JPH04332865A - Nucleic acid detection device - Google Patents

Abstract

PURPOSE:To achieve an automatic membrane hybridization assembly by placing a title item so that a syringe-type reaction container with a membrane filter for adsorbing a nucleic acid constituent can move between container groups. CONSTITUTION:A reaction container stand 4 and a piston stand 5 move up and down independently by each motor for enabling a reaction container 4 to travel up and down and at the same time the container stand 4 and the piston stand 5 move left and right between container groups consisting of a reagent pallet 8, a thermostat 9, and a detection portion 10 along a guide 7 along with a support stand 6. Then, an inner cylinder of a container 2 is moved up and down by a piston 3 which is fixed to the piston stand 5 for sucking and discharging a sample solution, a reagent, or a washing liquid etc., freely and each reaction of membrane hybridization is performed automatically in a membrane filter 1 which is added to an opening at a bottom of an outer cylinder, thus enabling a reaction liquid to be detected by the detector 10.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid detection device. More specifically, the present invention relates to a detection device for nucleic acid hybridization assay.

0002

BACKGROUND OF THE INVENTION Conventionally, membrane hybridization assays have been mainly used to examine the presence or absence of a nucleic acid component having a specific base sequence in a sample solution. In this method, a small amount of sample solution is placed on a membrane filter, the nucleic acid component is immobilized on the membrane filter, and the membrane filter is immersed in a prehybridization solution, a hybridization solution, a washing solution, and an enzyme reaction substrate solution in order to react. The presence or absence of a nucleic acid component with a base sequence of
"Probe" by Toyozo Takahashi, CMC Publishing, 1989).

0003

[Problems to be Solved by the Invention] In conventional membrane hybridization assays, the membrane filter must be manipulated and immersed in each reaction solution in order to perform each reaction, and the membrane filter is not physically strong, making the device difficult to use. Membrane hybridization assays could not be performed automatically using this method, and had to be performed manually.

[0004] Therefore, the object of the present invention is to provide a method for carrying out each reaction without directly handling the membrane manually.
An object of the present invention is to provide a detection device for nucleic acid hybridization.

[0005]

[Means for Solving the Problems] The present invention solves the above-mentioned problems, and provides a container group consisting of a reagent pallet, a constant temperature bath, and a detection unit, and a nucleic acid component movably arranged between the container group. a syringe-shaped reaction container equipped with a membrane filter that adsorbs the reaction container, and a means for holding the reaction container;
A nucleic acid detection apparatus comprising means for moving the reaction container in the vertical direction, means for moving the reaction container in the horizontal direction, and means for moving the inner cylinder in the reaction container in the vertical direction. Ru.

The present invention will be explained below with reference to the drawings. Figure 1
2 is a reaction vessel in the present invention, and FIG. 2 is a configuration explanatory diagram showing a nucleic acid detection apparatus using the reaction vessel. The membrane filter attached to the reaction container in the present invention is attached to the bottom opening of the outer cylinder as shown in FIG. 1, and the membrane filter is not particularly limited as long as it is used for membrane hybridization. Instead, membranes such as nylon membranes and nitrocellulose membranes are used. Further, the membrane filter can be attached to the bottom opening of the outer cylinder, for example, like a socket. The reaction container in the present invention is a syringe type, and the inner cylinder of the reaction container is movable in the vertical direction, so that the sample solutions and reagents necessary for each reaction of membrane hybridization can be collected by vertically moving the inner cylinder. Suction and discharge of cleaning liquid etc. can be performed in a membrane filter.

In the nucleic acid detection apparatus of the present invention, a container group consisting of a reagent pallet 8, a constant temperature bath 9, and a detection unit 10 arranged as shown in FIG. A pallet 8 is used, and a heating means such as a heating element or an electric heater is used as a constant temperature bath 9 to maintain the temperature at, for example, 60°C. The detection unit 10 is not particularly limited as long as it can detect the membrane hybridization reaction solution, and examples thereof include a method of measuring fluorescence intensity. An example of such a group of containers includes, but is not limited to, a tray or an aluminum block in which a plurality of sample tubes are arranged.

In the nucleic acid detection apparatus of the present invention, the reaction container 2 is fixed to a reaction container stand 4 as a means for holding the reaction container 2. The inner cylinder of the reaction vessel 2 is moved vertically by a piston 3, for example, and the piston 3 is fixed to a piston stand 5. Reaction vessel stand 4 and piston stand 5
can be moved up and down independently by motors, so that the reaction container 2 can be moved up and down, and the inner cylinder can be moved up and down independently of the reaction container 2. As means for moving the reaction container 2 in the horizontal direction, a reaction container stand 4 and a piston stand 5 are used.
This is because the support base 6 moves from side to side according to the guide 7.

[0009] The suction and discharge of the sample solution etc. in the reaction container 2 can be carried out, for example, by accommodating the piston 3 in the reaction container and sucking and discharging the solution by the operation of the piston as described above. The present invention is not limited to this. The piston is preferably actuated by a motor, but is not limited to this, and may be actuated by hydraulic pressure or gas pressure.

[0010] The means for moving the reaction vessels 2 between the groups of vessels is preferably a motor, but is not limited to this, and may also be a hydraulic or gas pressure mechanism. In the apparatus of the present invention, the operation and movement of the reaction vessel 2 are all programmed as a series of operations and controlled by a computer in the control section, and it is preferable that a part of the program can be easily changed, but the invention is not limited thereto.

[0011]

[Function] The reaction container in the present invention is a syringe-type reaction container, and the inner cylinder is movable in the vertical direction.
Sample solutions, reagents, cleaning solutions, etc. can be easily aspirated and discharged by simply moving the inner cylinder in the vertical direction, and each reaction of membrane hybridization can be performed in the membrane filter attached to the bottom opening of the outer cylinder. Can be done. That is, in addition to moving the inner cylinder vertically, the reaction container is moved vertically and horizontally,
Membrane hybridization assays can be performed automatically by moving between container groups consisting of a reagent pallet, a constant temperature bath, and a detection unit.

0012

[Examples] The present invention will be explained in more detail with reference to Examples below. Dupont membrane filter
Enzyme-labeled D
As an NA probe, an oligonucleotide with a length of 20 base pairs that has a complementary base sequence to the target DNA is used as a probe.
Kami et al.'s method (Nucleic Acid Res
Search, 17, 5587, (1989)) labeled with alkaline phosphatase was used.

The procedure is shown below. 20μl of the sample solution to be examined to see if it contains the desired DNA
．． In addition to 80 μl of 3N NaOH, the sample solution is sucked up into a reaction container equipped with a membrane filter according to the present invention, and the sample solution is left in contact with the membrane filter at room temperature for 30 minutes to adsorb the DNA to the membrane filter. After slowly spitting out and discarding the reaction solution, 2xSSC (0.6M NaCl, 0.06M
Slowly aspirate 100 μl of risodium citrate and exhale to wash the membrane filter and the inside of the reaction vessel.

[0014] After spitting out and discarding the 2xSSC, the temperature was 80°C.
The membrane filter and the inside of the reaction vessel are dried by breathing in and out air for 10 minutes in an aluminum block heated to . Next, add 100 μl of prehybridization solution (0.6N NaOH, 20mM EDT
A, 6%, PEG, 0.1% Tween20,
0.5mg/ml Salmon DNA, 1%
0.1M Tris containing skim milk
HCl (pH 8.0)) is slowly drawn in and out at room temperature for 10 minutes to perform prehybridization.

After spitting out the hybridization solution and discarding it, the membrane filter and the inside of the reaction container are
Phosphate buffered saline 20 containing 5% Tween20
Wash twice with 0 μl at room temperature. Subsequently, enzyme reaction buffer (1mM MgC12, 0.1mM ZnCl
0.1M glycine-NaOH buffer containing 2) 200
Wash the membrane filter and the inside of the reaction vessel with μl.

Next, add 200 μl of substrate solution (0.01 m
M 4-methylumbelliferyl p
37% of the enzyme reaction buffer (containing phosphate)
The enzymatic reaction is carried out by slowly sucking and pumping at ℃ for 30 minutes. After that, the substrate solution was mixed with enzyme reaction stop solution (50mM EDTA
0.5M K2 HPO4-KOH buffer (pH
10.3)) Add 200 μl to stop the enzyme reaction. The fluorescence intensity of the finally obtained solution is measured (excitation wavelength: 360 nm, detection wavelength: 450 nm).

[0017] Detecting the target DNA using the above procedure using a PCR reaction solution in which the target DNA was amplified and a PCR reaction solution in which DNA with a different base sequence from the target DNA was amplified as sample solutions. When the target DNA was present, the fluorescence intensity (arbitrary unit) was 6
7.6, whereas it was 26.9 when the target DNA was not present. From this result, the objective D
The fluorescence intensity of the PCR reaction solution containing NA is higher than that of the PCR reaction solution that does not contain the target DNA.
It can be seen that A is detected.

[0018]

[Effects of the Invention] When performing membrane hybridization assays, it has not been easy to handle membrane filters in the past, but with the reaction vessel of the present invention, all reactions can be carried out simply by moving the inner cylinder of the reaction vessel. . Furthermore, membrane hybridization assays can be easily automated by the nucleic acid detection device of the present invention using the reaction container of the present invention.

[Brief explanation of drawings]

FIG. 1 shows a schematic illustration of a reaction vessel in the present invention.

FIG. 2 shows a schematic diagram of the nucleic acid detection device of the present invention.

[Explanation of symbols]

1 Membrane filter 2 Reaction container 3 Piston 4 Reaction container stand 5 Piston stand 6 Support stand 7 Guide 8 Reagent palette 9 Constant temperature bath 10 Detection part

Claims (1)

[Claims] Claim 1: A container group consisting of a reagent pallet, a constant temperature bath, and a detection unit; a syringe-shaped reaction container equipped with a membrane filter that adsorbs a nucleic acid component, which is movably arranged between the container groups; The method comprises means for holding a container, means for moving the reaction container in the vertical direction, means for moving the reaction container in the horizontal direction, and means for moving the inner cylinder in the reaction container in the vertical direction. Nucleic acid detection device.