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Genotype inspection method and genotyping inspection method of staphylococcal enterotoxin

A staphylococcal intestinal and detection method technology, which is applied in the field of bacterial molecular detection, can solve the problems of high AT content, difficult detection and typing of enterotoxin genes, etc., and achieve high specificity, shortened detection time, and high accuracy.

Inactive Publication Date: 2010-06-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the AT content in the staphylococcal enterotoxin gene is relatively high, and the similarity between different types of enterotoxin sequences is also high, and there is a close linkage relationship between some specific toxin genotypes. bring difficulties

Method used

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  • Genotype inspection method and genotyping inspection method of staphylococcal enterotoxin
  • Genotype inspection method and genotyping inspection method of staphylococcal enterotoxin
  • Genotype inspection method and genotyping inspection method of staphylococcal enterotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 A kind of detection method of staphylococcus enterotoxin genotype comprises the steps:

[0062] 1) Sample processing:

[0063] Take 0.5ml suspected milk sample, centrifuge at 12000r / min for 1 minute;

[0064] 2) DNA extraction:

[0065] Discard the supernatant, add 500μl sterilized water to suspend the precipitate, centrifuge at 12000r / min for 30 seconds; discard the supernatant, add 200μl reagent A and act at 37°C for 25 minutes, add reagent B, and act at 50°C for 25 minutes, 12000r / min min, centrifuge for 8 minutes; take the supernatant and add an equal volume of tris-saturated phenol, shake and mix thoroughly, 12000r / min, and centrifuge for 5 minutes; take the supernatant and add an equal volume with a volume ratio of 25:24 : 1 mixed solution of tris-saturated phenol, chloroform and isoamyl alcohol, after mixing, centrifuge at 12000r / min for 8 minutes; take the supernatant and add 2.5 times the volume of absolute ethanol pre-cooled to 4°C , mix well, ...

Embodiment 2

[0076] Embodiment 2 A kind of detection method of staphylococcus enterotoxin genotype comprises the steps:

[0077] 1) Sample processing:

[0078] Take 0.1ml sample of suspected milk sample, centrifuge at 10000r / min for 2 minutes;

[0079] 2) DNA extraction:

[0080] Discard the supernatant, add 400 μl sterilized water to suspend the precipitate, and centrifuge at 10,000 r / min for 1 minute; discard the supernatant, add 100 μl of reagent A to act at 35°C for 30 minutes, add reagent B, and act at 45°C for 30 minutes, 10,000r / min min, centrifuge for 10 minutes; take the supernatant and add an equal volume of tris-saturated phenol, shake and mix thoroughly, 10000r / min, and centrifuge for 10 minutes; take the supernatant and add an equal volume with a volume ratio of 25:24 : 1 mixed solution of tris-saturated phenol, chloroform and isoamyl alcohol, after mixing, centrifuge at 10,000r / min for 10 minutes; take the supernatant and add 2 times the volume of absolute ethanol pre-cooled ...

Embodiment 3

[0090] Embodiment 3 A kind of detection method of staphylococcus enterotoxin genotype comprises the steps:

[0091] 1) Sample processing:

[0092] Take 0.3ml sample, centrifuge at 11000r / min for 1 minute;

[0093] 2) DNA extraction:

[0094] Discard the supernatant, add 700 μl sterilized water to suspend the precipitate, and centrifuge at 11000r / min for 1 minute; discard the supernatant, add 300μl reagent A to act at 40°C for 20 minutes, add reagent B, and act at 55°C for 20 minutes, 11000r / min min, centrifuge for 10 minutes; take the supernatant and add an equal volume of tris-saturated phenol, shake and mix well, and centrifuge at 11000r / min for 10 minutes; take the supernatant and add an equal volume with a volume ratio of 25:24 : 1 mixed solution of tris-saturated phenol, chloroform and isoamyl alcohol, after mixing, centrifuge at 11000r / min for 10 minutes; take the supernatant and add 2.2 times the volume of absolute ethanol pre-cooled to 4°C , mix well, and place at r...

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Abstract

The invention discloses a genotype inspection method and a genotyping inspection method of staphylococcal enterotoxin. The genotype inspection method of staphylococcal enterotoxin includes the steps of sample treatment, DNA extraction, PCR and PCR amplified product analysis. The amplified sample is put in agarose gel including ethidium bromide for electrophoresis and color development and observed under a transmissive uviol lamp; the inspected sample can be judged as a positive sample including corresponding staphylococcal enterotoxin genotype if the length of the amplified nucleic acid band is the same as the amplified length in table 2. The first method can fast inspect the particular staphylococcal enterotoxin genotype, with high sensitivity, high particularity and high accuracy. The second method can achieve systematic genotyping of staphylococcal enterotoxin genes from different sources and provide technical support for analysis of relationships among different enterotoxin types. The invention is significant in food security quarantine, disease diagnosis and molecular epidemiology research.

Description

technical field [0001] The invention belongs to the field of bacterial molecular detection, and in particular relates to a detection method of staphylococcal enterotoxin genotype and a detection method of genotyping. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important food-borne and iatrogenic pathogen, ubiquitous in nature, can be found in air, water, dust and human and animal excreta, and its nutritional requirements It is not high, has strong salt tolerance, and can survive in most types of food, so there are many opportunities for food to be contaminated by it. If the conditions are suitable, it will multiply in large numbers and produce a sufficient dose of enterotoxin. Staphylococcal poisoning is closely related and is the main pathogenic factor causing human diseases. Among the food safety accidents, the "Snowprint Milk" incident that caused more than 14,000 people to be infected in 2000 was the first to be promoted. It was a p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/45C12R1/445C12R1/44
Inventor 黄金海刘业兵李琳庄世文
Owner TIANJIN UNIV
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