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Kit capable of rapidly detecting staphylococcus aureus in meat and application

A staphylococcus and golden yellow technology, applied in the field of food safety, can solve the problems of difficult inhibition and low requirement of DNA template purity, achieve good non-specific amplification and ensure specificity

Inactive Publication Date: 2015-09-30
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, it is also reported that the LAMP reaction is not easily inhibited by the ingredients in the food matrix, and the purity of the DNA template is not high.

Method used

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  • Kit capable of rapidly detecting staphylococcus aureus in meat and application
  • Kit capable of rapidly detecting staphylococcus aureus in meat and application
  • Kit capable of rapidly detecting staphylococcus aureus in meat and application

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Experimental program
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Embodiment 1

[0033] 1. Experimental method

[0034] 1.1 Cultivation of different strains and extraction of DNA templates

[0035] Take Staphylococcus aureus ATCC13565 as the standard strain, 3 strains of Staphylococcus aureus (ATCC25923, CMCC26074, CMCC26075) as the reference strain and 26 other non-Staphylococcus aureus strains for specificity experiments. Staphylococcus aureus and other strains were activated and cultured in NB and compound enrichment medium BPW respectively, and cultured overnight at 37°C. Pipette 1 mL of the bacterial suspension in the logarithmic growth phase, extract bacterial DNA using a bacterial genome extraction kit, and store the obtained DNA template at -20°C for future use.

[0036] 1.2 Design and synthesis of LAMP primers

[0037] The thermostable nuclease gene (nuc) of Staphylococcus aureus was found in GenBank, which has good specificity and high conservation. Therefore, this article uses this specific gene nuc sequence to design primers. Using the onli...

Embodiment 2

[0071] Collect 10 samples of fresh pork, beef, mutton, chicken and their meat products, and use the following kits and detection methods for detection.

[0072] The kit of the present invention is composed of 25 parts of LAMP amplification reaction solution, 1 part of negative control solution and 1 part of positive control solution according to volume parts. The LAMP amplification reaction system consists of a LAMP primer set, Bst 2.0 WarmStart DNA polymerase and amplification reaction solution. In the LAMP primer set, the concentration ratio of the outer primer to the inner primer is 1:6. Each 25μL LAMP amplification reaction system consists of 0.4μM F3 and B3, 2.4μM FIP and BIP, 0.9μL 8U Bst 2.0 WarmStart DNA polymerase, 4mM MgCl2, 2mM dNTPs, 0.4M betaine, 2.5μL 10×Buffer and 2 μL of template DNA, and the rest was made up with sterilized double distilled water. The positive control solution is a genomic DNA solution extracted from the standard strain culture solution of S...

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Abstract

The invention discloses a kit capable of rapidly detecting staphylococcus aureus in meat and application and belongs to the technical field of food safety. The kit is composed of an LAMP amplified reaction system, negative control liquid and positive control liquid, and the LAMP amplified reaction system is composed of Bst 2.0WarmStart DNA polymerase and an SEQ IDNO1-4 primer group. The invention further provides the application of the kit capable of rapidly detecting the staphylococcus aureus in the food-grade fresh meat or meat products. The kit has the advantages of being sensitive, rapid, simple and convenient. The specificity is good, cold temperature condition is not needed to be created, the sensitivity is high, the sensitivity of the kit detection is 100 times the sensitivity of common PCR detection, and the kit is suitable for rapidly detecting the staphylococcus aureus in the food grade fresh meat or the meat products.

Description

technical field [0001] The invention relates to a kit for rapidly detecting Staphylococcus aureus in meat and its application, belonging to the technical field of food safety. Background technique [0002] At present, the method of detecting food-borne pathogenic bacteria based on nucleic acid amplification based on the principle of molecular biology has been developed rapidly. The detection methods for Staphylococcus aureus mainly revolve around the traditional method of combining isolation culture and biochemical identification, PCR method and fluorescent quantitative PCR method. In recent years, Bst large-fragment DNA polymerase has been used for LAMP amplification to detect Staphylococcus aureus, but it has not been detected from edible meat matrices. [0003] Biolab has recently developed Bst 2.0 WarmStart DNA polymerase, which can be used in LAMP amplification reactions. Compared with the traditional Bst large-fragment DNA polymerase, the enzyme has the advantage tha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
CPCC12Q1/6844C12Q1/689
Inventor 姜毓君满朝新赵玥明姜霞韩建春
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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