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Method for simultaneously detecting various pathogenic bacteria in raw milk and detection kit

A technology of pathogenic bacteria and raw milk, applied in the field of detection of bacteria in raw milk, can solve the problems of inability to detect multiple pathogenic bacteria at the same time, unsuitable detection of milk, and long time consumption, so as to save time for bacterial enrichment and save Steps and time, the effect of simple operation

Active Publication Date: 2009-03-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problems that the existing methods for detecting pathogenic bacteria in raw milk cannot detect multiple pathogenic bacteria at the same time, takes a long time, is expensive or is not suitable for detecting cow milk, and provides a method for simultaneously detecting pathogenic bacteria in raw milk. Method and detection kit for various pathogenic bacteria

Method used

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  • Method for simultaneously detecting various pathogenic bacteria in raw milk and detection kit
  • Method for simultaneously detecting various pathogenic bacteria in raw milk and detection kit
  • Method for simultaneously detecting various pathogenic bacteria in raw milk and detection kit

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specific Embodiment approach 1

[0013] Specific embodiment 1: In this embodiment 1, 200 mL of raw milk to be tested is centrifuged with a centrifugal force of 3000 g for 15 min to remove the fat in the upper layer to obtain a fat-free sample, and 30 mL of EDTA- 2Na solution, incubate in a water bath at 37°C for 30min; 2. Filter the sample after step 1 incubating through a Chua filter, cut the filter membrane into pieces, put it into a dry and sterile flask, and add 20mL 3. Collect the eluate and centrifuge at a centrifugal force of 10,000g for 5 minutes, remove the lower layer of bacterial sediment and resuspend it with 1mL of sterile double-distilled aqueous solution, and then use a centrifugal force of 10,000g Centrifuge for 5 minutes, discard the supernatant, and obtain the thallus; 4. Utilize the bacterial genome kit to extract bacterial genomic DNA as a multiplex PCR template, and finally perform multiplex PCR, and then identify the PCR amplification products with 2% gel electrophoresis, and use Gel ima...

specific Embodiment approach 2

[0015] Specific embodiment 2: In the kit for simultaneously detecting multiple pathogenic bacteria in this embodiment, 1 part of Taq enzyme solution with a concentration of 2.5U / μL, 20 parts of multiplex PCR reaction solution, and 1 part of negative The control solution and 1 positive control solution consist of 5 μL of 10×PCR buffer, 3 μL of 10 -2 mol / L dNTPs, 8 μL primer solution and 28 μL sterilized double distilled water, the primer solution contains 2.5×10 Listeria monocytogenes primers -11 mol, Salmonella primer 1.25×10 -11 mol, Staphylococcus aureus primer 2.5×10 -11 mol, Escherichia coli O157: H7 primer 1.25×10 -11 mol and Bacillus cereus primers 2.5 x 10 -11 mol; the positive control solution is the genomic DNA extracted from the culture solution of five standard strains of pathogenic bacteria; the negative control solution is sterilized double distilled water.

[0016] The kit of this embodiment is used for the multiplex PCR detection in step 4 of the specific em...

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Abstract

The invention discloses a method for synchronously detecting various pathogens in raw milk and a detection reagent kit, and relates to a method for detecting bacteria in the raw milk and a detection reagent kit. The invention solves the problems that the prior method for detecting the pathogens in the raw milk fails to synchronously detect the pathogens, has long time and high cost, or is not suitable to detect cow milk. The method for synchronously detecting the various pathogens in the raw milk comprises the following steps: 1. removing the fat in the raw milk to be detected; 2. filtering a sample which is subjected to thermal insulation in step 1; 3. collecting an eluent; and 4. performing the multiplex PCR, and analyzing a gel imaging system to obtain a detection result. The reagent kit consists of a Taq enzyme solution, a multiplex PCR reaction liquid, a negative control solution, and a positive control solution. The method has the advantages of short time, low cost, adaptability of detecting the cow milk and accurate detection results. The reagent kit is convenient to use.

Description

technical field [0001] The invention relates to a method for detecting bacteria in raw milk and a detection kit. Background technique [0002] Milk, as an important animal-derived food species that is widely eaten and is the raw material of countless processed foods, is one of the most ideal nutritional foods for human beings. People's demand for milk and dairy products is also increasing year by year. However, milk is also one of the foods that are most susceptible to contamination by germs and spoilage. The main pathogenic bacteria in dairy products that cause foodborne disease outbreaks in large-scale populations include Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella, Brucella, Shigella, and Bacillus cereus Wait. In 1994, an outbreak of ice cream contamination caused by Salmonella in the United States sickened about 22,400 people. In 2000, a dairy product poisoning event caused by Staphylococcus aureus broke out in Osaka City, Japan, with 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 姜毓君吕琦赵凤刘伟韩希妍毕宇涵王明娜张光辉相丽闫冰
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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