JPH03267000A - Method for detecting gene - Google Patents

Abstract

PURPOSE:To detect a specific gene by reacting single-stranded gene with a specific gene probe labeled with bacteriolysin to hybridize the gene and reacting the resultant hybrid with liposome reagent obtained by sealing a labeled substance in a phospholipid. CONSTITUTION:When gene of hepatitis virus (HBV) is detected from a human blood, double stranded DNA 2 is extracted from a sample 1 such as leukocyte and then cut with a restriction to fragmentate the DNA and the frangments are fractionate with electrophoresis and modified with an alkali to give a single- stranded DNA 3. Then after neutralizing the DNA 3, the single stranded DNA 3 on a nitrocellulose membrane 4 is reacted with methilin-labeled gene probe 5 to form a hybrid 6. The hybrid 6 is cleaned with a buffer to remove unreacted probe and the membrane 4 is reacted with a proper amount of liposome reagent 7. Thereby the reagent 7 is broken by reaction with methilin and a labeled substance 6 is allowed to flow and measured by a proper means to judge HBV gene.

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 100,000. If any abnormalities or changes occur in these genes, such as deletions or duplications, the characteristics, types, amounts, etc. of the proteins produced will change, resulting in an imbalance in the biological system and causing disease. . Therefore, by detecting genes that cause disease, it is 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 it allows diagnosis and prediction to 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, a general genetic diagnosis method will be briefly explained. That is, a gene is extracted from a sample, cut with an appropriate restriction enzyme, electrophoresed, Southern blotted, and a gene probe (usually labeled with a radioactive isotope) corresponding to the gene of interest is hybridized. The soybeans are grown and then 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.
Reagents must also 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 them have reached the point where they surpass radioisotopes 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.

(Problems to be Solved by the Invention) The present invention has been made to solve the above-mentioned problems, and has an object to detect a target gene with high sensitivity, which can be carried out in a short time with 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 labeling a single-stranded gene prepared from a sample with a hemolysin, and detecting a gene that specifically reacts with the gene of interest. After reacting and hybridizing the probe, a liposome reagent containing at least one of phospholipids and glycolipids and encapsulating a labeling substance is reacted, and the labeling substance flowing out from the liposome reagent that has collapsed due to the action of hemolysin is removed. It is characterized by detection.

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. After neutralizing this, it is blotted onto a nitrocellulose membrane.

In the present invention, the gene probe specifically reacts with the gene of interest to form a hybrid when the gene of interest is present in the single-stranded gene prepared as described above. In the present invention, the gene probe is
Labeled by hemolysin. Examples of the hemolysin include melittin, streptolysin S1-leptolysin 01 titanolysin, alpha element, and the like. In order to immobilize hemolysin on the gene probe, methods commonly used for immobilizing enzymes and the like can be applied. There is no need to use radioactive isotopes as labeling agents for this gene probe.

For example, melittin, a type of hemolysin, is a type of bee venom produced by bees, etc., and is composed of 26 amino acids.
Molecule N2846 is a polypeptide of 54 and has very strong basicity. Melittin has the effect of directly lysing red blood cells and the like, and is said to attack lipid bilayers as phospholipase A. It is also known that melittin acts on liposomes and causes them to collapse.

In the liposome reagent used in the present invention, at least one of phospholipids and glycolipids is used as the main component of the liposome. Phospholipids and glycolipids are not particularly limited, and include, for example, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE),
Dimyristoylphosphatidylethanolamine (DM
PE), distearoylphosphatidylethanolamine (DSPE), and the like. These phospholipids,
The fatty acid carbon chain in the glycolipid preferably has 12 to 18 carbon atoms, more preferably an even number. Note that, if necessary, cholesterol may be added to the phospholipids and glycolipids at a ratio of 10 to 500 mol%, thereby making it possible to prepare a stable lipid bilayer membrane.

In the liposome reagent used in the present invention, the labeling substance encapsulated within the liposome is selected from a substance that is hydrophilic and can be quantified when it flows out of the liposome. Examples of such substances include fluorescent substances such as carboxyfluorescein, which does not exhibit fluorescence due to self-quenching at high concentrations, but emits very strong fluorescence at low concentrations (10-3 M or less); Luminescent substances such as luminol and luciferin; light-absorbing compounds (water-soluble dyes, etc.) with specific absorption bands in the visible or ultraviolet range; decomposed by the action of oxidative enzymes, consuming oxygen or producing hydrogen peroxide Sugars such as glucose and sucrose, and their oxidizing enzymes: Relatively large ionic compounds such as tetrapentylammonium; Coenzymes such as nicotinamide adenine dinucleotide (NAD); Radical compounds such as methyl viologen, etc. can be mentioned. These compounds are appropriately selected in consideration of factors such as detection method, sensitivity, and stability of liposomes.

A method for producing the liposome reagent used in the present invention will be explained. First, a lipid mixture (usually an organic solvent such as chloroform is used) containing an appropriate amount of a suitable phospholipid or glycolipid and, if necessary, cholesterol or other lipids, is prepared.

An appropriate amount of this lipid mixture is placed in a pear-shaped flask with an appropriate volume, and the solvent is removed using a rotary evaporator.

Add an appropriate amount of solvent again, and remove the solvent again using a rotary evaporator. By repeating this operation several times,
A uniform thin lipid film forms on the wall of the pear-shaped flask.

Next, add an appropriate amount of an aqueous solution of a labeling substance at an appropriate concentration into a pear-shaped flask, heat it to an appropriate temperature, and then shake vigorously for an appropriate time using a portex mixer to prepare multilamellar liposomes. .

Hereinafter, the gene detection method of the present invention will be explained in more detail with reference to FIG. Here, the case of detecting the hepatitis virus (HBV) gene from human blood will be explained as an example. Note that melittin was used as the hemolysin, modified in advance with a bifunctional reagent, and reacted with a gene probe for the HBV gene to create a melittin-labeled gene probe (also, by the method described above, Prepare the liposome reagent in advance.

First, double-stranded DNA 2 is extracted from a sample (white blood cells, etc.) 1 according to a conventional method, cut into fragments by cutting with an appropriate restriction enzyme, fractionated by electrophoresis, and denatured with alkali to become single-stranded DNA. After further neutralization, the DNA was prepared as DNA 3, and then proteased onto a nitrocellulose membrane 4. Next, the nitrocellulose membrane 4
Melittin-labeled gene probe 5 is added to the single-stranded DNA 3 above.
react. If the HBV gene is present in sample 1, hybrid 6 is formed by this reaction.

Thereafter, it is thoroughly washed with an appropriate buffer solution to wash away unreacted probe. Finally, an appropriate amount of liposome reagent 7 is reacted with the nitrocellulose membrane 4. When the hybrid 6 is formed, melittin that labels the gene probe remains, and its action destroys the liposome reagent 7, causing the labeling substance (e.g. fluorescent substance) sealed inside. ) flows out. Therefore, by measuring this using an appropriate method (eg, fluorescence spectrophotometer), the presence or absence of the HBV gene can be determined.

The reaction conditions such as time, temperature, and pH required for each of the above-mentioned reactions vary depending on the gene to be tested, the type of corresponding gene probe (length, base sequence, etc.), the characteristics of the Lipotum reagent, etc. Therefore, it is desirable to set the optimal reaction conditions and buffer solution depending on each individual case.

0 (Function) In the gene detection method of the present invention, when the liposome reagent is destroyed by the action of the hemolysin that labels the hybridized gene probe, a large amount of labeling substance flows out from inside the liposome, so that the amplification is delayed. Effects can be obtained. Therefore, sensitivity comparable to conventional methods using radioisotopes as labeling agents for gene probes can be obtained. Moreover,
Since it does not use radioactive isotopes, it can be operated safely without the need to select a measurement location.

Furthermore, since there is no need to perform an operation to separate gene hybrids, the measurement time is short and the device is easy to implement. Furthermore, regardless of the type of gene of interest,
Since common liposomes can be used, a significant reduction in reagent development costs can be expected.

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

Among the reagents used in this example, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE)
, cholesterol and melittin were those manufactured by Sigma. As for other reagents, commercially available products (special grade) were used without purification.

Note that all water used was ion-exchanged water.

(2) Preparation of carboxyfluorescein-containing liposome reagent All the lipids used were dissolved in chloroform or a chloroform/methanol (2/I) mixed solvent.

200μi of 5mM DPP, 100μg of 10mM cholesterol and 25μD of 5mM stearylamine were placed in a pear-shaped flask with a capacity of 10mΩ, and 2mg of chloroform was added and mixed well.

The solvent was then removed by rotary evaporation in a water bath at approximately 40°C. After about 2 mg of chloroform was added again and thoroughly stirred, the solvent was removed again using a rotary evaporator. When this operation was repeated several times, a thin lipid film was formed on the flask wall. Subsequently, the flask was transferred to a desiccator and suctioned with a vacuum pump for about 1 hour to completely remove the solvent.

Next, 0.2M carboxyfluorescein (manufactured by Eastman Kodak, pH 7.4: hereinafter referred to as CF)
After adding 100 μg and purging the inside of the flask with nitrogen, the flask was tightly stoppered and immersed in a water bath at about 60° C. for about 1 minute. Subsequently, the flask was vigorously shaken using a portex mixer until the lipid thin film on the flask wall completely disappeared. A multilayer liposome suspension was prepared by this operation. Additionally, 0.01M HEP was added to the liposome suspension.
Contains ES buffer (0.85% NaC4), pH 7.4
5: After adding a small amount of HBS (hereinafter referred to as HBS), transfer everything to a centrifuge tube and centrifuge at 15,000 rpm for 20 minutes at 4°C.
The operation of centrifugation for 1 minute was repeated several times. Finally, while washing the centrifuge tube with HBS, transfer the liposome suspension to a serum tube (Corning), centrifuge once to remove the supernatant, resuspend in 2 mg of HBS, and resuspend until use. Stored in the refrigerator.

■Preparation of melittin-labeled gene probe A commercially available HBV probe (100 μg) was added with lomM 5PDP (manufactured by Pharmacia) as a bifunctional 3 reagent.
/HBS solution was reacted at 87°C for 80 minutes.
0.1M dithiothreitol/acetate buffer (pH 4,
5) was reduced. On the other hand, Img melittin was similarly 5P
Treated with DP. A melittin-labeled HBV gene probe was prepared by mixing the two and reacting overnight at room temperature. The product was analyzed by high performance liquid chromatography (HPLC).
, manufactured by Shimabara Seisakusho).

(2) Detection of HBV gene in human blood Using the aforementioned liposome reagent and melittin-labeled HBV gene probe, an attempt was made to detect the HBV gene from genes in human blood.

Conventional method (clinical examination, Vol. 32. No. 4. p. 40
(1988)), double-stranded DNA was extracted from human blood.
A was extracted, fragmented with restriction enzyme Pst1, denatured with alkali to prepare single-stranded DNA, further neutralized, and then blotted onto a nitrocellulose membrane.

This nitrocellulose membrane was reacted with a melittin-labeled HBV gene probe at 65° C. for 30 minutes and subjected to Vibe 4 rehydration. Then, the nitrocellulose membrane was coated with HBS.
Washed thoroughly. On the surface of this nitrocellulose membrane,
Suspension of liposome reagent diluted 10 times with HBS 10
0 μg was added and incubated for 30 minutes at 37°C.

Then, the amount of CF that flowed out was measured using a fluorescence spectrophotometer (MTP'-
32, Corona Electric) at an excitation wavelength of 490 nm.

Measurement was performed under the condition that the fluorescence wavelength was 520 nm, and the relative fluorescence intensity was calculated based on the following formula.

Here, Fe - actually measured fluorescence intensity, Fo = fluorescence intensity when no liposomes are destroyed, and fluorescence intensity when all liposomes are destroyed by adding Faz deionized water.

Table 1 shows the relative fluorescence intensities of the 10 samples. this house,#
Numbers 1 to 7 are blood samples of hepatitis patients and #8 to 10 are blood samples of normal people.

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

In addition, when we examined the detection sensitivity of standard HBV genes using liposome reagents, we found that it was possible to detect DNA from an amount of approximately 1 pg, which is approximately the same sensitivity as the conventional method using a gene probe labeled with a radioactive isotope. Shown.

[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.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the gene detection method according to the present invention. DESCRIPTION OF SYMBOLS 1... Sample, 2... Double-stranded DNA, 3... 1 channel 6 NA, 4... Nitrocellulose membrane, 5... Melittin labeled gene probe, 6... Hybrid, 7...・・・
Liposome reagent, 8... labeling substance.

Claims (1)

[Claims] After reacting and hybridizing a single-stranded gene prepared from a sample with a gene probe labeled with hemolysin that specifically reacts with the target gene, at least one of phospholipids and glycolipids is added. 1. A gene detection method, which comprises reacting a liposome reagent having the same composition and encapsulating a labeling substance, and detecting the labeling substance flowing out from the liposome reagent which has been disintegrated by the action of hemolysin.