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Kit for detecting vibrio parahaemolyticus in food and detection method

A detection kit and technology for vibrio hemolyticus, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc. Pollutant impact, aquaculture economic damage and other issues, to achieve the effect of reducing false positive and false negative rates, shortening detection time, and shortening culture time

Inactive Publication Date: 2016-02-17
中山鼎晟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vibrio parahaemolyticus is currently the most important pathogenic bacteria in bacterial food poisoning, which poses a great threat to human health and has caused huge economic damage to the aquaculture industry
[0003] The traditional identification and detection methods of Vibrio parahaemolyticus include microscopic examination, biochemical identification, halophilicity test, immunochromatography and DNA hybridization, etc., but all have their own shortcomings, such as microscopic examination, biochemical identification, halophilicity test, etc. The operation is complex, the detection time is long, and the sensitivity is low, while the immunochromatography and DNA hybridization methods require high equipment, and the detection process is easily affected by pollutants

Method used

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  • Kit for detecting vibrio parahaemolyticus in food and detection method
  • Kit for detecting vibrio parahaemolyticus in food and detection method
  • Kit for detecting vibrio parahaemolyticus in food and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0025] A fluorescent quantitative PCR detection kit for Vibrio parahaemolyticus in food, the detection kit includes a culture medium and a detection solution, and the culture medium is selective for halophilic bacteria containing 2% sodium chloride crystal purple bacterial growth liquid Agar medium, the detection solution is made of the following components:

[0026]

[0027] The surfactant is Tween 20 with a concentration of 5 mg / ml.

[0028] The alkaline solution is NaOH with a concentration of 1 mg / ml; the acid solution is HCL with a concentration of 2 mg / ml.

[0029] The DNA extraction solution is 0.5mmol / L Tris-HCl.

[0030] The BstDNA polymerase concentration is 0.35U / ul

[0031] The 10× reaction buffer is a mixture of 20ml Tris-HCl, 6ml0.2mg / mlKCl, 6ml1.8mg / mlNH4SO4, 10ml2.8mg / mlMgSO4.

[0032] The PCR primers are marked 5' by FAM, 3' by TRAMA, upstream: 5-CGGTAGTAAACACACT-GTCAG-3; downstream: 5-GTTTCAGGCTCACCATGACG-3;

[0033] A method for detecting Vibrio parah...

Embodiment 2

[0040] A fluorescent quantitative PCR detection kit for Vibrio parahaemolyticus in food, the detection kit includes a culture medium and a detection solution, and the culture medium is selective for halophilic bacteria containing 2% sodium chloride crystal purple bacterial growth liquid Agar medium, the detection solution is made of the following components:

[0041]

[0042] Described surfactant is betaine, and concentration is 10mg / ml.

[0043] The alkaline solution is KOH with a concentration of 1.8 mg / ml; the acid solution is one or more of H2SO4 with a concentration of 3 mg / ml.

[0044] The DNA extraction solution is 0.8mmol / L Tris-HCl.

[0045] The BstDNA polymerase concentration is 0.4U /

[0046] The 10× reaction buffer is a mixture of 50ml Tris-HCl, 10ml0.5mg / mlKCl, 10ml3.5mg / mlNH4SO4, 15ml5.5mg / mlMgSO4.

[0047]The PCR primers are marked 5' by FAM, 3' by TRAMA, upstream: 5-CGGTAGTAAACACACT-GTCAG-3; downstream: 5-GTTTCAGGCTCACCATGACG-3;

[0048] A method for det...

Embodiment 3

[0055] A fluorescent quantitative PCR detection kit for Vibrio parahaemolyticus in food, the detection kit includes a culture medium and a detection solution, and the culture medium is selective for halophilic bacteria containing 2% sodium chloride crystal purple bacterial growth liquid Agar medium, the detection solution is made of the following components:

[0056]

[0057] Described tensio-active agent is the mixture of Tween 20, Tween 40, betaine, and the volume ratio of described Tween 20, Tween 40, betaine is (2-5): (1-8): ( 2-6), the surfactant concentration is 8mg / ml.

[0058] Described alkali solution is KOH, and concentration is 1.5mg / ml; Described acid solution is the mixture of HCL, H2SO4, and the volume ratio of described HCL, H2SO4 is (2-5): (1-8), so Said acid solution concentration is 2.5mg / ml.

[0059] The DNA extraction solution is 0.65mmol / L Tris-HCl.

[0060] The BstDNA polymerase concentration is 0.38U / ul

[0061] The 10× reaction buffer is a mixtur...

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Abstract

The invention relates to the field of food detection, in particular to a kit for rapidly detecting vibrio parahaemolyticus in food and a detection method. The kit comprises a culture medium and detection liquid, and is characterized in that the culture medium is a halophilic bacteria selective agar culture medium containing 2% sodium chloride violet purple enrichmen, and the detection liquid is prepared from 5-10ml of surfactants, 2-5ml of alkaline solution, 2-5ml of acid solution, 5-8ml of protective solution, 20-50 microliters of DNA (deoxyribonucleic acid) extract solution, 1-5ml of 10*reaction buffer, 3-6ml of ethidium monoazide bromide, 0.35-0.4U / microliter BstDNA polymerase and 5-10 microliters of PCR (polymerase chain reaction) primers. The kit for detecting vibrio parahaemolyticus in the food is simple and convenient to operate without special equipment and instruments and short in detection time, and lower detection limit can reach 2cfu / g, so that requirements of the food safety detection departments on food safety supervision can be met.

Description

technical field [0001] The invention belongs to the field of food detection, and in particular relates to a detection kit and a detection method for Vibrio parahaemolyticus in food. Background technique [0002] Vibrio parahaemolyticus is a Gram-negative polymorphic bacillus that is widely distributed in nature, and its carrier rate is the highest in seafood such as sea snails, yellow mud snails, and crabs. In addition, eggs, fresh fruits and vegetables, and meat products are also more common. common. When people directly or indirectly eat food containing this bacteria, it can cause intestinal diseases, such as diarrhea, gastroenteritis, and other systemic diseases, such as arthritis and heart disease. Vibrio parahaemolyticus is currently the most important pathogenic bacteria in bacterial food poisoning, which poses a great threat to human health and great economic damage to the aquaculture industry. [0003] The traditional identification and detection methods of Vibrio ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12R1/63
Inventor 郭狄
Owner 中山鼎晟生物科技有限公司
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