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Dual polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) detection method for staphylococcus aureus in aquatic products

A PCR-DHPLC, staphylococcus technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc. Long cycle and other problems, to achieve the effect of simplifying reaction conditions, easy operation and high sensitivity

Active Publication Date: 2013-09-25
中华人民共和国舟山出入境检验检疫局
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Problems solved by technology

However, the problem of the bacteria exceeding the standard often plagues export aquatic product processing enterprises, which has become a major obstacle restricting the development of enterprises and the improvement of economic benefits, and has also made it one of the key monitoring items of inspection and quarantine departments.
[0004] The traditional detection methods of Staphylococcus aureus in aquatic products include bacterial enrichment, biochemical identification, serological identification, etc., not only the detection cycle is long, the operation is cumbersome, but also it is not suitable for the detection of large batches of samples. In other fields such as aquaculture environment monitoring, large batches of detection are required. In terms of the traditional detection method exposed its various drawbacks

Method used

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  • Dual polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) detection method for staphylococcus aureus in aquatic products
  • Dual polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) detection method for staphylococcus aureus in aquatic products
  • Dual polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) detection method for staphylococcus aureus in aquatic products

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Embodiment Construction

[0039] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0040] 1. Take 25g shrimp samples, cut them into pieces, mix them with 225mL enrichment solution, and incubate at 36°C±1°C for 18h-24h;

[0041] 2. Take 1 mL of the above enrichment solution, centrifuge at 12000 rpm for 3 minutes, remove the supernatant, pretreat the obtained precipitate with lysostaphin for 1 hour, and then extract the DNA of Staphylococcus aureus according to the instructions of the bacterial DNA extraction kit;

[0042] 3. Primer: sea-F: 5'-AGTCTGAATTGCAGGGAAC-3',

[0043] sea-R: 5'-CACCACCATACATACAAGC-3',

[0044] nuc-F: 5'-CTTGCTATGATTGTGGTAGC-3',

[0045] nuc-R: 5'-CTAGCAAGTCCCTTTTCCAC-3';

[0046] 4. Amplification reaction system: 10×PCR Buffer (containing 20mM Mg 2+ ) 5μL, dNTP (10mM) 2μL, two pairs of primers (50μM) each 0.5μL, Ex Taq enzyme (2u·μL -1 ) 1 μL, template DNA 2 μL, and finally ddH 2 O Make up the to...

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Abstract

The invention relates to a dual polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) detection method for staphylococcus aureus in aquatic products, which is characterized by comprising the following steps of: 1, staphylococcus aureus culture: aquatic product samples to be tested are mixed with enrichment liquid for culture; 2, staphylococcus aureus genome deoxyribonucleic acid (DNA) extraction; 3, primer design; 4, dual PCR amplification by using the staphylococcus aureus genome DNA as templates; and 5, DHPLC peak type map obtaining through PCR product DHPLC detection for analysis and identification. The integral detection method has the advantages that the reaction condition is simplified, the detection sensitivity is improved, in addition, the operation is simpler and more convenient, and the goal of high flux is reached.

Description

technical field [0001] The invention relates to a method for detecting Staphylococcus aureus. Background technique [0002] Staphylococcus aureus is an important class of food-borne pathogens, which widely exist in the air, soil, water, and mucous membranes of humans and animals. According to the US CDC report, food poisoning caused by Staphylococcus aureus is second only to Escherichia coli, accounting for 33% of bacterial food poisoning, and food poisoning incidents caused by Staphylococcus aureus pollution are also high in my country Frequently. Obviously, the pollution of Staphylococcus aureus has become a worldwide public health problem. Therefore, the establishment of specific, sensitive, rapid and convenient detection technology has become the key to effectively control the contamination and spread of Staphylococcus aureus in food. [0003] Staphylococcus aureus can produce a variety of toxins, such as enterotoxin, exfoliation toxin, leukocidin and toxic shock syndr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/14G01N30/02C12R1/445
Inventor 周向阳万婧沈飚胡兴娟邵宏宏张静颜建波
Owner 中华人民共和国舟山出入境检验检疫局
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