Indirect competitive ELISA (Enzyme Linked Immunosorbent Assay) method based on multi-epitope tandem peptides and used for synchronously detecting staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin G (SEG)
A simultaneous detection and tandem peptide technology, applied in the field of immune analysis, can solve the problems of unsatisfactory detection specificity and sensitivity, blind spots in the detection of new enterotoxins, and limited detection range, so as to reduce subjectivity and reagent storage time The effect of long and simple result judgment
Active Publication Date: 2015-06-24
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology
High-quality ELISA kits have been developed abroad for the detection of SE in foods such as milk and meat products, but they are expensive
In 2010, my country also designated the indirect ELISA method as the national standard method for detecting classic SE, but the detection range is limited, and only classic enterotoxins can be detected. There are blind spots in the detection of new enterotoxins, and the detection specificity and sensitivity are not satisfactory. false positive
At present, the relevant SE antigen detection methods declared in China can only detect classical enterotoxin, and the quality of detection reagents needs to be further improved
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[0048] Below by embodiment the present invention will be further described, but do not limit the present invention.
[0049] The experimental methods in the following examples are conventional methods unless otherwise specified.
[0050] The percentages in the following examples are all mass percentages unless otherwise specified.
[0051] The proportions in the following examples are volume proportions unless otherwise specified.
[0052] 1. Preparation of multi-epitope tandem peptide SE A / G of Staphylococcus aureus type A and type G enterotoxins
[0053] 1.1 Design and gene synthesis of multi-epitope tandem peptide SE A / G
[0054] Design the S. aureus type A and G type enterotoxin multi-epitope tandem peptide SE A / G gene, that is, use the nucleotide sequence (SEQ ID NO: 8) of AAY (alanine-alanine-tyrosine) to combine SE The nucleotide sequences of the 4 B cell linear epitope regions of A and the 3 dominant B cell linear epitopes of SE G are sequentially concatenated into ...
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Abstract
The invention belongs to the technical field of immunoassay and particularly relates to an indirect competitive ELISA (Enzyme Linked Immunosorbent Assay) method based on multi-epitope tandem peptides and used for synchronously detecting staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin G (SEG). The method mainly comprises preparation of SEA and SEG multi-epitope tandem peptide peptides (including antigens), preparation of anti-rabbit SEA and SEG antibodies, sample pretreatment and establishment of the indirect competitive ELISA detection method based on multi-epitope tandem peptides. The indirect competitive ELISA method comprises the following steps: (1) enveloping; (2) plate washing; (3) sealing; (4) plate washing; (5) adding standard substances, samples to be detected and specific antibodies in sequence; (6) plate washing; (7) adding ELIAS secondary antibodies; (8) plate washing; (9) developing and ending reaction. The method provided by the invention has the characteristics of high specificity, high sensitivity and good repeatability, is suitable for quick, synchronous and quantificational detection of SEA and SEG in foods such as milk and dairy products.
Description
technical field [0001] The invention belongs to the technical field of immune analysis, and relates to a rapid, specific, sensitive and synchronous detection method for Staphylococcus aureus type A and type G enterotoxins in foods such as milk and dairy products, specifically an indirect competition method based on multi-epitope tandem peptides ELISA method. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, referred to as Staphylococcus aureus) is an important food-borne pathogen, which is widely found in air, water, dust, and human and animal excrement. Staphylococcal Enterotoxin (SE) is easy to grow and reproduce in foods containing more water, protein and starch, especially milk and dairy products. Due to the strong thermal stability and toxicity of SE, after heat treatment of food contaminated by Staphylococcus aureus, although the bacteria can be killed, the SE produced by it is still pathogenic, and a small amount of SE can contaminate food ...
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IPC IPC(8): G01N33/569G01N33/531
Inventor 李槿年梁明燕刘雪兰张婷婷李冠青
Owner ANHUI AGRICULTURAL UNIVERSITY