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Solid support

a solid support and support technology, applied in the field of solid support, can solve the problems of low cell-collection efficiency, cumbersome operation, and difficulty in performing cell-collection of a specified bacterial cell in high yield, and achieve the effects of high yield, high accuracy, and safe operation

Inactive Publication Date: 2009-05-21
KONICA MINOLTA MEDICAL & GRAPHICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Therefore, it is an object of the present invention to provide a solid support for performing the steps of isolation and collection of bacterial cell or extraction and purification of nucleic acid, safely, easily, efficiently, and with high yield, in the genetic test for investigation of the presence of pathogenic bacterial infection.
[0012]The present inventors have studied intensively to solve the above-described problems. As a result, the present inventors have found that, by using a solid support comprising a polypeptide having a binding capability with mycolic acid-containing glycolipids or nucleic acid contained in the cell wall of pathogenic bacteria which is immobilized on the solid support, isolation of bacterial cell or extraction and purification of nucleic acid in the genetic test can be performed safely, easily, efficiently, and with high yield, and have thus completed the present invention.
[0015]According to one aspect of the solid support of the present invention, isolation of pathogenic bacteria containing mycolic acid-containing glycolipids in the cell wall from clinical sample can be performed safely, easily, efficiently, and with high yield. To be brief, high-sensitive and highly accurate test can be performed without apprehension of contamination and biohazards or requirement of cumbersome operations, in contrast to the traditional methods.
[0016]In addition, according to another aspect of solid support of the present invention, extraction and purification of nucleic acid can be performed safely, easily, efficiently, and with high yield. That is, since use of hazardous reagents such as chaotropic reagents and organic solvents in the traditional methods is avoided, the purification step is safe as a whole, and substantially no denaturation of isolated nucleic acid is observed. Further, high-sensitive and highly-accurate test can be performed without carrying out such operations as centrifugal separation, filtration, and treatment under reduced pressure.

Problems solved by technology

However, the cell-collection method using a solid support described in US-A-2003 / 153028 has a problem that the cell-collection efficiency is lower as compared to the high-speed centrifugal separation method because the method utilizes nonspecific interaction between proteins on the cell wall of bacteria and a polysaccharide immobilized on a carrier, and has a difficulty in performing cell-collection of a specified bacterial cell in high yield.
In addition, in the method for purifying a nucleic acid, there also remain problems such as cumbersome operation and a difficulty to attain a desired yield.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of MDP1 and its Immobilization on Magnetic Beads

(MDP1 Protein)

[0075]MDP1 protein (hereinafter, referred to as “MDP1”) was prepared from BCG Tokyo strain (hereinafter, referred to as “BCG”) according to the method described in WO 20 00 / 44905. General description is as follows.

[0076]The BCG was resuspended in 50 ml of TMNSH (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 60 mM NH4Cl, and 6 mM 2-mercaptoethanol), and disrupted by ultrasonication. The pellet obtained by centrifugation at 30,000×g for 2 hours was resuspended in 0.25 N HCl by stirring overnight at 4° C., then centrifuged at 20, 000×g for 20 minutes. To the supernatant solution, 0.1 times volume of 100% (w / v) TCA was added under vigorous stirring. The precipitate formed by standing at 4° C. for 4 hours was recovered by centrifugation, and washed once with acidic acetone prepared by adding 0.01 ml of concentrated hydrochloric acid into acetone (20 ml), then washed twice with acetone, and then dried in a vacuum desiccator....

example 2

Recovery of Tuberculosis Bacteria (BCG) Using MDP1 and rMDP1 Immobilized Magnetic Beads

[0083]A 50 μl of luciferase expressing BCG (BCG-Luc) solution (5×104 cfu) was mixed by stirring with 7.5 μg of MDP1-Epoxy BEADS or rMDP1-His-TALON BEADS suspended with binding buffer (PBS). On the side, the same amount of BCG-Luc was mixed by stirring in 50 μl with 7.5 μg of magnetic beads of BUGS'n BEADS version TB (Genpoint AS) under the recommended condition of solution. After standing at 25° C. for 15 minutes, the magnetic beads and the supernatant solution were separated using a magnetic sorting machine, and thereby BCG-Luc was recovered on the magnetic beads. Recovery efficiency of the BCG-Luc was calculated by determination of cell number using Luciferase assay kit (Promega Corp.). The results are shown in Table 1.

TABLE 1Recoveredbacterial numberRecoveryMagnetic beads(cfu)efficiency (%)MDP1-Epoxy BEADS1.4 × 10428rMDP1-His-TALON2.6 × 10452BEADSBUGS' n BEADS0.65 × 104 13

[0084]Harvest efficien...

example 3

Recovery of Tuberculosis Bacteria (BCG) and Extraction of Nucleic Acid Using MDP1-Immobilized Magnetic Beads

[0085]A 50 μl of BCG solution (7.5×104 cfu) was mixed by stirring with 7.5 μg of MDP1-Epoxy BEADS suspended with binding buffer (PBS). On the side, the same amount of BCG was mixed by stirring in 50 μl with 7.5 μg of magnetic beads of BUGS'n BEADS version TB (Genpoint AS) under the recommended condition of solution. After standing at 25° C. for 15 minutes, the magnetic beads and the supernatant solution were separated using a magnetic sorting machine, and thereby BCG was recovered on the magnetic beads. Next, the recovered BCG was re suspended in 50 μl of dissolution buffer (TE), and treated by ultrasonication of 23 kHz at 25° C. for 10 minutes, and thereby, cells were lysed, and DNA was extracted. The obtained DNA was subjected to quantitative PGR, and extraction efficiencies of nucleic acid were compared. The quantitative PCR was carried out using a Mycobacterium nucleic aci...

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Abstract

The present invention provides a solid support for performing steps of isolation of cell or extraction and purification of nucleic acid, safely, easily, efficiently, and with high yield in the genetic test for investigating the presence of pathogenic bacterial infection. A solid support for binding with cell as an embodiment of the above-described solid support, comprises a polypeptide having capability of binding with mycolic acid-containing glycolipid which is immobilized on the surface of a carrier. In addition, a solid support for binding with nucleic acid as another embodiment of the above-described solid support, comprises a polypeptide having capability of binding with nucleic acid which is immobilized on the surface of a carrier.

Description

BACKGROUND OF THE INVENTION [0001]1. Field of the Invention[0002]The present invention relates to a solid support. More specifically, the present invention relates to a solid support comprising a polypeptide which is immobilized on the surface of a carrier.[0003]2. Description of Related Art[0004]Genetic test for investigating the presence of pathogenic bacterial infection is carried out by determining the presence of nucleic acid derived from the pathogenic bacteria in a clinical sample collected from patient. The testing process comprises mainly the following 3 steps: (1) collection of pathogenic bacteria (isolation of cell); (2) bacteriolysis (extraction and purification of nucleic acid); and (3) gene amplification and detection. Yield of the pathogenic bacterial cell and / or the nucleic acid in each step affects greatly on sensitivity and accuracy of the test.[0005]In the above-described step (1) of collecting a pathogenic bacteria, when, for example, tuberculosis bacteria is col...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N1/02C07H21/00
CPCG01N33/5308G01N2333/916G01N33/5695G01N33/54326
Inventor YAMAMOTO, NORIAKITAMAKI, KEIGOMIYAZAKI, KOJINAKAJIMA, AKIHISA
Owner KONICA MINOLTA MEDICAL & GRAPHICS INC