JPH02119799A - Method for detecting nucleic acid - Google Patents

Abstract

PURPOSE:To rapidly and readily detect a nucleic acid with high sensitivity by hybridizing the nucleic acid having a specific base sequence with an enzyme- labeled probe for hybridization assay and detecting an enzymic reaction product of the labeled enzyme using an electrochemical detector. CONSTITUTION:An enzyme is reacted with a label in hybridization assay to detect a nucleic acid having a specific base sequence. In the process, an enzyme- labeled probe prepared by linking a nucleic acid having a base sequence complementary to the nucleic acid having a specific base sequence to be detected with an enzyme (e.g., an alkaline phosphatase) and a binder, etc., is hybridized with the nucleic acid to be detected in a specimen and the labeled enzyme is enzymically reacted with a substrate (e.g., phenyl phosphate) to detect the product thereof (e.g., phenol) using an electrochemical detector, such as an amperometric detector. Thereby, the target nucleic acid is detected.

Description

[Detailed description of the invention] (b) Industrial application field The present invention is a method for detecting nucleic acids having a specific base sequence.

More specifically, the present invention relates to a detection method using nucleic acid hybridization.

(Example) Prior Art Nucleic acid hybridization is used as a method for detecting all nucleic acids having a specific base sequence of interest in a specimen sample. In this analysis method, a single-stranded nucleic acid forms a hypurinode with another single-stranded nucleic acid containing a complementary base sequence under appropriate conditions through sequence-specific hydrogen bonds (hy7ridized). It takes advantage of the fact that

That is, in hybridization assay, a nucleic acid having a known sequence is used as a probe to examine whether a sample has a complementary sequence that serves as a target. Labeling the hybrid nodes formed by the probe and target allows detection and quantification of complementary sequences in the sample.

One way to label probes to detect hybrids is to label them with radioisotopes (e.g.
2P or I)k bonding. However, due to safety, stability, and equipment issues, non-radioactive labeling systems have recently been developed.

The first type of non-radioactive labeling system is
A functional group that produces fluorescence or chemiluminescence is directly introduced into the probe through a covalent bond.

The first type is to covalently introduce a site recognized by a macromolecule such as an antibody or biotin into a probe, and then recognize it with a fluorescent label or an r+c chemiluminescent label macromolecule to detect the presence of the probe. It is something to do.
The third type utilizes the amplification effect of enzymes in addition to the systems of the first and second types.

Hybridization assays are often used for disease diagnosis, and are therefore quick and simple.

There is a need for a method that is highly sensitive and does not require the special equipment required to handle radioactive isotopes.

For these reasons, a third type of non-radioactive labeling system has recently been developed.

A third type of non-radioactive system is one in which an enzyme is covalently introduced directly into the probe (Ren
z, M., and Kurz, C. (1984).
Nucl, Ac1dsRes, 12.3435-3
444) is known.

The outline of conventional nucleic acid hybridization assays is as shown below.

First, the nucleic acid in the specimen sample is denatured to become single-stranded, and then 2n
After it has been slightly burnt, fix it.

Next, in order to suppress the non-specific absorption of the labeled probe, pre-hybridization vanofur (for example,
EDTA, sodium salts, sodium dodecyl sulfate, polyethylene glycol, Ficoll.

Polyvinylpyrrolidone, bovine serum albumin, salmon sperm D
Prehybridization is performed in Tris-HCl buffer containing NA and formamide).

Prehybridization is usually carried out at 37°C with 30 pairs.
After prehybridization, the membrane on which the sample nucleic acid is immobilized is incubated overnight in a hybridization buffer (same composition as the prehybridization buffer) containing a labeled probe.

Then add sodium dodecyl sulfate and sodium citrate to remove labeled probes that have not hybridized to the sample nucleic acids that have been fixed.

Wash with a lithium-hydrochloric acid buffer containing sodium chloride.

When the washed solution is incubated in a buffer containing an enzyme reaction substrate that generates precipitable dyes, soluble dyes, fluorescent substances, etc. by enzymatic reaction, complementary substances to the labeled probe are added to the sample sample. The membrane becomes colored only when it contains a nucleic acid with a specific sequence.

Alternatively, fluorescence or luminescence is observed. In this way, a specific base sequence in the nucleic acid of the specimen sample is detected.

(c) Problems to be solved by the invention However, currently it is sufficiently sensitive, quick, and simple.

A non-radioactive labeling system for quantitatively detecting target nucleic acids has not been established.

The reason for this is that a system for detecting enzymes quickly, simply, quantitatively, and with high sensitivity has not been available.

Currently, the following systems are known as systems for hanging enzymes. 1. Colorimetric method, 2. Deposition pigment method. 3 Fluorescence method. 4 Bioluminescence method. 5 Chemiluminescence method.

We will discuss these problems in turn.

1 Colorimetric method. Although this is currently the most popular method, it generally has poor sensitivity. In the 1001Lin assay, alkaline phosphatase (hereinafter abbreviated as AT, P) was 200a ttomol (Eiji Ishikawa et al., "Enzyme immunoassay j p58. 1987, published by Igaku Shoin), β-
D-galactosidase, IQQ attomol
(Eiji Ishikawa et al., “Enzyme immunoassay j p58.1987
Published by Igaku Shoin).

Peroxidase can only be detected up to 5 attomol (Bos, E., 8., van der
Doeien, A., A., lan ROOY.

N,, 5chuurs, A, W, M, : J-
Immunoassay, 2: 187-204°1981). On the other hand, a method using enzymatic cycling using NADP as a substrate uses alkaline phosphatase (hereinafter abbreviated as ALP).
It can be detected by m oli (Johannsson
,A., 3tanley.

C, J, and 5elf, C, H,: C11n
, Chim, Acta, 1481119-124,
]9ss) In addition to Ka-AbP, it requires diaphorase and alcohol dehydrogenase, making the system complex, and it is not practical because the stability of these enzymes and the inhibition of the enzyme reaction due to the presence of impurities affect the detection sensitivity. There is a problem.

2 Deposition pigment method. The immobilized ALP was converted into 5-bromo-4-
Detection method using chloro-3-indolylphosphate l- etc. (D, A-Knecht, R, L, Dimon
d: Anal, Biochem, 136.180
-184 (1984)) etc. are known. It's Kade.
Products that apply the pigment deposition method to nucleic acid hybridization (5NAP from DUPONT)
Hybridization kit is this kit.
Using t f 4. Qattomol DNA can be detected (DUPONT 5NAP HYBRIDIZAT
ION SYSTEM TBCNICAL DATA 5
HEET). These methods have relatively good sensitivity because the dye is deposited only around the immobilized enzyme, but there is a problem in that it is difficult to perform quantitative measurements. In addition, it uses a 3-fluorescence method that makes it difficult for the automatic f-light to be damaged by the reading device. In general, it is more sensitive than colorimetric methods and can be used to measure θ-D-galactosidase with Q of 100 without using enzymatic cycling
, Q up to 2 attomol can be detected (Imaga
wa, M, , Hasbida, S, , 0h
ta, Y. and Ishikawa, E.:
Ann, CI in, Biochcm, +2
1: 310-317 +1984), but this enzyme cannot be used in hybridization assays because it is unstable to heat.

Horseradish peroxidase can be detected up to Q, 5 attomol in a 100 m assay (Zaits
u, K, and 0hkura, Y: Ana
! , Biochem, , 109:109-113
．． 1980). ``And ALP can be detected down to 1 attomol in a 100ain assay.These are not sensitive enough to be used in hybridization.Another problem is that the detectors are expensive.

4 Bioluminescence method. ATP, NADH, etc. can be detected with high sensitivity by a bioluminescence method using luciferase, and enzymes that catalyze the production of ATP, NADH, etc. can also be detected with high sensitivity. 100ai
Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase ff1o, 005attomo in the ++ assay
It has detected up to l('I'anaka,'l(an
d ishikawa.

E,: Anal, Lett,, 17:2025-
2034.1984) ・But.

In this method, N is the enzymatic reaction product of the enzyme to be detected.
Enzymatic cycling must be used to detect A, DH, making the system complex.

It also takes time to detect. Sensitivity is greatly affected by the activity of the enzyme used in enzymatic cycling, and this may be impractical.

5 Chemiluminescence method. Unlike the bioluminescence method, the system is not complicated because it does not use many enzymes, but currently no system has been established to detect enzymes with sufficient sensitivity. For example, luminol and H2O2' (peroxidase k when used with i7)
5 fg (0,125 attomol) i (Puget*K, , Miche
l5on + A, M, and Avrameas
T3: Anal, Biochem-, 79:4
47. ]977). V. Glucone oxidase has been detected up to 30 attomol using bis(2,4,6-)lichloropheny/I/)oxalate and 8-anilinonaphthalene-1-nulfonic acid (Δra
kawa, H., Maeda, M., and Ts.
uj i, A, : Chem, plarm, Bu
ll, 30.3036.1982).

Among the enzyme detection methods mentioned above, the ones that have been put to practical use and are used to detect nucleic acids include the colorimetric method and the deposited dye method, but the colorimetric method does not have a sensitivity problem, and the deposited pigment method does not have a quantitative method. However, other detection methods have not been put into practical use due to the problems mentioned above.

The present invention has been made to overcome the following two situations. In other words, it is quick, simple, quantitative, and highly sensitive.
The purpose is to enable rapid, simple, quantitative, and highly sensitive detection of a target nucleic acid W1 by establishing a system that can detect it well and easily carry out automatic testing.

) means to solve the problem The present invention is a means for solving the above problems.

The present invention provides a method for detecting all nucleic acids in which a specific base sequence is detected by detecting a labeled enzyme using an electrochemical detector.

Electrical fL detectors are generally highly sensitive, and high-performance liquid chromatography systems incorporating amperometric detectors can detect up to IQQ ferntomol of pheno~/l/, which is considered to be an enzymatic reaction product of AT, P. Can be done (Wehmeycr, ni, R, ;Ha
lsall, H, B,; l-11-1eine.

W, R, Cl1n, Chem, 31 (9),
1546-1549 (1985)). At this time, sensitivity increases if as much of the enzyme reaction solution is injected as possible. The present invention aims to increase sensitivity by reducing the volume of the enzyme reaction solution as much as possible and injecting as much of the enzyme reaction product produced by the enzyme reaction as possible.

That is, in the conventional method, the amount of the enzyme reaction solution was several ml, but in the present invention, the amount of the enzyme reaction solution is set to be 10 times or less (several μ4) the amount of the solution injected at the time of detection.

In addition, by using an electric high-performance liquid chromatography system, enzymatic reaction products such as diphenols can be detected with high sensitivity and quantitatively, eliminating the need for amplification methods such as enzymatic cycling. , nucleic acids easily,
Rapid, highly sensitive, and quantitative detection is possible.In addition, this system does not require the reading of the cydot pattern, which is different from the pigment deposition method, and is considered to be a system suitable for automatic detection.

Note that the electron X chemical detector used in the present invention is preferably an amperometric detector or a Coulomb IJ detector. However, it is not limited to this.

In addition, the labeled enzyme used for hybridization is one that changes the electrostatic properties of the enzyme reaction substrate through an enzymatic reaction, or one that changes the electrostatic properties of the enzyme reaction solution through an enzymatic reaction. Preferably something that changes.

When an electrodynamics detector is incorporated into a high performance liquid chromatography system, it is not necessarily necessary to change the electrodynamics of the enzyme reaction substrate or enzyme reaction melt. Moreover, it is not limited to these. The labeled enzyme used in the present invention is preferably one that is directly bound to a probe or one that is bound to a macromolecule that recognizes some kind of label bound to the probe, but is not limited thereto.

(e)) Examples Hereinafter, the present invention will be explained in detail based on examples. However, the present invention is not limited to the examples.

Example 1 Detection of AT, P by amperometric detector (
Preparation of 1:l ALP solution: Dilute with commercially available 50mM soft sodium carbonate-sodium solution (pH 9.8) (hereinafter abbreviated as CB buffer) derived from bovine small intestine to 0.0. 1.0.5.1 , 5
,]0°50.10G attOmoI/]0μ6.

(2) ALP solution prepared by AT, P enzyme reaction (]) was added to 100μ4 of CBM solution containing 1mM phenyl phosphate and 15mM magnesium perchlorate, and incubated at 37°C for 1 h.
Incubated. After reaction, immediately cool on ice.

1 ttlJ (D O, 75M phosphate Nt2 and
The enzymatic reaction was stopped by adding 20p(1(D 0.2M EDTA, -2Na).

(3) Detection of phenol using an amperometric detector The amount of phenol produced by the enzymatic reaction in (2) was detected using a high performance liquid chromatography system incorporating an amperometric detector as shown in FIG. Column i SH],, M -PΔCK ODS inner diameter 4.
6fl length 5Qm, power 7mm temperature i40'c, mobile phase; 0
1M potassium-phosphate buffer (pH 6,8): meta/-
/l/=4: J, flow rate; 1.0 m67 m
, detection potential; 0.8()V vs, Ag/A+<(J
! , Detection temperature: ・10°C, Injiek)iiiOμ
Analyze under conditions such as e1 or higher.

Using the chromatogram obtained with a reaction solution with an ALP amount of (l attomol) as a baseline, the peak current 1ifj k of the peak appearing near the 3 gates was read and plotted on a graph as shown in Figure 2.In other words, this system By using Q, l attomol i
ALP could be detected. I cut it. In this system, the injection amount can be increased up to t100till, so A L P f O,0] attomol
In other words, the enzymatic reaction only needs to be performed once.

The time is 1h1. It does not require much time, the operation is simple, it is quantitative, and it is highly sensitive.

Example 2 (])M]3 primer (5°-()TAAAACGA
CGGCCAGT-3′) ALP-labeled M]3 primer (5°-()TAAAACGACGG
ALP labeling of CCAGT-3') was carried out according to the method described in Tada et al., Japanese Patent Application No. 191087/1987.

That is, a phosphoric acid residue was introduced into the 5' position of the 9M]3 primer, and further reacted with cystamine to introduce a cystamine residue into the phosphoric acid residue.

H2N-(CH2)2-8 5-(CH2)2-NH-
P-05° (M13 primer) 3゛An M]3 primer whose terminal end was converted to a thiol group by further reduction using dithionuretol was obtained.

H8-(CH2)2-NH-P-0 53゛Also, by reacting ALP with 25 times the amount of 5PDP,
A 2-pyridyl disulfide foundation was introduced into AT and P.

(2) The M13 primer whose 5' end obtained by these steps was converted into a thiol group was reacted with ALP into which a 2-pyridyl disulfide group was introduced.

AT, P-labeled M]3 primer was obtained.

ALP NHCo (CH2)2 S 5-(
CH2) 2-NHP-053' (3)M]3m1) Hybridization of 8-heavy chain 77-di DNA and ALP-labeled M13 primer M13
mp8-heavy chain 77-diDN & (approximately 7000 bp) f:T
B buffer (pH 7,5,] OmM Tris-CI,
Serial dilution with 1mM EDTA) and 0.0.5.5 in 1111 increments were applied to a nylon membrane cut into squares with -5fi sides.
, 50.5005QQ(l attomol M13m
l) 8-heavy chain phage DNA was dotted. After air drying, 5
DNA'f: was fixed by mUV irradiation. This membrane was fixed in a 96-well plate, and 0 μl of a total of 1 F-5 w/v% skimmed aqueous solution was added to each chamber and shaken for 30 i at room temperature on a microplate mixer. After discarding the skim mill solution 1'lf', 1170μe of skim mill solution iv'1 was added again, and the mixture was shaken at room temperature on a microplate mixer at 30W. Thereafter, the skim milling solution was discarded, and 170 μβ of phosphate buffered saline (abbreviated as PBS) was added to each chamber and shaken for 1 m on a microplate mixer to wash the membrane. This washing operation was further repeated once.

Next, add prehybridization solution (4XS) to each chamber.
ET, 0.04W/V% BSA, 0.0
4W/V%Pv p, 0.04W/V% Ficot)
Iy, 5 w/v% polyethylene glycol:? -/L
/; 0.1 w/v%8DS)k170μβ each, 40°C on a microplate mixer, 15UI
+ Shake. After discarding the prehybridization solution, add 0.6 pmol/ALP-labeled probe prepared in (1).
Hybridization solution (other!ll) containing ml
The composition is the same as the prehybridization solution. )k1
Add 70 μβ at a time and place on a microplate mixer for 40
'C 30i shaking hybridization was performed at room temperature on a microplate mixer using 170 μl each of 1X SSC, 0.2% SDS solution.

The membrane was washed with shaking: twice at 40°C, once at 5°C, and twice at room temperature. Next, ice-cold 1X8
The membrane was washed with 170 μa of sc solution on a microplate mixer, shaking once and twice, followed by ice-cooled C containing 1.5 mM magnesium perchlorate.
The membrane was washed with 170 μβ of buffer B while shaking once and twice on a microplate mixer.

Transfer each membrane to a 1.5 ml sampling tube and add 1 mM phenylphosphate and 1.5 mM
100 μe of CB buffer containing magnesium perchlorate
and incubated at 37°C for 1 hour.

After that, immediately cool it on ice and give each one O, 15M! J acid! 5 μ/, 20 p of 0.2 MEDTA-2Na aqueous solution (I
f was added to stop the enzyme reaction.

In order to detect phenol produced by the enzymatic reaction, 10 μe of each reaction solution was injected from an autoinjector of a high performance liquid chromatography system equipped with an amperometric detector shown in FIG. Analysis conditions: Column: SI-IIM-PAC
K OD S inner diameter 4.6 ff length 50 m.

Column temperature: 40°C9 Mobile phase: 01M potassium-phosphate buffer (pH 6,8): methanol-4.1 flow rate;
l Q mJ/mi, detection potential; 0.80V v
s, Ag/AgC4 detection temperature; 40°C. Using the chromatogram obtained from the fixed DNA amount of Oattomol as the baseline, the peak current of the peak that appears around 3 mix was read and plotted on a graph as shown in Figure 3.In other words, this system By using 5 Q Q attomo
The present invention has the following effects.

(1) Nucleic acids can be detected with high sensitivity.

(2) Nucleic acids can be detected quantitatively.

(3) Nucleic acids can be detected rapidly.

(4) Nucleic acids can be detected easily.

(5) Since no amplification means such as chemical cycling is required, fewer types of reagents are required.

(6) Does an electrochemical detector have a simpler mechanism than a fluorescence detection aH system? It is small, easy to install, and can be made inexpensive.

[Brief explanation of the drawing]

FIG. 1 is a diagram of a high performance liquid chromatography system for carrying out the method according to the present invention, and FIG. 2 is a diagram of a high performance liquid chromatograph system for carrying out the method according to the present invention. FIG. 3 is a diagram showing the detection characteristics of ALP according to the method of the present invention. FIG. 3 is a diagram showing the detection characteristics of M13mp88 S DNA according to the method of the present invention.

Claims (1)

[Claims] 1. In a nucleic acid detection method in which a nucleic acid having a specific base sequence is detected using an enzyme as a label in a hybridization assay, the labeled enzyme is subjected to an enzymatic reaction and the product is detected using an electrochemical detector. 1. A method for detecting a nucleic acid, characterized in that the method comprises: 2. The method for detecting nucleic acids according to claim 1, characterized in that the volume of the enzyme reaction solution used for the enzymatic reaction of the labeled enzyme is 10 times or less the volume of the solution injected during detection.