Acinetobacter baumannii infection fast detection method based on multiple epitope fusion antigens

A technology of Acinetobacter baumannii and fusion antigen, applied in the direction of fusion polypeptide, hybrid peptide, measuring device, etc., can solve the problems of long time, difficult to meet rapid identification, unsuitability, etc.

Active Publication Date: 2020-01-24
HUBEI UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, the method for detecting the pathogen in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. Difficult to meet the needs of rapid identification; the PCR technology developed in recent years is a fast, sensitive, and specific technology, bu

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  • Acinetobacter baumannii infection fast detection method based on multiple epitope fusion antigens
  • Acinetobacter baumannii infection fast detection method based on multiple epitope fusion antigens
  • Acinetobacter baumannii infection fast detection method based on multiple epitope fusion antigens

Examples

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Example Embodiment

[0062] Example 1

[0063] Preparation of Acinetobacter baumannii multiple epitope fusion antigen (Fhue+OmpA) antibody:

[0064] 1.1) Cloning of FhuOmp fusion gene of Acinetobacter baumannii

[0065] Obtain the most abundant peptide fragments in the extracellular domain of Acinetobacter baumannii surface proteins Fhue and OmpA (the accession numbers in the NCBI protein database are KMV27515 and AJF83030, respectively), find the gene coding sequence, and optimize the gene coding sequence , And the two sequences are connected with the coding sequence of flexible connecting peptide (ggsggsggsggs) to form a fusion gene. At the same time, at the 5'end of the fusion gene, the restriction site NdeI was introduced, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene, and the complete gene sequence was chemically synthesized, which was recorded as FhuOmp. The full sequence of the gene and the encoded amino acid sequence are shown in the...

Example Embodiment

[0093] Example 2

[0094] Preparation of Acinetobacter baumannii multiple epitope fusion antigen (Pilf+ponA) antibody:

[0095] 2.1) Cloning of PilPon fusion gene of Acinetobacter baumannii

[0096] Acquire Acinetobacter baumannii surface proteins Pilf and ponA (the accession numbers in the NCBI protein database are AJF80497 and ADX05080, respectively) peptides with the most abundant epitopes in the extracellular domain, find the gene coding sequence, and optimize the gene coding sequence , And the two sequences are connected with the coding sequence of rigid linking peptide (eaaakeaaak) to form a fusion gene. At the same time, at the 5'end of the fusion gene, the restriction site NdeI was introduced, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene, and the complete gene sequence was chemically synthesized, which was recorded as PilPon. The full sequence of the gene and the encoded amino acid sequence are shown in the sequ...

Example Embodiment

[0125] Example 3

[0126] Preparation of Acinetobacter baumannii multiple epitope fusion antigen (Fhue+OmpA) antibody latex microsphere marker:

[0127] 3.1) Activation of latex microspheres

[0128] Take 1mL of 10% red carboxylated polystyrene latex microspheres (100nm) solution, add 9mL 2-(N-morpholinyl)ethanesulfonic acid (MES) buffer (0.1mol / LMES, pH8.5) After mixing; prepare 10mg / mL N-hydroxysuccinimide (NHS) and 10mg / mL 1-(3-dimethylaminopropyl)-3-ethyl 1-(3-bis Methylaminopropyl)-3-ethylcarbodiimide hydrochloride hydrochloride (EDC) solution;

[0129] Add 1mL NHS solution and 1mL EDC solution to polystyrene latex microspheres (100nm) solution in turn, mix slowly at room temperature for 30 minutes, centrifuge at 19000g for 20 minutes after incubation, remove the supernatant, and use 10mL borax buffer for precipitation ( 0.1mol / L Na 2 B 4 O 7 , PH8.5) resuspension, shaking, ultrasonic treatment (ultrasonic breaker product model: YJ92-IIDN, power 50W, working time 2s, interval t...

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Abstract

The invention relates to a preparation method of an acinetobacter baumannii infection fast detection card based on multiple epitope fusion antigens. The detection card consists of a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC plate, wherein colored latex microsphere coupled anti-acinetobacter baumannii surface protein multiple epitope fusion antigen polyclonal antibodies are sprayed and coated on the combination pad; and the nitrocellulose membrane comprises detection lines coated with anti-acinetobacter baumannii surface protein multiple epitopefusion antigen polyclonal antibodies and quality control lines coated with goat anti rabbit IgG antibodies. When acinetobacter baumannii is contained in an added sample, the acinetobacter baumannii firstly forms a compound with latex-rabbit anti-acinetobacter baumannii surface protein polyclonal antibodies, and is captured when migrating to detection lines coated with the anti-acinetobacter baumannii surface protein polyclonal antibodies under the capillary action; and the detection lines show corresponding colors, so that whether the acinetobacter baumannii is contained in the sample or not can be detected. The detection card has the advantages of high speed, simplicity, high speed, high sensitivity and high specificity.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a rapid detection method for Acinetobacter baumannii infection based on multiple epitope fusion antigens. Background technique [0002] Acinetobacter baumannii (Ab) is a non-fermenting gram-negative bacillus that widely exists in nature and is an opportunistic pathogen. This bacterium is an important pathogen of nosocomial infection, mainly causing respiratory tract infection, and can also cause bacteremia, urinary tract infection, secondary meningitis, surgical site infection, ventilator-associated pneumonia, etc. The rate of resistance to commonly used antibiotics is increasing year by year, and has caused serious concern for clinicians and microbiologists. Domestic data show that A.baumannii accounts for more than 70% of clinically isolated Acinetobacter. The drug resistance rate of A.baumannii to the third and fourth generation cephalosporins has reached 63.0%-89.9%. My...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/569G01N33/531
CPCC07K14/212C12N15/70G01N33/56911G01N33/531C07K2319/00Y02A50/30
Inventor 杨波胡征王毅
Owner HUBEI UNIV OF TECH
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