Method for detecting food-borne pathogenic bacteria in microecological active bacterial preparation through multiplex PCR

A technology of food-borne pathogenic bacteria and live bacteria preparations, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, which can solve problems such as cumbersome procedures, complicated detection methods, and reduced accuracy

Inactive Publication Date: 2017-07-04
JILIN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Due to the cumbersome procedures and time-consuming of these traditional detection methods
However, the micro-ecological living bacteria preparation itself contains

Method used

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  • Method for detecting food-borne pathogenic bacteria in microecological active bacterial preparation through multiplex PCR
  • Method for detecting food-borne pathogenic bacteria in microecological active bacterial preparation through multiplex PCR
  • Method for detecting food-borne pathogenic bacteria in microecological active bacterial preparation through multiplex PCR

Examples

Experimental program
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Embodiment 1

[0052] 1) Rehydration of a solid probiotic preparation: accurately weigh 0.05g of solid probiotic preparation (including solid particles, bacterial powder, etc.), add 0.95 mL sterile PBS buffer to the EP tube, and shake at 37°C The bed was slowly shaken at 100 rpm for 30 minutes.

[0053] 2) Genome extraction: Take out 1 ml of the fermentation solution prepared in step (1), centrifuge at 12,000 rpm for 1 minute, and discard the supernatant. First resuspend the bacteria with 500μL 70% ethanol, ice bath for 20min, add lysozyme to resuspend, incubate at 37℃ for 60min, add protease, incubate at 55℃ for 15min, add RnaseA and let stand for 2min, add 400μL of Binding Buffer to mix, add to centrifuge In the column, discard the flow-through, wash twice with Wash Buffer and Clean Buffer, and elute DNA with Elution Buffer. The extracted genome was detected by electrophoresis using 1% agarose gel.

[0054] 3) Primer synthesis: The designed primer pairs of various common pathogenic bacteria a...

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Abstract

The invention provides a method for detecting food-borne pathogenic bacteria in a microecological active bacterial preparation through multiplex PCR. For the method provided by the invention, the multiplex PCR technology is mainly adopted, and the method adopts the PCR reaction in which multiple specific primers are added in the same reaction system, and multiple nucleic acid fragments are simultaneously amplified. According to the method, the specific primers of seven common food-borne pathogenic bacteria including salmonella, escherichia coli, bacillus pumilus, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and aspergillus niger are designed, seven target bands are simultaneously amplified in the same system, meanwhile, the specificity among each other is good, the limit of detection of the method achieves 200fg/[mu]l (template concentration), the corresponding concentration of bacterium is 120CFU/mL, the standard of the food safety can be achieved, the common pathogenic bacteria can cause the pathogenic effect when the concentration is 1000CFU/mL, therefore, the method is high in sensitivity, and is rapid, simple and convenient, and the cost is very low.

Description

[0001] 1 Technical field [0002] The invention relates to the field of food safety detection, in particular to a method for detecting whether food-borne pathogenic bacteria in a microecological live bacteria preparation exceeds the standard by multiple PCR technology. [0003] 2 Background technology [0004] Food-borne diseases refer to diseases caused by pathogenic factors such as toxic and harmful substances (including biological pathogens) that enter the human body through ingestion. Generally can be divided into infectious and toxic, including common food poisoning, intestinal infectious diseases, zoonotic diseases, parasitic diseases and diseases caused by chemical toxic and harmful substances. Food-borne pathogens can cause food-borne diseases or food poisoning, and even affect human safety, cause major economic and property losses, and reduce people's quality of life. [0005] According to the World Health Organization (WHO) report, there are up to 150 million cases of diarrh...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12Q1/10C12Q1/14C12R1/19C12R1/42C12R1/445C12R1/125C12R1/385C12R1/07C12R1/685
CPCC12Q1/686C12Q1/689C12Q1/6895C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 房学迅李庆超于大海杨吉雨
Owner JILIN UNIV
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