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Immunity colloidal gold test paper strip for detecting and staphylococcal enterotoxin A and its production method

A colloidal gold test paper and colloidal gold technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that the detection process is limited in the laboratory, the detection takes a long time, and the operation process is complicated, so as to prevent the spread of the epidemic , maintaining human health and ensuring food safety

Inactive Publication Date: 2008-09-24
SHENZHEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of EIISA are: (1) special equipment such as a microplate reader is required for use; (2) operators need to be specially trained; (3) the operation process is relatively complicated, and the detection time is relatively long; (4) The detection process is limited in the laboratory and is not suitable for the analysis of on-site samples

Method used

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  • Immunity colloidal gold test paper strip for detecting and staphylococcal enterotoxin A and its production method
  • Immunity colloidal gold test paper strip for detecting and staphylococcal enterotoxin A and its production method
  • Immunity colloidal gold test paper strip for detecting and staphylococcal enterotoxin A and its production method

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: The connection relationship of the staphylococcus aureus enterotoxin A immune colloidal gold test strip is:

[0039] according to figure 1 It can be seen that it includes a sample pad 3, a gold standard pad 4 coated with a colloidal gold-labeled anti-SEA antibody, a nitrocellulose membrane coated with an anti-SEA antibody and goat anti-mouse IgG at a detection line 5 and a quality control line 6, Absorbent pad 7, a staphylococcus aureus enterotoxin A immune colloidal gold test strip whose connection relationship is: a PVC base plate 1 of appropriate size; at both ends of the PVC base plate 1, a sample pad 3 and an absorption pad 7 are respectively provided; The middle part of the PVC bottom plate 1 is provided with a nitrocellulose membrane detection layer 2, and at the junction of the nitrocellulose membrane detection layer 2 and the sample pad 3, a gold standard pad 4 coated with a gold-marked anti-SEA antibody is provided, and the gold standard pad 4 O...

Embodiment 2

[0040] Embodiment 2: produce the isolation of SEA staphylococcus aureus standard strain

[0041] In this example, Staphylococcus aureus (Zhang Wenli., etc. Different culture methods have influence on the results of Staphylococcus aureus enterotoxin. Chinese Journal of Health Inspection, 2004, 14(1): 108) was selected as the screening strain. According to the known gene sequence of Staphylococcus aureus enterotoxin A (SEA) (GenBank accession number: M28521), use Primer5.0 to design the upstream primer sea F: 5′GCC GCT AGC ATG AAA AAA ACA GCA TTT ACA TTA C 3′, (the underline is the Nhe I restriction site); downstream primer sea R: 5′CGC C GT CGA C TT AAC TTG TAT ATA AAT ATATAT CAA 3', (the underline is the Sa / I restriction site).

[0042] Inoculation of Staphylococcus aureus (Zhang Wenli., etc. Different culture methods have effects on the results of Staphylococcus aureus enterotoxin. Chinese Journal of Health Inspection, 2004, 14 (1): 108) in 7.5% sodium chloride broth, 37 ...

Embodiment 3

[0043] Embodiment 3: the preparation of SEA:

[0044] Extract the genomic DNA of the enterotoxigenic Staphylococcus aureus standard strain, and use it as a template to amplify the product by PCR. After the PCR product is purified by DNA Extraction Kit (Fermentas Company), it is carried out with the pGEM-T Easy cloning vector (Promega Company). Ligation, transformation, sequencing, the recombinant plasmid containing the correct sequence of SEA was digested by Nhel and SalI, and then ligated and transformed with the pET28a (Novagen Company) expression vector of the same double digestion, and the recombinant plasmid pET-SED was transformed into Escherichia coli BL21 ( DE3), induced by 1 mM IPTG at 37°C for 6 hours, and the cells were collected by centrifugation. Add 50mM pH8.0 Tris-HClbuffer according to the amount of bacterial weight 1 (g): 10 (ml), and ultrasonically destroy the bacteria on ice water. 4°C, 5000rpm, centrifuge for 10min, collect supernatant and precipitate resp...

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Abstract

The invention discloses an immune colloidal gold test paper strip for detecting staphylococcus aureus enterotoxin A and a preparation method, comprising the construction of engineering bacteria of expression recombinant SEA protein, the separation and the purification of the SEA protein, the preparation of an anti-SEA specific antibody and the preparation of the SEA colloidal gold test paper strip. The test paper strip consists of a sample pad, a colloidal gold marker pad which is coated by a colloidal gold marker-labeled anti-SEA antibody and a cellulose nitrate film which is coated by an anti-SEA antibody and an anti-mouse IgG at a detection line and a quality control line; furthermore, the sample pad, the colloidal gold marker pad, the cellulose nitrate film and a water absorbent pad are sequentially adhered on a PVC bottom plate. The test paper strip has convenient, rapid and accurate operation, the whole process only needs 10 minutes and is not interfered by the environmental conditions, the specificity is good, the detection sensitivity is high and the lowest detection limit is 1ng / ml. The test paper strip is applicable to customs, hospitals and inspection quarantine institutions, etc., which can further realize the rapid detection of the staphylococcus aureus enterotoxin A in foods or in clinical samples.

Description

technical field [0001] The invention relates to an immune colloidal gold test strip for detecting Staphylococcus aureus enterotoxin A, and also relates to a preparation method of the immune colloidal gold test strip, which can realize rapid diagnosis of Staphylococcus aureus enterotoxin A and is suitable for a large number of On-site detection of samples, rapid diagnosis and emergency treatment of food poisoning and nosocomial infection. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus), referred to as Staphylococcus aureus, is one of the main pathogens of common food poisoning and hospital pollution. It is widely distributed in the skin and mucous membranes, especially in the nasopharynx of birds and mammals. When the resistance of the animal body decreases or the skin and mucous membranes are damaged, germs can take advantage of the weak point and cause various suppurative diseases of livestock and poultry. In recent years, Staphylococcus aureus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/532
Inventor 胡章立张仁利甄茵
Owner SHENZHEN UNIV
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