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Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis

A technology for Listeria and Salmonella, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. The effect of simple result determination, reduced detection cost, and improved sensitivity

Active Publication Date: 2015-01-21
WUXI ZODOLABS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Listeria monocytogenes can still grow and multiply in an environment of 4°C, which is an important hidden danger of refrigerated food food safety
[0006] The existing methods for detecting these three pathogenic bacteria are mainly through the traditional culture method, which has the disadvantages of long time period and complicated operation, and most of them are detected separately, and the efficiency is low
At present, simultaneous detection using multiplex PCR technology has also appeared, but it is often only the simultaneous detection of two pathogenic bacteria, and when more strains are detected simultaneously, it is prone to poor specificity and low accuracy. In addition, due to the complex factors affecting multiple PCR, templates, primers, reaction systems and cycle parameters, etc. will have a great impact on the final detection results, and at the same time limit the selection of target genes, so multiple PCR in When detecting multiple strains at the same time, there are many difficulties in the selection of target genes and primer design.

Method used

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  • Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis
  • Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis
  • Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, Preparation and Purification of Polyclonal Antibody

[0044] 1.1 Preparation of bacteria for immunization

[0045] LB solid plate was streaked to activate Salmonella typhimurium and Escherichia coli O157:H7, pick a single colony and carry out shaking culture in LB liquid at 37℃; Shake culture. Collect bacteria by centrifugation at 4000g for 5 minutes; resuspend and wash the bacteria twice in PBS, and finally resuspend them in PBS and perform ultrasonic crushing; centrifuge at 13000g and 10 minutes to enrich the bacteria fragments, resuspend in PBS, wash twice, and resuspend ; Add formalin solution (37% formaldehyde) at 0.7:100, mix well, place at room temperature, and inactivate the bacteria for 24 hours; centrifuge at 13000g for 10 minutes, take the precipitate, wash it twice with PBS (to remove formaldehyde); collect by centrifugation Precipitate, store at -80°C for future use (you can weigh it first and divide it into equipment).

[0046] 1.2 Preparatio...

Embodiment 2

[0049] Embodiment 2, the preparation of immunomagnetic beads

[0050] 2.1 Accurately prepare 1mg / mL BSA solution with 0.005M borate buffer.

[0051] 2.2 Take 5 mg of magnetic beads and wash them once with ethanol (to remove the surfactant).

[0052] 2.3 Wash twice with sterile MEST (pH5.5~6.0), remove the supernatant after magnetic separation.

[0053] 2.4 Add EDC / NHSS (each 5 mg) and shake to activate at room temperature for 30 minutes.

[0054] 2.5 Remove the supernatant after magnetic separation, resuspend with sterile MEST (pH5.5~6.0), transfer to a new centrifuge tube, and wash three times.

[0055] 2.6 Take a 1.5mL centrifuge tube, add 100μL 1mg / mL purified polyclonal antibody, make up to 1mL with 0.005M borate buffer, adjust the pH to 8.5~9.0 with NaOH, quickly add to the freshly activated magnetic beads and shake at room temperature Reaction 3h.

[0056] 2.7 After magnetic separation, the supernatant was aspirated, and the magnetic beads coupled to the antibody wer...

Embodiment 3

[0057] Embodiment 3, sample pretreatment

[0058] Let us take a food sample as an example to illustrate the process of sample processing in the method of the present invention.

[0059] Weigh 1g of food sample, add 9mL of PBS, grind to make a homogenate, centrifuge at 900g for 5min, remove the enlarged food residue, and take the supernatant (this process must be performed aseptically).

[0060] Step 4. Immunomagnetic beads capture target bacteria in food samples

[0061] Take 1 mL of food sample liquid, add 2.0 mg of immunomagnetic beads, incubate with gentle shaking at 37°C for 45 min, separate with a magnetic frame, wash twice with PBST, and resuspend the separated magnetic beads in 50 μL of PBS.

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Abstract

The invention discloses a method for simultaneously and quickly detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis in food. The method comprises the following steps of: preparing nano immunomagnetic particles of the targeted typhimurium, escherichia coli O157:H7 and listeria monocytogenesis so as to capture target bacteria from a detection sample; obtaining the target bacteria by magnetic field separation; treating by adopting propylmercuric bromide azide (PMA) to eliminate the interference of killed bacteria; and finally, extracting DNA (Deoxyribose Nucleic Acid) to carry out multiple PCR (Polymerase Chain Reaction) detection. Compared with a classical bacteria separation and identifying and serological typing method, the method disclosed by the invention is quick and accurate, can be used for only detecting viable bacteria and can be used for simultaneously detecting and identifying the three pathogens.

Description

technical field [0001] The invention belongs to the field of microbial detection, and relates to a detection method, in particular to a simultaneous and rapid detection of Salmonella typhimurium (Salmonella typhimurium), Escherichia coli (EnteroheamorrhagicE.coli) O157:H7, and Listeria monocytogenes method. Background technique [0002] Salmonella typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes are pathogenic bacteria that are often found in food and drinking water, and are also the three most common sources of human infection, causing serious harm to human health . [0003] Salmonella is the most important pathogenic bacteria causing gastroenteritis in the world, which can cause 1.4 million people to suffer from illness every year. At the same time, salmonellosis is also one of the most important zoonotic diseases, which can adapt to almost any type of host. Salmonella-contaminated food and drinking water is a major source of human infection. Among them...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/04C12N15/11
CPCY02A50/30
Inventor 李林杨晓慧许恒毅徐锋
Owner WUXI ZODOLABS BIOTECH
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