ELISA (enzyme-linked immuno sorbent assay) detection kit and method both for detecting streptococcus equi subsp. zooepidemicus

An enzyme-linked immunosorbent reagent, the technology of Streptococcus equi, is applied in the field of epidemiology and sanitation detection, and can solve the problems of lack of rapid and sensitive serological detection methods

Inactive Publication Date: 2015-02-04
NANJING AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still a lack of rapid a

Method used

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  • ELISA (enzyme-linked immuno sorbent assay) detection kit and method both for detecting streptococcus equi subsp. zooepidemicus
  • ELISA (enzyme-linked immuno sorbent assay) detection kit and method both for detecting streptococcus equi subsp. zooepidemicus
  • ELISA (enzyme-linked immuno sorbent assay) detection kit and method both for detecting streptococcus equi subsp. zooepidemicus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the preparation of class M protein

[0044] 1.1PCR amplification of the coding gene fragment of M-like protein

[0045] According to the gene sequence of M protein of Streptococcus equi subsp. zooepidemicus ATCC35246 published by GeneBank (accession number is AY263781), a pair of primers were designed by Primer5.0 software, and XholI and NdeI restriction sites were added to both ends of the primers respectively. and protective bases, the primers were synthesized by Shanghai Yingjun Biotechnology Company.

[0046] Upstream primer (P1) 5'-GCATGCGCATATGGCCCTCTTGGTTGGTGTGGCAGCTG-3'

[0047] Downstream primer (P2) 5'-GGCACGTCTCGAGGTTTTCTTTGCGTCTTGTTGACACTGC-3'

[0048] The PCR cycle parameters are as follows: after denaturation at 94°C for 4 minutes, enter the cycle, 94°C for 30s, 55°C for 30s, and 72°C for 30s for a total of 30 cycles, and finally extend at 72°C for 10 minutes. A gene fragment with a size of 1140bp was obtained. Sequencing showed that its nu...

Embodiment 2

[0065] Example 2 Detection of M-like protein immunogenicity

[0066] Perform immunoblotting analysis on the purified M-like protein, the specific steps are as follows:

[0067] (1) Perform 10% SDS-PAGE electrophoresis on the purified M protein-like protein.

[0068] (2) Cut 1 piece of PVDF membrane and 4 pieces of filter paper to match the size of the 2-hole gel. The filter paper was equilibrated in transfer buffer for 10 min, and the PVDF membrane was equilibrated in methanol solution.

[0069] (3) Put 4 pieces of filter paper, PVDF membrane, gel, and 4 pieces of filter paper on the splint surface from bottom to top, and all layers are exhausted of air bubbles and aligned accurately.

[0070] (4) Put the upper electrode on the interlayer, connect the power supply, and transfer for 15 minutes at 15V. Then disconnect the power, disassemble the transfer device from top to bottom, and lift off each layer one by one.

[0071] (5) Put the PVDF membrane into a plate filled with ...

Embodiment 3

[0076] Embodiment 3: Establishment of the optimal condition of ELISA reaction and establishment of detection method

[0077] 3.1 Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0078] Using the square array titration method, use 0.05mol / L pH9.6 carbonate buffer solution to make the M protein antigen of Streptococcus equi zooepidemic subspecies 20μg / ml, 10μg / ml, 5μg / ml, 2.5μg / ml, 1.25μg / ml ml, 0.625μg / ml, and 0.312μg / ml were diluted, respectively added to the 1-6 rows of the 1-12 column on the left side of the 96-well ELISA plate, 100μL per well, and reacted at 37°C for 1 hour, then placed in 4 ℃ overnight. Remove and wash with washing solution (phosphate buffered saline containing 0.5% Tween-20) for 3 times, add 200 μL of blocking solution (phosphate buffered saline containing 5% skimmed milk) to each well, act at 37°C for 2 hours, discard the well The blocking solution inside; the positive serum and negative serum of Strepto...

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Abstract

The invention belongs to the field of eqidemiology and sanitation detection, and discloses an ELISA (enzyme-linked immuno sorbent assay) quick detection kit and a method both for quickly detecting streptococcus equi subsp. zooepidemicus; particularly, the kit provided by the invention includes a recombined streptococcus equi subsp. zooepidemicus type M protein antigen and an ELIAS secondary antibody. The invention also discloses an indirect ELISA method for detecting streptococcus equi subsp. zooepidemicus. The method is quick, simple and convenient, and can be used for directly detecting to-be-detected blood serum. The detection kit and the detection method provided by the invention have high sensitiveness and strong specificity.

Description

technical field [0001] The invention belongs to the field of epidemiology and sanitation detection. Specifically, the present invention relates to an enzyme-linked immunosorbent assay (ELISA) rapid detection kit for rapidly detecting Streptococcus equi subsp. zooepidemicus, and a detection method using the kit. Background technique [0002] Streptococcus equi subspecies zooepidemicus is the main pathogen of Streptococcus suis in my country, belonging to Group C Streptococcus in Lambert Group, which mainly causes lower respiratory tract infections in horses, pigs, cattle, dogs, cats and other animals, and causes Symptoms of sepsis, meningitis, arthritis, pneumonia, and sudden death in severe cases. Humans can also get sick when they come into contact with infected animals or eat food contaminated by this bacteria, so this bacteria is an important pathogen of zoonotic diseases. However, there is still a lack of rapid and sensitive serological detection methods. Therefore, th...

Claims

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Application Information

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IPC IPC(8): G01N33/569
Inventor 范红结周红周瑾陆承平
Owner NANJING AGRICULTURAL UNIVERSITY
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