus aureus in food simultaneously. The method includes the following steps: 1), diluting a to-be-tested food sample according to GB4789 to obtain sample homogenate of 1:10, adding 50-65wt% of nitrilotriacetic acid solution into the sample homogenate according to the volume ratio of the sample homogenate to the nitrilotriacetic acid solution being (5-7):1, vibrating the mixture, adding phosphate buffer solution or 0.05% tween-20 contained phosphate buffer solution, performing centrifugal layering, and discarding an upper layer; 2), mixing a sample obtained after processing of the step 1) with immunomagnetic beads, vibrating the mixture, subjecting the mixture to standing and layering on a magnetic frame, discarding an upper layer, and washing a lower layer of the sample, wherein the immunomagnetic beads are obtained by modifying salmonella and staphylococcus aureus antibodies, which are labeled by magnetic bead coupled biotin, by streptavidin; 3), adding the phosphate buffer solution to the washed sample, vibrating the mixture, applying the mixture to an LB plate, and performing cultivation and observation. The method is simple and convenient to operate, accurate, efficient and low in cost, two target bacteria are enriched, and detection time can be shorten from 72h to 25h.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular 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, and it can cause a series of diseases after livestock and poultry infection. Eating infected food can cause food poisoning. In my country, 70% to 80% of bacterial food poisoning incidents are caused by Salmonella. About 90% of the foods poisoned by Salmonella are meat, eggs, milk and other livestock products. [0003] Staphylococcus aureus can cause multi-tissue infections, and can spread widely among people to cause a variety of diseases, sometimes even life-threatening, such as bacteremia, endocarditis and pneumonia. Moreover, Staphylococcus aureus is very prone to drug resistance, especially the emergence of a large number of methicillin-resistant strains, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/10C12Q1/14G01N33/569
CPCY02A50/30
Inventor 王永王法云周莉贾广乐朱海华关炳峰任钊平洋智军丽张亚勋谭静李栋
Owner HENAN BUSINESS SCI RES INST
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