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Method for detecting staphylococcus aureus live bacteria in milk

A staphylococcus and aureus technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of overestimation of the risk of pathogenic bacteria, inability to effectively distinguish dead bacteria from live bacteria, etc., to avoid mistakes Evaluate and detect effects with high sensitivity

Inactive Publication Date: 2012-01-11
黑龙江省乳品工业技术开发中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the problem that the existing LAMP detection method cannot effectively distinguish between dead bacteria and live bacteria, resulting in an overestimation of the risk of pathogenic bacteria, and provides a method for detecting live Staphylococcus aureus in milk

Method used

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  • Method for detecting staphylococcus aureus live bacteria in milk
  • Method for detecting staphylococcus aureus live bacteria in milk
  • Method for detecting staphylococcus aureus live bacteria in milk

Examples

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specific Embodiment approach 1

[0010] Specific embodiment 1: the method for detecting Staphylococcus aureus viable bacteria in milk according to this embodiment is carried out according to the following steps: 1. Pretreatment of the tested milk sample: add PMA to 1 mL of the tested milk so that the concentration of PMA is 3 μg / mL, then placed on ice in a dark place for 5-30min, then placed 15-20cm away from a 650W halogen lamp, and exposed for 3-5min; 3. Use the extract as a template to carry out LAMP amplification to obtain the LAMP amplification product; 4. Identification: Take 5 μL of the LAMP amplification product, spot it on an agarose gel with a mass concentration of 2%, and use a voltage of 100V at 1 Electrophoresis in ×TAE electrophoresis buffer for 30 minutes, and then imaged in a gel imaging system, the detection is completed.

specific Embodiment approach 2

[0011] Embodiment 2: This embodiment differs from Embodiment 1 in that: in step 1, place it on ice in a dark place for 5 minutes. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0012] Embodiment 3: This embodiment is different from Embodiment 1 in that: in step 1, place it on ice in a dark place for 30 minutes. Others are the same as in the first embodiment.

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Abstract

The invention relates to a method for detecting staphylococcus aureus live bacteria in milk. The invention aims to solve the problem of overestimation of pathogenic bacteria risk due to the ineffective distinction between dead bacteria and live bacteria by present LAMP detection methods. The method provided by the invention comprises the following steps of: 1, pretreating a tested milk sample; 2, extracting DNA of the tested milk sample treated by Step 1 to obtain an extract; 3, performing LAMP amplification by using the extract as a template; 4, loading a LAMP amplification product into agarose gel for electrophoresis and imaging in a gel imaging analysis system to finish the detection. The method provided by the invention has high detection sensitivity with the minimal measminimal detectable limit of staphylococcus aureus being 100fg, and can be widely applied in the field of foodborne pathogens detection.

Description

technical field [0001] The invention relates to a method for detecting viable staphylococcus aureus in milk. Background technique [0002] Loop-mediated isothermal amplification technology (LAMP) is a simple, rapid, specific and economical new nucleic acid amplification technology developed by Japan's Eiken Co., Ltd. in 2000. The method is characterized by six regions designed for the target gene Four specific primers can be completed by using a strand-displacing DNA polymerase to react under constant temperature conditions for tens of minutes. With the characteristics of high specificity and isothermal amplification, the target gene can be amplified 10 times within 1 hour 9 ~10 10 times. During DNA extension synthesis, pyrophosphate ions precipitated from deoxynucleoside triphosphate substrates (dNTPs) combine with magnesium ions in the reaction solution to produce a white precipitate of magnesium pyrophosphate derivatives. It can be judged whether to expand or not. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14
Inventor 姜毓君卢雁刘春波胡惠秩满朝新蒋亚男刘颖杨士芹郎友董鑫悦
Owner 黑龙江省乳品工业技术开发中心
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