JPH03264863A - Detecting method of gene - Google Patents

Abstract

PURPOSE:To detect an object gene in a short time and with high sensitivity by a safe and simple operation by a method wherein two or more kinds of gene probes connecting specifically with the object gene at different portions are immobilized by one kind. CONSTITUTION:A single stranded gene is prepared from a sample, two or more kinds of particles prepared by immobilizing, by one kind, two or more kinds of gene probes connecting specifically with an object gene at different portions and a particle prepared by immobilizing an antibody or part thereof recognizing a DNA-RNA hybrid are made to react with the gene, and the object gene existing in the sample is detected by utilizing the agglutination of the particles. The gene probes are not limited particularly, provided that they are hybridized specifically with a part of the gene sequence of the single stranded gene prepared in this way, when the object gene exists therein. Besides, the gene probes of two kinds or more which connect specifically with the object gene at different portions are used.

Description

Detailed Description of the Invention [Object of the Invention] (Industrial Application Field) The present invention relates to a method for specifically detecting a particular gene present in a sample.

(Prior Art) Genetic information imprinted in genes (DNA) is expressed as proteins or enzymes via messenger RNA. Through the action of these proteins and enzymes, various compounds are produced, and living things exist as aggregates of these compounds. The total number of human genes is said to be 50,000 to 10,000. If any abnormalities or changes occur in these genes, such as deletions or duplications, the characteristics, type, amount, etc. of the proteins produced will change, and as a result, the balance of the biological system will be disrupted, causing diseases. . Therefore, conversely, by detecting genes that cause disease, it becomes possible to identify and prevent diseases.

As described above, with recent advances in genetic engineering, diagnosis based on genes themselves (referred to as genetic diagnosis) has become possible. This genetic diagnosis has several features compared to conventional diagnostic methods.

First, considering the mechanism of gene expression, it is presumed that genetic changes occur prior to changes at the biochemical level. Therefore, a major feature of genetic diagnosis is that diagnosis and prediction can be made prior to the phenotypic change of the disease, that is, before the onset of the disease, during the latent period of the disease, or at an extremely early stage.

Another feature of genetic diseases is that they do not depend on the organ or tissue being analyzed, since the genes are the same in all cells within the body.

This is particularly important in fetal diagnosis, which can be diagnosed simply by collecting amniotic fluid from a pregnant woman and examining the fetal cells floating in the fluid.

Below, common genetic diagnosis methods will be briefly explained. That is, a gene is extracted from a sample, cut with an appropriate restriction enzyme, electrophoresed, and then subjected to Southern blotting to generate a gene probe (usually labeled with a radioactive isotope) corresponding to the gene of interest. After hybridization, the DNA is exposed to X-ray film at low temperatures to confirm the presence or absence of the target gene. However, since this method uses radioactive isotopes, detection locations are limited and the reagents must be handled with great care.

In order to improve this point, it is desired to develop a safe labeling agent to replace radioactive isotopes. Already, avidin-
Proposals include methods that utilize biotin binding, and methods that use enzymes and fluorescent substances. However, none of these methods has reached the point where it exceeds that of radioactive isotopes in terms of sensitivity. Furthermore, it takes at least 2 to 3 days to detect the gene, and the measurement operation is also quite complicated.

On the other hand, in patent application publication No. 83-502875,
A nucleic acid detection method is described in which particles to which a nucleic acid sequence complementary to a target nucleic acid sequence is bound are reacted with a sample, and the target nucleic acid sequence present in the sample is detected using particle aggregation. has been done. This publication also illustrates a method using two or more types of particles on which one of two or more types of gene probes each complementary to different sites of the target nucleic acid sequence is immobilized. There is. However, according to the results of studies conducted by the present inventors on this method, the aggregation rate of particles is slow when using particles bound with a complementary nucleic acid sequence to the nucleic acid sequence, and genes labeled with radioactive isotopes are There is a problem in that several hours of reaction time are required to obtain the same level of sensitivity as conventional methods using probes.

(Problems to be Solved by the Invention) The present invention has been made to solve the above-mentioned problems, and is capable of detecting a target gene with high sensitivity while being able to be carried out in a short time using safe and simple operations. The purpose of this study is to provide a gene detection method that allows for the detection of genes.

[Structure of the Invention] (Means for Solving the Problems) The gene detection method of the present invention involves preparing a single-stranded gene from a sample, and detecting two types of genes that specifically bind to the target gene at different sites. Two or more types of particles on which one type of each of the above gene probes is immobilized and particles on which an antibody or a part of the antibody that specifically recognizes a DNA-RNA hybrid is immobilized are reacted to prevent aggregation of the particles. This method is characterized in that it is used to detect a gene of interest present in a sample.

In the present invention, operations up to the preparation of single-stranded genes from samples are performed according to conventional methods (e.g., clinical tests, Vo
l, 32. No, 4. p. 401 (1988)).

That is, a double-stranded gene is extracted from a sample, fragmented with a restriction enzyme, and then denatured with an alkali, for example, to prepare a single-stranded gene.

In the present invention, when the gene of interest is present in the single-stranded gene prepared as described above, the gene probe is particularly limited as long as it specifically hybridizes with a part of the gene sequence. Not done. Furthermore, in the present invention, two or more types of gene probes that specifically bind to the gene of interest at different sites are used.

In the present invention, the antibody or a part of the antibody that specifically recognizes DNA-RNA7\ibrynd is IgG, IgE
, IgD, IgA, IgM, etc. In addition, from the point of view of improving sensitivity,
It is preferable to use monoclonal antibodies rather than polyclonal antibodies.

In addition, F (ab') obtained by removing the Fc part of the antibody
Two antibodies may be used.

In the present invention, the materials constituting the particles for immobilizing the gene probe and the particles for immobilizing the antibody or a part of the antibody that specifically recognizes the DNA-RNA hybrid are heated at a temperature for hybridization. Although there is no particular limitation as long as it is stable, latex particles made mainly of methyl methacrylate or the like are generally preferred. Latex particles can be prepared by ordinary emulsion polymerization using methyl methacrylate or the like as a main raw material.

In addition, RNA gene probes and antibodies can be immobilized on the surface of latex particles using, for example, the carbodiimide method (rNew use cases of immunoassay methods and applications to the development of diagnostic reagents and therapeutic drugs" (published by Management Education Publishing) , pp.
, 105-113.1986).

Hereinafter, the gene detection method of the present invention will be explained in more detail.

Here, the case of detecting the hepatitis virus (HBV) gene from human blood will be explained as an example. In addition, in advance, H
Two types of latex particles on which one of two types of RNA gene probes for the BV gene are immobilized, and latex particles on which an antibody that specifically recognizes a DNA-RNA hybrid is immobilized are prepared in advance.

First, double-stranded DNA was extracted from a sample (human serum) according to a conventional method.
A is extracted, fragmented by cutting it with an appropriate restriction enzyme, fractionated by electrophoresis, and denatured with alkaline to obtain single-stranded DNA.
Prepare A. At this time, in order to prevent self-hybridization between single-stranded DNAs, the reaction was
It is preferable to carry out the test at temperatures above ℃. After neutralizing this, 50
Cool to ~60°C and test with two types of R against the HBV gene.
Two types of latex particles each immobilized with one type of NA gene probe are reacted in the presence of 50% formamide. If HBV genes are present in the sample, a DNA-RNA hybrid is formed by this reaction,
Latex agglomeration occurs. Next, after cooling to an appropriate temperature, latex particles immobilized with an antibody that specifically recognizes the DNA-RNA hybrid are added and reacted for a certain period of time.

This reaction further causes latex aggregation if a DNA-RNA hybrid is formed. Therefore, the presence or absence of the HBV gene can be determined by measuring the change in turbidity of the reaction solution (corresponding to the aggregation of latex particles) using a suitable spectrophotometer after the reaction.

The reaction conditions such as time, temperature, and pH required for each of the above-mentioned reactions depend on the gene to be tested, the type of corresponding gene probe (length, base sequence, etc.), the characteristics of the latex particles (particularly particle size), and even the latex. D immobilized on the surface of the particle
It varies depending on the type, amount, purity, etc. of the antibody or part of the antibody that specifically recognizes the NA-RNA hybrid. Therefore, it is desirable to set optimal reaction conditions and buffer solutions depending on each individual case.

(Function) The gene detection method of the present invention uses only two or more types of particles on which one of two or more types of gene probes that specifically bind to the target gene at different sites is immobilized. However, since particles on which an antibody or a portion of an antibody that specifically recognizes a DNA-RNA hybrid is immobilized are used, aggregation of particles occurs extremely quickly as a result of the reaction. In addition, since there is no need to perform an operation to separate hybrids, the measurement time is shortened and the device is easy to implement.

Furthermore, it is possible to obtain the same level of sensitivity as conventional methods that use radioactive isotopes as labeling agents for gene probes. Furthermore, since no radioactive isotopes are used, there is no need to select a measurement location, and operation is safe.

(Example) The present invention will be explained in detail below.

■Preparation of latex particles Latex particles were prepared by emulsion polymerization using methyl methacrylate as the main raw material. The particle size of the latex particles was about 10 Or+m.

■Modification of monoclonal anti-DNA-RNA/hybrid-recognizing antibody and its immobilization on latex particles Prepare monoclonal anti-DNA-RNA hybrid-recognizing antibody (subclass: IgG), and 100 μg of it
The mixture was dialyzed against 0.1 M acetate buffer (pH 4,5), 10 μg of pepsin (manufactured by Sigma) was added, and the mixture was reacted at 37° C. for 1 hour. Next, only the F (ab') 2 fraction was collected by high performance liquid chromatography. The volume of this protein fraction solution is 9.5mj7゜Concentration is 0D280
It was a car.

ε-Aminocaproic acid and water-soluble carbodiimide were added to the latex particle suspension (50 mg/mD), and the mixture was stirred at 4° C. for 2 hours to react. After thorough centrifugal washing, the solution of the modified antibody was added and stirred at 10° C. for 24 hours to react. After the reaction, it was washed three times with gelatin veronal buffer (hereinafter referred to as GVB-). Finally, the obtained latex particles were suspended in GVB 2mρ and stored at 4°C until use.

(2) Immobilization of RNA gene probes on latex particles Two types of RNA gene probes for the HBV gene were prepared.

ε-aminocaproic acid and water-soluble carbodiimide were added to latex particle suspension a (50 mg/ml), and the mixture was stirred at 4°C for 2 hours to react. After thorough centrifugal washing, two types of RNA gene probe solutions were added separately and stirred at 10°C for 24 hours to react. After the reaction,
Gelatin veronal loose? It was washed three times with 1M (hereinafter referred to as GVB-). Finally, the obtained latex particles were suspended in 2rrl of GVB and stored at 4°C until use.

■Detection of HBV genes in human serum Detection of HBV gene from genes in human serum using the aforementioned RNA gene probe immobilized latex particles and monoclonal anti-DNA-RNA hybrid recognition antibody immobilized latex particles. I tried.

Conventional method (clinical examination, Vol. 32. No. 4. p. 40
(1988)), double-stranded DNA was extracted from human serum.
A was extracted, fragmented using the restriction enzyme Pstl, and then denatured with alkali to prepare single-stranded DNA, which was neutralized. 1
ml of GVB containing 50% formamide (GVB containing 0, 5 m M MgCj!2 and 0.15 m M Ca
Two types of RNA gene probe-immobilized latex particles were added to the solution (added with Cl2), and 6
Hybridization was carried out by reacting at 0°C for 30 minutes. Next, the reaction solution was diluted 10 times with GVE+, and a suspension of latex particles immobilized with a monoclonal anti-DNA-RNA hybrid recognition antibody previously diluted 10 times with GVB'' was added.
0 μρ was added and incubated at 37° C. for 10 minutes. After this reaction, the increase in absorbance at 850 ni (corresponding to agglomeration of latex particles) was measured using a spectrophotometer. As a control, the absorbance of a reaction solution obtained from a sample (human serum) excluding only the gene was used.

Table 1 shows the relative fluorescence intensities of the IO samples. this house,#
Samples #1 to 7 are serum samples from patients with hepatitis, #8 to #10 are serum samples from normal subjects.

As is clear from Table 1, it can be seen that the HBV gene was detected only from hepatitis patients.

Furthermore, when the detection sensitivity for the standard HBV gene was investigated, it was shown that it was possible to detect from about 5 pg of DNAJit, which was approximately the same sensitivity as the conventional method using a gene probe labeled with a radioactive isotope.

[Effect of the invention] As detailed above, according to the gene detection method of the present invention,
Genes of interest can be detected with high sensitivity in a short time using safe and simple operations.

Claims (1)

[Claims] A single-stranded gene is prepared from a sample, and two or more particles each have immobilized one of two or more gene probes that specifically bind to the target gene at different sites;
and a particle on which an antibody or a part of the antibody that specifically recognizes the DNA-RNA hybrid is immobilized is reacted, and the target gene present in the sample is detected using the aggregation of the particles. Gene detection method.