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Cell isolation method

A cell and target cell technology, applied in the field of separation of bacteria present in samples, can solve the problems of inability to lyse, block filters, inaccuracy, etc., and achieve the effect of favorable binding ability, flexibility and cost advantages

Inactive Publication Date: 2003-02-05
挪威诊断联合股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present technology allows for flexible and affordable ways to separate substances that have different properties or characteristics from each other without requiring multiple types of materials with unique chemical structures. This makes it possible to use this new material more efficiently than current options like silica gel columns.

Problems solved by technology

This patents describes various ways to quickly separate small organisms like yeast cells found within certain types of biological material into their own liquid phase during laboratory testing procedures. However current methods require multiple steps and involve complicated processes due to high concentrations of solids generated after each process.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0136] Preparation of carbohydrate-coated particles and testing of cell-binding properties

[0137] Compounds were coupled to M-PVA OCN-activated beads (Chemagen AG). reaction:

[0138] Bead-NCO + R-OH →

[0139] Activated beads Sugars with hydroxyl groups Carbamates of sugars (urethar)

[0140] The modified beads were incubated with different bacterial cultures, either pure undiluted cultures (o.n.) or diluted in water or buffer (PBS). To investigate cell binding, DNA was isolated from bacteria-bead complexes and subjected to PCR analysis. Reference: WO98 / 51693. Chemagen is used for this coupling reaction and the resulting modified beads are used in the separation method of the invention as described herein.

Embodiment 2

[0142] Method for isolating bacteria on beads and identifying specific bacteria

[0143]Bacteria were grown overnight at 30°C without stirring in 100 ml of tryptone soy broth. 800 microliters of PBS (binding buffer) and 10 microliters of beads of the invention (10 mg / ml) were mixed, then 100 milliliters of the overnight culture was added, pipetting with a pipette, and stirred gently. The supernatant was removed using a magnetic separator, and the bead-bacteria complex was resuspended in 50 microliters of lysis buffer (4M guanidinium thiocyanate-1% sodium lauryl sarcosine). The sample was opened and incubated at 65°C for 5 minutes.

[0144] Then add 100 microliters of 96% ethanol, and continue to keep the lid closed for 5 minutes. Remove the supernatant with a magnetic separator and wash the DNA sample twice with 1 mL of 70% ethanol.

[0145] Resuspend the bead-DNA sample in 45 μl water and incubate uncovered at 65°C for 15 minutes to remove all traces of ethanol (or add 5 μ...

Embodiment 3

[0152] The protocol of Example 2 was followed with M-PVA beads on which D-mannose was coupled.

[0153] Beads are shown to bind Bacillus, Escherichia coli (pathogenic and non-pathogenic), Listeria, Salmonella, Yersinia.

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Abstract

The present invention relates to a method of isolating cells from a sample which method comprises binding said cells to a solid support by means of a non-specific ligand immobilised on said solid support, particularly to a method of isolating microorganisms from a sample. Preferred ligands for use in such methods include carbohydrates and derivatives thereof. Also described is a kit for isolating microorganisms from a sample comprising: (a) a solid support having immobilised thereon a ligand which is capable of non-specific binding to microorganisms; (b) means for binding microorganisms to said solid support; optionally (c) means for lysing said cells; and optionally (d) means for binding nucleic acid released from said lysed cells to a solid support.

Description

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Claims

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Application Information

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Owner 挪威诊断联合股份有限公司
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