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Method for preparing nucleic acid probe-coated porous solid support strip

A porous solid and nucleotide technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/testing, etc., can solve the difficulty of controlling the level of cross-linking of thymine bases, long-term cycle, high cost and other issues to achieve the effect of mass production and automated processing

Active Publication Date: 2012-07-11
LG CHEM LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, the problem with UV irradiation of membranes is that it is quite difficult to control the level of cross-linking by thymine bases
[0024] However, conventional techniques employing direct covalent attachment between probes and membranes are generally limited to amine-labeled probes and carboxyl-activated (using carbodiimide (e.g., 1-ethyl-3-[3-dimethylamino Covalent linkage between membranes of propyl]carbodiimide hydrochloride (EDC or EDAC reagent))
[0025] Furthermore, the method of immobilizing nucleotide probes to membranes in the form of controlled ligation on membranes is a batch process in nature, which is difficult for large-scale production and automated processing.
Furthermore, these methods require long periods of time and high cost
[0026] Due to the physical and chemical advantages and disadvantages associated with the production of nucleotide probe-coated membrane strips for nucleotide hybridization assays, large-scale production of normally hybridizing membrane strips can be performed in a simple and rapid manner, as described above. production is limited

Method used

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  • Method for preparing nucleic acid probe-coated porous solid support strip
  • Method for preparing nucleic acid probe-coated porous solid support strip
  • Method for preparing nucleic acid probe-coated porous solid support strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Embodiment 1: Preparation polyaldehyde dextran (PAD)

[0110] (1) Preparation of required reagents and instruments

[0111] Using dextran (Sigma, D-4133, Mr ~ 43kDa), KIO 4(Aldrich, 32242-3, F.W230.00), cellulose dialysis membrane cut-off 12000: Spectra / Por, MWCO 12-14000), hydroxylamine hydrochloride (NH 2 OH-HCl=69.49), methyl orange powder, 10N hydrochloric acid, acidity meter, 0.1N sodium hydroxide (NaOH), freeze dryer and vacuum centrifugal evaporation concentrator, etc.

[0112] (2) Preparation of oxidized dextran

[0113] 0.5 g of dextran (Sigma, D-4133, Mr~43 kDa / equivalent to 62.5 mmol of glucose) was completely dissolved in autoclaved D.W to adjust the total volume to 10 ml. Add 0.72g of KIO to it 4 (equivalent to the same equivalent of 62.5 mmol) (reference: Azzam T et al., J.Med.Chem., 45, 1817-1824, 2002; Azzam T et al., Macromolecules, 35, 9947-9953, 2002; Azzam T et al. People, J. Controlled Release, 96, 309-323, 2004; Hosseinkhani H. et al,. G...

Embodiment 2

[0135] Embodiment 2: the preparation of nucleotide probe

[0136] The probes used in this experiment were designed to detect: (i) Mycobacterium tuberculosis and identify resistance to rifampicin and isoniazid drugs, (ii) detect mycobacteria, i.e. distinguish non-tuberculous mycobacteria ( NTM) and Mycobacterium tuberculosis (TB) species.

[0137] First, for aspect (i), when the probe goes to detect M. tuberculosis and recognizes M. tuberculosis resistance to rifampicin and isoniazid, it will target the transcript specific for the M. tuberculosis complex Probes for the interspacer (ITS) locus were used for the identification of Mycobacterium tuberculosis.

[0138] When using probes to identify susceptibility and resistance to rifampicin (antituberculosis drug), use an anti-tuberculosis drug that is wild-type or mutated in the gene encoding rpoB (ie, RNA polymeric beta-subunit) Oligonucleotides of type base sequence.

[0139] In addition, when probes were used to detect sus...

Embodiment 3

[0153] Embodiment 3: Preparation of nucleotide probe-polyaldehyde dextran conjugate

[0154] (1) Preparation of required reagents

[0155] Prepare 0.5 M sodium borate buffer (pH 11.0) and use it as buffer for binding reaction, prepare 0.1 mM NaBH 4 solution to serve as a mild reducing agent for the conversion of Schiff's bases through the reaction between the aldehyde group of polyaldehyde dextran as a support and the amino group at the 5'-terminus of the nucleotide probe via Stable covalent linkages are formed.

[0156] (2) Preparation of the main mixed solution for the binding reaction

[0157] The main mixed solution consisted of polyaldehyde dextran (1 pmol / μL), 0.5M sodium borate buffer (pH 11.0) and sterilized distilled water. These components were mixed in the proportions listed in Table 5 below.

[0158] [table 5]

[0159]

[0160] (3) Binding reaction

[0161] 40 sheets of films having a size of 16.7 cm x 5.5 cm and a thickness of each line of 0.8 mm ...

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Abstract

Disclosed is a method for preparing a nucleic acid probe-coated porous solid support strip. The method includes linking a nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support by vacuum transfer.

Description

technical field [0001] The present invention relates to a method of preparing a porous solid support strip coated with nucleotide probes. More specifically, the present invention relates to a method for preparing a nucleotide probe-coated porous solid support test strip, which method comprises linking the nucleotide probe to a carrier to prepare a conjugate and transferring the The conjugate is immobilized on a porous solid support. Background technique [0002] Nucleotide diagnostics are utilized in determining genetic factors of disease, adequacy of treatment, bioassays, and the like. [0003] In general, techniques for analyzing specific nucleotide sequences in samples of nucleotides isolated from bacteria or cells or in products obtained by amplifying such samples by nucleotide polymerization are used to confirm the presence of nucleotides, which A nucleotide fragment (ie, a nucleotide probe) having a complementary base sequence specifically linked to a part of the nuc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/32
CPCC12Q1/6834C12Q2537/125C12Q2537/143C12Q2565/518C12Q1/6876C12Q2565/625
Inventor 权准徹朴荣石姜镇锡吴宰勋俞恩珠
Owner LG CHEM LTD
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