Rapid detection method of enterobacter sakazakii in dairy products

A technology for the detection of Enterobacter sakazakii, which is applied in the field of rapid detection of Enterobacter sakazakii in dairy products, can solve the problems of increased production costs for manufacturers, and achieve the effects of saving time and cost and avoiding false positive interference

Pending Publication Date: 2020-07-10
西安市食品药品检验所 +1
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  • Abstract
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Problems solved by technology

At present, the method used by the dairy factory is still the direct bacterial culture method, and it takes 4-5 days to issue a report, resulting in a large number of products having to be placed in the warehouse for a few days before they can be sold out of the factory, resulting in an increase in the production cost of the manufacturer

Method used

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Embodiment Construction

[0018] The technical solutions of the present invention will be described in detail below through specific examples, but the scope of the present invention is not limited by these examples.

[0019] The invention provides a rapid detection method for Enterobacter sakazakii in dairy products, the steps of which are as follows:

[0020] (1) First extract the dairy sample, and extract the RNA with more dairy samples;

[0021] (2) In a 0.5ml microcentrifuge tube, add 1-5μg of RNA in step 1, supplemented with DEPCH 2 O to make the total volume up to 11ul, add 10μM Oligo(dt) 12-18ul to the tube, then shake evenly for centrifugation;

[0022] (3) After heating at 70°C for 10 minutes, immediately insert the microcentrifuge tube in step 2 into an ice bath for 1 minute;

[0023] (4) Take a 0.5ml PCR tube and add the following reagents in sequence: first strand cDNA 2ul; upstream primer (10pM) 2ul; downstream primer (10pM) 2ul; dNTP (2mM) 4ul; 10×PCRbuffer 5ul; Taq enzyme (2u / ul) 1ul,...

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Abstract

The invention discloses a rapid detection method of enterobacter sakazakii in dairy products. The rapid detection method comprises the following steps: firstly, extracting dairy product samples and extracting RNA (ribonucleic acid) of more dairy product samples; adding 1-5 micrograms of the RNA in step 1 into a 0.5ml microcentrifuge tube and supplementing DEPCH2O to enable the total volume to reach 11 mu l; adding 10 microns Oligo (dt)12-18 microliters into the tube; then uniformly shaking and centrifuging; after heating at 70 DEG C for 10 min, immediately inserting the microcentrifuge tube instep 2 into an ice bath for 1 min. According to the rapid detection method of the enterobacter sakazakii in the dairy products, disclosed by the invention, mRNA (messenger ribonucleic acid) is used as a target gene for detection and can be detected through methods including PCR (polymerase chain reaction) and the like; the mRNA can be rapidly degraded after bacteria are dead, so that the proper mRNA is selected as a target gene and only the bacteria with activity can be detected, and false positive interference caused by dead bacteria is avoided; and meanwhile, a PCR primer is improved and designed and a real-time fluorescent RT-PCR technology is applied, so that combined detection of various bacteria can be realized and the time and the cost are saved.

Description

technical field [0001] The invention relates to a dairy product detection method, in particular to a rapid detection method for Enterobacter sakazakii in dairy products, and belongs to the technical field of dairy product detection. Background technique [0002] In recent years, with the development of PCR technology, PCR detection methods for a certain type or type of pathogenic bacteria have been established and widely used. In the field of food safety testing, the detection time of pathogenic bacteria by PCR based on DNA amplification takes about two days, and if the test result is positive, it cannot indicate whether the detected pathogenic bacteria are dead or alive, and need to be further sent to the laboratory The inspection report can only be issued after the bacterial culture is carried out in accordance with the national standard method. At present, the method used by the dairy factory is still the direct bacterial culture method, and it takes 4-5 days to issue a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10
CPCC12Q1/686C12Q1/689C12Q2600/158C12Q2563/107C12Q2527/149C12Q2527/125Y02A50/30
Inventor 高安成胡军杨晓东李宏铎李亚楠沙淼
Owner 西安市食品药品检验所
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