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Method for detecting staphylococcus aureus and enterotoxin genotypes thereof through multiple PCR (polymerase chain reaction)

A staphylococcus, aureus technology, applied in the detection of Staphylococcus aureus and its enterotoxin genotype in food, food-borne pathogenic microorganisms, can solve the problem of no internal control, etc., achieve good accuracy, strong specificity, The effect of high sensitivity

Active Publication Date: 2013-12-18
嘉兴实践医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is no specific multiplex PCR method for the common enterotoxin genotypes of Staphylococcus aureus, and there is no internal control

Method used

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  • Method for detecting staphylococcus aureus and enterotoxin genotypes thereof through multiple PCR (polymerase chain reaction)
  • Method for detecting staphylococcus aureus and enterotoxin genotypes thereof through multiple PCR (polymerase chain reaction)
  • Method for detecting staphylococcus aureus and enterotoxin genotypes thereof through multiple PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Sample: Raw milk sample from a dairy farm

[0044]The milk samples collected from dairy farms were tested and labeled as a, b, c, d, e, f, g, h respectively. Among them, a, b, and c are mastitis milk samples, d, e, f, and g are milk samples from randomly selected cows, and h is a comprehensive sample after milk is poured into the cooling tank.

[0045] 1. Sample processing

[0046] (1) Take 1mL raw milk, add 200uL absolute ethanol, 200uL petroleum ether (or diethyl ether) and 200uL acetone to it, mix well, and centrifuge the mixture at 12000rpm for 10min.

[0047] (2) Discard the supernatant, dissolve the remaining precipitate with 300uL TE (pH8.0), add 10uL (50mg / mL) lysozyme solution, and incubate at 37°C for 1h.

[0048] 2. DNA extraction

[0049] Use the Bacteria Genomic DNA Extraction Kit to extract the total DNA of the bacteria in the raw milk (some steps have been improved), and dissolve the extracted DNA in 84uL 1×TE buffer solution to obtain the DNA template...

Embodiment 2

[0079] Sample: a commercially available milk powder

[0080] A certain commercially available milk powder purchased was tested. Before the artificial contamination of Staphylococcus aureus, the product was sterilized and tested according to the national standard method to confirm that it does not contain Staphylococcus aureus. Artificially pollute the milk powder with the standard strains of Staphylococcus aureus producing A, B, C, and D, that is, take 25g of the sample and add it to 225mL of buffered peptone water for homogeneous mixing, and then artificially pollute the homogeneous solution. Incubate with shaking at 37°C for 12 hours.

[0081] 1. Sample processing

[0082] (1) Take 2mL of emulsion, add 200uL absolute ethanol, 200uL petroleum ether (or diethyl ether) and 200uL acetone to it, mix well, and centrifuge the mixture at 12000rpm for 10min.

[0083] (2) Discard the supernatant, dissolve the remaining precipitate with 300uL TE (pH8.0), add 10uL (50mg / mL) lysozyme s...

Embodiment 3

[0115] Sample: a certain commercially available pork

[0116] A certain commercially available pork was tested. Before the artificial contamination of Staphylococcus aureus, the product was sterilized, and tested according to the national standard method to confirm that it does not contain Staphylococcus aureus. The standard strains of Staphylococcus aureus producing A, B, C, and D were artificially contaminated into pork, that is, 25g of the sample was added to 225mL of buffered peptone water for homogeneous mixing, and then the homogeneous solution was artificially contaminated and cultured with shaking at 37°C for 12h.

[0117] 1. Sample processing

[0118] Take 10 mL of the homogenate and centrifuge at 800 rpm for 5 min to remove the pork and leave the bacterial liquid in the slurry. Draw the supernatant into another sterilized centrifuge tube and centrifuge at 10000rpm for 10min, then discard the supernatant.

[0119] 2. DNA extraction

[0120] Use the Bacteria Genomic...

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Abstract

The invention provides a method for detecting staphylococcus aureus and enterotoxin genotypes of the staphylococcus aureus by utilizing multiple PCR (polymerase chain reaction) technology. The main technical scheme is as follows: designing primer sequences and optimizing a multiple PCR system and conditions. Enterotoxins and invasive enzymes of staphylococcus aureus affect the pathogenicity of staphylococcus aureus and the enterotoxins which have been identified already are A, B, C1-C3, D, E, G, H, I, J, L, M and N. Generally speaking, food poisoning is commonly caused by A and D and less commonly caused by B and C, the occurrence rate of food poisoning related to the toxin E is the lowest, and the enterotoxins have different toxicity, wherein the toxin A has stronger toxicity and D has weaker toxicity. Aiming at the common enterotoxin genotypes of staphylococcus aureus, the invention overcomes the defects in the prior art and provides the method for detecting staphylococcus aureus and enterotoxin genotypes of the staphylococcus aureus in food by utilizing the multiple PCR technology. The method can be used for simultaneously detecting staphylococcus aureus generating enterotoxins A, B, C and D in the same reaction system.

Description

technical field [0001] The invention relates to a bacteria inspection technology, specifically a technology for detecting staphylococcus aureus and its enterotoxin genotype in food, especially food-borne pathogenic microorganisms, by using multiple PCR technology. Background technique [0002] Staphylococcus aureus ( Staphylococcus aureus ) is an important pathogenic bacteria belonging to the genus Staphylococcus ( Staphylococcus ), which can cause a variety of serious infections. Under the microscope, they are arranged in bunches of grapes, with a diameter of about 0.8um. No spores, flagella, most without capsules, Gram-positive, easy to distinguish from other bacteria. At the same time, it has low nutritional requirements and can grow under aerobic or facultative anaerobic conditions. The optimum growth temperature is 37°C and the optimum growth pH is 7.4. The pathogenicity of Staphylococcus aureus mainly depends on the toxins and invasive enzymes it produces. The ent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
Inventor 胡萍
Owner 嘉兴实践医学科技有限公司
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