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Universal rapid magnetic separation method and kit for pathogenic organism

A technology of pathogenic microorganisms and magnetic separation, applied in the direction of analysis materials, measuring devices, instruments, etc., can solve the problems of complex preparation process of immunomagnetic beads, insufficient amount of conjugated antibody, time-saving and labor-saving, etc., to achieve fast and accurate The effects of diagnosis, promotion of industrialization development, and reduction of work intensity

Inactive Publication Date: 2019-10-08
珠海市德灏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] But up to now, the isolation of pathogenic bacteria by immunomagnetic bead method has not been recognized and popularized. The main reasons are: 1) To isolate a pathogen, a special immunomagnetic bead needs to be specially prepared, and the preparation process of immunomagnetic beads is complicated and technically difficult. The loss of antibody activity during the coupling process is large, the amount of conjugated antibody is insufficient, and the quality is difficult to guarantee; 2) The purity of pathogen isolation only reaches the "enrichment" level, and it is still necessary to separate and culture by streaking to obtain pure target bacteria, which does not save It does not save time and effort, but increases the detection cost; 3) It is impossible to synchronize and automatically separate samples of various pathogens

Method used

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  • Universal rapid magnetic separation method and kit for pathogenic organism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of universal bridged magnetic beads

[0035] 1. Prepare the following reagents

[0036] Reaction buffer: 0.05mol / L MES, pH5.0;

[0037] Washing solution: HEPES (hydroxyethylpiperazine ethanesulfonic acid) of 0.02mol / L, pH7.4;

[0038] Diluent: 0.01 mol / L, pH 8.0 PB (phosphate buffer, made of NaH 2 PO 4 ·2H 2 O, Na 2 HPO 4 ·12H 2 O preparation);

[0039] Blocking solution: 1mol / L NH4Cl;

[0040] EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) activation solution: the concentration is 20mg / ml, prepared with the aforementioned reaction buffer;

[0041] NHS (N-hydroxysuccinimide) activation solution: the concentration is 20mg / ml, prepared with the aforementioned reaction buffer;

[0042] Preservation solution: 0.1% BSA (bovine serum albumin), 0.02% sodium azide, 0.1% Tween-20, prepared with 0.02mol / L HEPES;

[0043] Streptavidin solution: the concentration is 0.35mg / ml, prepared with 0.01mol / L, pH8.0 PB to dissolve streptav...

Embodiment 2

[0062] Example 2 Separation and Purification of Salmonella Paratyphi A by Specific Capture Magnetic Beads

[0063] 1. To prepare magnetic beads specific for Salmonella paratyphi A, follow the steps below:

[0064] a) Take the universal bridging magnetic beads prepared in Example 1 and dilute to 2 mg / ml with washing liquid; then take 1 ml of murine Salmonella paratyphi A monoclonal antibody diluted with washing liquid to 1 mg / ml, and mix with 1 ml of 2mg / ml universal bridging magnetic beads for mixing, and react at 37°C for 1 hour;

[0065] b) Step a) After the reaction is completed, use washing liquid to magnetically separate and wash 3 times, and then use washing liquid to dilute to 1 mg / ml to obtain specific capture magnetic beads.

[0066] 2. Capturing test of Salmonella paratyphi A

[0067] Follow the steps below:

[0068] a) Take newly amplified Salmonella paratyphi A (ATCC9150) and adjust to 10 with sterile washing solution 4 CFU / ml;

[0069] b) Take 1ml of the abov...

Embodiment 3

[0080] Embodiment 3 four kinds of pathogens are separated synchronously

[0081] 1. Preparation of specific capture magnetic beads

[0082] a) Get the universal bridging magnetic beads of Example 1 diluted to 2mg / ml by washing liquid, 1ml / tube, 4 tubes in total; , Shigella dysenteriae monoclonal antibody, Candida albicans monoclonal antibody, and Bacillus cereus monoclonal antibody were mixed, and reacted at 37°C for 1 hour;

[0083] b) Magnetic separation and washing with washing liquid for 3 times, and then diluting to 1 mg / ml to make specific capture magnetic beads.

[0084] 2. Synchronous separation test:

[0085] a) Mixed sample preparation: prepare a mixed bacterial solution with Salmonella paratyphi A, Shigella dysenteriae, Candida albicans, and Bacillus cereus, and dilute it with sterile washing liquid to make the A The concentrations of Salmonella paratyphi, Shigella dysenteriae, Candida albicans and Bacillus cereus were all 10 4 CFU / ml, divide the mixed bacterial...

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Abstract

The invention discloses a universal rapid magnetic separation method for a pathogenic organism. The method comprises the steps that (1) a universal bridging magnetic bead is prepared by a) preparing amagnetic bead and streptavidin crosslinked agent, b) preparing a biotin-Ab2 crosslinked agent, and c) mixing the magnetic bead and streptavidin crosslinked agent and the biotin-Ab2 crosslinked agentfor incubation to prepare a universal bridging magnetic bead; (2) the universal bridging magnetic bead is mixed with a murine AB1 of the pathogenic organism to be measured to prepare a specific capturing magnetic bead; and (3) the specific capturing magnetic bead is used to carry out pathogen capturing separation purification and preliminary discrimination on a sample to be detected. The inventionalso discloses a kit including the specific capturing magnetic bead prepared via the method. The immune magnetic bead is prepared simply, performance is optimized and automation is easy to realize, single targeted bacterium can be obtained by separation, and different pathogens in the sample can be detected synchronously and automatically.

Description

technical field [0001] The invention relates to the technical field of in vitro biological detection and diagnosis, in particular to a universal rapid magnetic separation method of pathogenic microorganisms and a kit. Background technique [0002] Pathogenic microorganisms include virus, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, actinomycetes, fungi and so on. Viruses, chlamydia, and rickettsia are parasitic microorganisms that need to proliferate in specific living cells; mycoplasma, bacteria, spirochetes, actinomycetes, and fungi are independent growth microorganisms that can be propagated in cell-free medium, which is a microbiological test the main object. [0003] Microbiological testing is a confirmatory method for judging pathogenic infection and hygienic status, and the testing results are related to clinical drug selection and hygienic decision-making. The detection process mainly includes three major links: pathogen isolation, identification, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531G01N33/543
CPCG01N33/56911G01N33/531G01N33/54326
Inventor 农高惠何林声
Owner 珠海市德灏生物科技有限公司
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