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Method for detecting pathogenic bacteria in foods by isothermal rapid amplification

A pathogenic bacteria and food technology, applied in the field of food safety testing, can solve the problems of limited application range, difficulty in basic level, high testing cost, etc., and achieve the effect of simple sample pretreatment, short time, simple principle and operation

Active Publication Date: 2017-09-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The relevant PCR method detects Staphylococcus aureus at the molecular level, which has high specificity and sensitivity, especially the real-time fluorescent quantitative PCR method, which can detect the reaction process of the system in real time and avoid the contamination of carcinogens such as EB during the experiment , so it has certain advantages in the detection method, but the reaction process requires expensive and complicated instruments, the detection cost is high, it is difficult to achieve basic level, and the scope of application is limited
[0005] In addition, there is currently a lack of methods that can simultaneously and rapidly quantitatively detect multiple food-borne pathogens such as Staphylococcus aureus, Salmonella, enterohemorrhagic Escherichia coli, Shigella, etc.

Method used

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  • Method for detecting pathogenic bacteria in foods by isothermal rapid amplification
  • Method for detecting pathogenic bacteria in foods by isothermal rapid amplification
  • Method for detecting pathogenic bacteria in foods by isothermal rapid amplification

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: CSDPR amplification principle

[0043] Such as figure 1 Shown is a schematic diagram of the principle of CSDPR amplification.

[0044] The CSDPR reaction opens the stem structure of the molecular beacon probe through the hybridization of the target nucleic acid fragment and the molecular beacon loop sequence, and forms the molecular beacon-cDNA structure under the action of the primer and DNA polymerase, and retains the opened molecular beacon probe. Needle signal, the release of target nucleic acid fragments during cDNA formation triggers the next cycle, and signal amplification is achieved through the amplification of non-target nucleic acids.

Embodiment 2

[0045] Embodiment 2: the design of probe and primer

[0046] (1) Design of the pathogenic bacteria probe loop sequence in the molecular beacon probe

[0047] (1) Design of the loop sequence of the molecular beacon probe of Staphylococcus aureus

[0048] Search for Staphylococcus aureus ATCC6538, Staphylococcus epidermidis, Staphylococcus caprae, Staphylococcus capitis, Staphylococcus pasteuri by keywords in NCBI's nucleic acid database The 16S rDNA sequence of the above 5 sequences was compared using DNAMAN, and the fragments in the 16S rDNA sequence of Staphylococcus aureus that differed from the other four sequences by more than 3 nucleotides were selected (5'-GGA CGA GAA GCT TGC TTC -3', ie SEQ ID NO:11) as the target sequence for Staphylococcus aureus detection. Design a Staphylococcus aureus probe loop sequence complementary to the target sequence (5'-GAA GCA AGC TTC TCG TCC-3', namely SEQ ID NO:3).

[0049] (2) Design of Escherichia coli Molecular Beacon Probe Loop Se...

Embodiment 3

[0066]Embodiment 3: the method for detecting Staphylococcus aureus based on real-time fluorescent CSDPR

[0067] (1) Activation and enrichment culture of Staphylococcus aureus

[0068] Prepare nutrient agar (1L) before activating Staphylococcus aureus: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar, and the final pH is 7.3±0.2. After autoclaving at 121°C for 15 minutes, cool to about 45°C for pouring into a plate or pouring into a sterilized plate for solidification before use. Add 0.3mL of the supporting Staphylococcus aureus resuscitation solution dropwise to the freeze-dried strains, blow and suck repeatedly with a sterile dropper until the freeze-dried strains dissolve into a bacterial suspension; The suspension was inoculated on a nutrient agar plate, and the plate was placed in a 37°C constant temperature incubator and cultured upside down for 12 hours; prepare brain-heart infusion broth (BHI) medium (1L): tryptone 10g, sodium chloride 5g,...

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Abstract

The invention discloses a method for detecting pathogenic bacteria in foods by isothermal rapid amplification and belongs to the technical field of food safety detection. The method comprises the following steps: determining detection target nucleic acid aiming at each bacterium through multiple sequences; designing a specific molecular beacon probe and a primer; taking a genome DNA (Deoxyribonucleic Acid) of each strain as a template and establishing real-time fluorescence CSDPR; carrying out the isothermal nucleic acid amplification and identifying the specificity, sensitivity and minimum limit of detection of a CSDPR method. The detection of an actual sample determines that the method established by the invention has practical feasibility. The method disclosed by the invention has commonality on detection of various pathogenic bacteria in the foods and the pathogenic bacteria comprise staphylococcus aureus, salmonella, enterohemorrhagic escherichia coli, shigella and the like; only if a specific part in the molecular beacon probe needs to be replaced, effective detection of the different pathogenic bacteria can be realized; and the method has high detection sensitivity and high specificity, simple principle and operation and the pretreatment of the sample is simple.

Description

technical field [0001] The invention relates to a method for isothermal rapid amplification detection of pathogenic bacteria in food, belonging to the technical field of food safety detection. Background technique [0002] In recent years, in the context of globalization, with the rapid growth of global food trade, the global flow of population, and the emergence of drug-resistant strains, the incidence and fatality rate of food-borne diseases have gradually increased, and have become the world's food Important issues in the fields of safety and public health. Microorganisms (including bacteria, viruses, fungi, and parasites) that spread through contaminated food or water and cause foodborne illness are called foodborne pathogenic microorganisms. In foreign countries, in the cases of foodborne diseases caused by pathogenic microorganisms, the main pathogenic factor is pathogenic bacteria (or foodborne pathogens), and the most common foodborne pathogens include enterohemorrh...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2545/114Y02A50/30
Inventor 沈晓芳陈万明庞月红
Owner JIANGNAN UNIV
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