A rapid and simultaneous method for the detection of Escherichia coli O157:H7 and Salmonella typhimurium in food

A technology of Escherichia coli and O157, which is applied in the field of microbial detection, can solve problems such as artificial pollution, inability to take samples, and long detection time, and achieve strong anti-interference ability, shorten detection time, and improve efficiency

Active Publication Date: 2022-04-26
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method needs to extract pathogenic bacteria DNA from food. During the extraction process of bacterial DNA, gene fragments will be broken and some genes will be lost. Not only the detection time is long and the efficiency is low, but also for the detection of low-concentration pathogenic bacteria, It will affect the accuracy of detection results; in addition, this method needs to artificially contaminate food samples with high-concentration pure cultures of pathogenic bacteria, and then extract DNA after artificial enrichment steps, and cannot directly sample low-concentration contaminated samples

Method used

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  • A rapid and simultaneous method for the detection of Escherichia coli O157:H7 and Salmonella typhimurium in food
  • A rapid and simultaneous method for the detection of Escherichia coli O157:H7 and Salmonella typhimurium in food
  • A rapid and simultaneous method for the detection of Escherichia coli O157:H7 and Salmonella typhimurium in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] In this example, the detection sensitivities of qPCR and ddPCR to detect Escherichia coli O157:H7 and Salmonella typhimurium in the bacterial suspension, and the detection accuracy under different background bacteria conditions were compared.

[0045] ddPCR reaction system and conditions: ddPCR amplification system is prepared as follows:

[0046]

[0047]In order to avoid air bubbles during droplet transfer, 22 μL of reaction system was added to each sample well. The prepared reaction system was transferred to the micro-droplet generation card, and 70 μL of special oil for micro-droplet generation was added, and the sample in the micro-droplet generation card was generated into micro-droplets in the micro-droplet generator. Take 40 μL of the generated sample droplet to a 96-well plate and seal the film. Amplify in a PCR machine. The amplification program was: pre-denaturation for 10 min at 95°C; denaturation at 95°C for 20 s, annealing at 60°C for 25 s, extension ...

Embodiment 2

[0058] In this example, to determine the detection limit of Escherichia coli O157:H7 and Salmonella typhimurium in apple juice, 100 μL of pathogenic bacteria with different concentration gradients were taken and added to 900 μL NFC apple juice for artificial contamination, so that the pathogenic bacteria in apple juice The final concentration gradient was 10 6 cfu / mL~10 2 cfu / mL, the apple juice was centrifuged (12000rpm, 15min), the supernatant was discarded, resuspended with 1mL LPW solution, and 2μL was taken as a DNA template for ddPCR detection as described in Example 1.

[0059] Figure 4 In the middle, (a) ddPCR detects E.coli O157:H7 in apple juice alone, (b) Salmonellatyphimurium; in the figure, the black column is the plate count, and the gray column is the estimated concentration of E.coli O157:H7 copy number by ddPCR, oblique Striped columns are ddPCR calculated concentration of Salmonella typhimurium copy number. As a result, the minimum detection limit of E.co...

Embodiment 3

[0062] This example is to determine the detection limit of Escherichia coli O157:H7 and Salmonella typhimurium in pasteurized fresh milk: take 100 μL of pathogenic bacteria with different concentration gradients and add them to 900 μL of milk for artificial contamination, so that the pathogenic bacteria in milk The final concentration gradient of 10 6 cfu / mL~10 2 cfu / mL, centrifuge the milk (12000rpm, 15min), discard the upper fat layer, wipe the remaining protein on the tube wall with a sterile cotton swab, and wash the tube wall with 1mL LPW solution for 2-3 times, wait until there is no protein residue on the tube wall, and re- Resuspend with 1m LPW solution, take 2 μL as DNA template for ddPCR detection as shown in Example 1.

[0063] Figure 4 In the middle, (c) ddPCR alone detects E.coli O157:H7 and (d) Salmonellatyphimurium in fresh milk; in the figure, the black column is the plate count, and the gray column is the estimated concentration of E.coli O157:H7 copy numbe...

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Abstract

The invention provides a method for rapid and simultaneous detection of Escherichia coli O157:H7 and Salmonella typhimurium in food. The food sample to be detected is directly amplified as a template, and ddPCR is used to simultaneously detect Escherichia coli O157:H7 and Salmonella typhimurium in food. Salmonella typhi; the conversion formula for the bacterial liquid concentration in the food sample to be tested is cfu / mL=copies / μL×20μL / sample volume×1000μL. Compared with the prior art, the method of the present invention omits the DNA extraction step, and directly uses bacteria as a template to carry out ddPCR detection, which improves the detection efficiency of pathogenic bacteria in food; Non-extracted DNA can also have strong anti-interference ability and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, in particular to a method for rapid and simultaneous detection of Escherichia coli (E.coli) O157:H7 and Salmonella typhimurium in food. Background technique [0002] Droplet digital polymerase chain reaction (ddPCR), also known as "third-generation PCR technology", is a new detection method that can qualitatively and quantitatively target nucleic acids. ddPCR technology can be divided into three steps: droplet generation, droplet PCR amplification, and droplet fluorescence signal interpretation. First, dilute the sample reaction system with fluorescent labels, and generate tens of thousands of tiny water-in-oil droplets through the droplet generation card. Each droplet contains at least one nucleic acid molecule to be detected, or there is no nucleic acid molecule to be detected. ; During the PCR reaction process, each droplet can be regarded as an independent reaction system for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/10C12Q1/06C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2563/159C12Q2537/143C12Q2563/107Y02A50/30
Inventor 李会孔嘉琪赵靓廖小军陈爱亮
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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