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Method for detecting bacillus cereus in raw milk

A technology of Bacillus cereus and raw milk, applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of low sensitivity, easy pollution, time-consuming and labor-intensive, etc. Simple operation effect

Inactive Publication Date: 2012-05-16
张文龙
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  • Abstract
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  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the problems that the traditional Bacillus cereus detection method is time-consuming, labor-intensive, low-sensitivity and the existing genetic detection technology must carry out pre-enrichment, which cannot quickly detect Bacillus cereus in raw milk and is easy to contaminate. Provided is a method for rapid detection of Bacillus cereus in raw milk

Method used

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specific Embodiment approach 1

[0010] Specific embodiment one: the method for detecting Bacillus cereus in raw milk in this embodiment is realized according to the following steps: one, the raw milk to be detected is centrifuged to skim; two, the skim milk and EDTA-Na solution are mixed at 5-7 Add EDTA-Na solution with a volume concentration of 60% to the skim milk at a ratio of 1, and bathe in water for 20-30 minutes at a temperature of 37°C; 3. Sterile filter the sample after the water bath; 4. Elute and filter with normal saline For the bacteria on the membrane, centrifuge twice, discard the supernatant, and collect the bacteria; 5. Use the bacterial DNA extraction kit to extract the genomic DNA of the bacteria as a template, and perform Real Time RT-PCR detection. According to the amplification curve and melting curve Analyze; get the detection result; wherein the 20ul reaction system in step 5 is composed of 10ul of 2×SYBR buffer, 2.0ul of DNA template, 1.0ul of primers and 7.0ul of sterilized double di...

specific Embodiment approach 2

[0013] Embodiment 2: This embodiment is different from Embodiment 1 in that the centrifugation in step 1 is performed at 3000 g for 20 minutes. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0014] Specific embodiment three: the difference between this embodiment and specific embodiment one is that in step two, skim milk and EDTA-Na solution are added to the skim milk with a volume concentration of 60% EDTA-Na solution in a ratio of 6:1. Other steps and parameters are the same as those in Embodiment 1.

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Abstract

The invention relates to a method for detecting bacillus cereus in raw milk. The invention solves the problems of time consuming and low sensitivity of the traditional bacillus cereus detection method and the problems that bacterium pre-culturing is needed so that bacillus cereus in raw milk can not be quickly detected and pollution is easy to occur in the traditional gene detection technology. The detection method comprises the following steps of: (1) degreasing the raw milk to be detected; (2) adding an EDTA-Na (Ethylene Diamine Tetraacetic Acid Sodium) solution for water bath; (3) filtering the degreased milk after water bath; (4) collecting the eluent; and (5) extracting the DNA of thalli, and carrying out Real-Time RT-PRC (Polymerase Chain Reaction) detection. The method provided by the invention has the advantages of simple operation, no need of bacterium pre-culturing and quickness. Through the method, the thalli in the raw milk can be quickly filtered. In the method, a primer is designed for a pathogenic gene of bacillus cereus, and the method has strong pertinence and good specificity.

Description

technical field [0001] The invention relates to a method for detecting bacteria in raw milk. Background technique [0002] Bacillus cereus is a Gram-positive bacterium that can form spores, so it widely exists in nature, and some of these strains can easily contaminate food and cause food poisoning, especially in foods rich in protein and carbohydrates. In food, such as meat, rice, especially raw milk and dairy products. [0003] Raw milk, as a kind of raw and auxiliary material widely eaten and processed by many dairy products, is one of the very rational nutritional foods for human beings. With the gradual improvement of people's living standards, people's demand for milk and dairy products is also increasing at a relatively high speed. However, due to the pollution of raw milk by Bacillus cereus, food poisoning incidents of crowds occur frequently. Therefore, in order to improve food safety, it is imperative to detect pathogenic Bacillus cereus in raw milk and dairy pro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张文龙
Owner 张文龙
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