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A food-borne pathogen and detection kit technology is applied in the fields of biotechnology and pathogen detection, which can solve the problems of inflexible detection methods and inconvenient operation.
Inactive Publication Date: 2018-06-29
HANGZHOU ZHUNXIN BIOTECHNOLOGY CO LTD
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[0015] The technical problem to be solved by the present invention is to provide a detection kit for 16 kinds of food-borne p
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Embodiment 1
[0109] Example 1 Optimization of amplification system, taking Salmonella as an example
[0110] 1. Primer concentration optimization
[0111] In the PCR system, too high a primer concentration may cause mismatches and lead to non-specific amplification, while too low a concentration will affect the generation of PCR products, so it is necessary to optimize the primer concentration. In the experiment, we set 3 different primer concentrations, using plasmid bacteria (1×10 5 CFU / ml and 1×10 3 CFU / ml) and negative control are used as the test samples, the template dosage is 5μl, and the final volume of each reaction system is 25μl, to detect the amplification difference of different concentrations. When the primer concentration is low, the amplification efficiency is slightly worse, and the fluorescence response intensity is also low. When the amount of invA gene primer (10μmol / L) is 0.5μL or more, there is no significant difference in amplification efficiency. Considering comprehens...
Embodiment 2
[0123] Example 2 Operational specification for detection of food-borne pathogens
[0124] 1. Sample preparation in the sample processing area
[0125] 1.1 Food specimen: select 1g of suspicious food, place it in a centrifuge tube containing 1ml of saline, invert and mix 3 times, let it stand for 2 minutes, transfer the supernatant to another centrifuge tube, and centrifuge at 12,000 rpm for 2 minutes. The supernatant is removed and the pellet is used for sample nucleic acid extraction.
[0126] 1.2 Food culture specimens: The food samples are enriched in broth medium for 6 hours, 1ml of enrichment liquid is taken, centrifuged at 12,000 rpm for 2 minutes, the supernatant is discarded, and the precipitate is used for sample nucleic acid extraction.
[0127] 1.3 Stool specimen: Pick about 0.2g of feces (take the mucus, pus and blood part as much as possible) and place it in a centrifuge tube containing 0.5ml of normal saline, shake and mix well, and centrifuge at 12,000rpm for 2 minutes....
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Abstract
The invention discloses a detection kit of 16 food born pathogenic bacteria. The detection kit comprises a PCR amplification reagent and reference reagents; the PCR amplification reagent is composed of PCR buffer solution A, enzyme system B, and a primer probe mixed solution of the 16 food-borne pathogenic bacteria; the reference reagents are mainly composed of a negative control and a positive control; the PCR buffer solution A contains 5*Buffer, 25mM dN(U)TPs; the enzyme system B is mainly composed of HotStart Hi Taq DNA polymerase and UNG anti-pollution enzyme; the primer probe mixed solution of the 16 food-borne pathogenic bacteria contains a mixed solution of detection primer probes of salmonella, Shigella, enteropathogenic Escherichia coli, enterotoxigenic E. coli, enteroinvasive escherichia coli, adherent escherichia coli, Escherichia coli O157, Staphylococcus aureus, listeria monocytogenes, Bacillus Cereus, campylobacter jejuni, vibrio parahaemolyticus, Vibrio cholerae group O1, Vibrio cholerae group O139, Yersinia enterocolitica, and enterobacter sakazakii. The detection kit is high in detection sensitivity, is simple, and is convenient.
Description
Technical field [0001] The invention relates to the field of biotechnology and pathogen detection, in particular to a detection kit for 16 kinds of food-borne pathogens. Background technique [0002] Food is food to safety first. But in recent years, food safety incidents have occurred frequently, posing a greater threat to people's health. The problem of food microbial contamination has become increasingly prominent and has become an important public health problem worldwide. According to the statistics of the Bureau of Economic Research of the United States Department of Agriculture, Salmonella, pathogenic Escherichia coli (EPEC, ETEC, EIEC, EAEC, EHEC), Campylobacter, Vibrio parahaemolyticus, Staphylococcus and toxins, pathogenic wax Several important types of bacteria such as Bacillus, Proteus, Listeria, Shigella, etc. will cause huge economic losses. [0003] Salmonella is an important type of Gram-negative bacteria in the Enterobacteriaceae. It is very easy to contaminate ...
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