Method for detecting staphylococcus aureus and enterotoxin A gene in food

A Staphylococcus aureus technology, applied in the detection of target genes, primer pairs and probes, can solve the problems of limited detection of Staphylococcus aureus, false negatives, false positives, etc., to achieve practical, specific, and results. Determining simple effects

Active Publication Date: 2015-06-24
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the variation of the target sequence, the transfer of the gene level and other factors, it often leads to false negative, false positive and other false detection results. The specificity and quantity of the detection target limit the ability to detect Staphylococcus aureus based on nucleic acid molecules.

Method used

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  • Method for detecting staphylococcus aureus and enterotoxin A gene in food
  • Method for detecting staphylococcus aureus and enterotoxin A gene in food
  • Method for detecting staphylococcus aureus and enterotoxin A gene in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Establishment of PMA-qPCR detection method

[0056] Step 1, design of detection primers and related probes

[0057] Through comparative genomics analysis, the specific detection target gene RpiR of Staphylococcus aureus was obtained. Download all the nucleic acid sequences of the RpiR gene from NCBI GenBank, and perform homology comparison analysis to find the conserved region of the RpiR gene. The base sequence information of the target sequence is shown in SEQ ID NO:1. At the same time, the nucleotide sequence of the Staphylococcus aureus enterotoxin A gene (sea) was downloaded from GenBank, and then homologous comparison analysis was performed to find the conserved region of the gene. The base sequence information of the enterotoxin A gene is shown in SEQ ID NO:2.

[0058] Input the base sequences of RpiR and sea into Beacon designer7.0 respectively, design primer pairs and probes in the conserved regions of the two genes, set the GC% range to 40-60%, and the prod...

Embodiment 2

[0076] Performance Evaluation of PMA-qPCR Assay

[0077] The Staphylococcus aureus standard strain ATCC13565 (sea+) and the standard strain ATCC25923 (sea-) cultured overnight were serially diluted after plate counting. Weigh 25g of quick-frozen pork dumplings negative for Staphylococcus aureus, add 225mL of normal saline, beat with a homogenizer for 100s, take 900μL of homogenized solution, add 100μL of diluted pure culture, and make 3 parallels for each concentration gradient. Contaminate food samples with standard strains ATCC 13565 and ATCC 25923 alone, so that the concentration of the strains that contaminate the food is about 10 2 cfu / g, 10 3 cfu / g, 10 5 cfu / g and 10 7 cfu / g and mixed strains contaminated food samples (choose two concentration gradients, approximately: 10 7 cfu / g ATCC25923 and 10 3 cfu / g ATCC 13565, 10 7 cfu / g ATCC 25923 and 10 5 cfu / g ATCC 13565, 3 parallels per concentration gradient). Then the food samples were treated with PMA, and the genomi...

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Abstract

The invention discloses a method for detecting staphylococcus aureus and enterotoxin A gene in food, as well as a detecting target gene, a primer and a probe thereof. The method comprises the following steps: processing a food sample to be tested by using the PMA to eliminate the over high quantification problem caused by the dead bacterium DNA; creating a detecting method for rapidly and quantitatively detecting the staphylococcus aureus in the food, screening the enterotoxin A gene and indicating the false-negative PMA-qPCR of PCR reaction. By adopting the detecting method, the staphylococcus aureus with 10<3>-10<8> cfu / g of pollution load in the food can be accurately quantified within 5-6h and the enterotoxin A gene can be screened. The detecting method provided by the invention is strong in specificity, accurate in quantification and rapid in detecting speed and the viable bacteria also can be detected.

Description

technical field [0001] The invention relates to the field of food safety, in particular to an internal standard PMA-qPCR method for quantitatively detecting live Staphylococcus aureus bacteria and enterotoxin A gene in food, and detection target genes, primer pairs and probes thereof. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important food-borne and clinical pathogen. Food poisoning caused by it is a worldwide health problem and poses a huge threat to public health and food safety. In 2011, my country revised the "National Food Safety Standard" for quick-frozen rice and flour products, in which Staphylococcus aureus was adjusted from non-detection to limited detection, and the maximum detection limit of Staphylococcus aureus in raw and cooked products was 10 4 cfu / g and 10 3 cfu / g. Although the national limit standard has been newly adjusted, the supporting detection method is still the traditional selective plate culture method, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/6851C12Q2537/143C12Q2563/173C12Q2545/114
Inventor 史贤明宋明辉施春雷张易崔妍
Owner SHANGHAI JIAO TONG UNIV
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