JPH07303499A - Quantification of gene - Google Patents

Abstract

PURPOSE:To easily quantify in high accuracy a gene in many specimens by subjecting a gene to be detected and an internal standard gene to PCR reaction and independently determining the resultant respective amplified genes from the above two kinds of gene by enzyme immunoassay and by using the signal ratio for both the two kinds of the amplified gene. CONSTITUTION:In amplifying a gene to be detected by PCR with a pair of primers, a ligand A-labeled primer P1 and a ligand B-labeled primer P2, a specified amount of an internal standard gene is made to coexist with the above gene and both the former and latter genes are simultaneously subjected to PCR by using a pair of primers, a ligand C-labeled primer P3 and ligand B-labeled primer P4, and part of the resultant reaction liquor is reacted with a solid phase with a receptor Ra corresponding to the ligand A immobilized thereon and another part of the reaction liquor with another solid phase with a receptor Rc corresponding to the ligand C immobilized thereon, and a receptor Rb corresponding to the ligand B labeled with a detecting marker E is reacted with said solid phases each, and the signals corresponding to the marker E on the respective solid phases are measured to determine the signal ratio for the marker on both the two kinds of solid phase, thus easily quantifying the gene in high accuracy.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for quantifying a trace amount of a gene. Its main fields of use include quantitative gene measurement in fields such as medicine, pharmacology, biology, and veterinary medicine.

[0002]

2. Description of the Related Art A polymerase chain reaction (hereinafter abbreviated as "PCR") sandwiches a specific region in a gene to be detected (hereinafter sometimes abbreviated as "detection target gene"). It is a reaction in which a gene fragment having the specific base sequence is amplified exponentially using two kinds of primers, and has been widely used in recent years for detecting a trace amount of gene. When PCR is used, a trace amount of gene can be easily amplified 10,000 to 1,000,000 times, but the unknown reaction promoter and reaction suppressor are present, resulting in poor reproducibility of amplification efficiency and quantification thereof. Measurement is extremely difficult.

Genes amplified by PCR (hereinafter,
Abbreviated as "amplified gene". ) Can be quantitatively measured by the "competitive PCR method" developed by Wang et al. [Proc. Natl. Acad. Sci. USA], 86. [Proceedings of the National Academy of Sciences of USA], 86 Vol., Page 9717 (1989)]. In this method, a gene to be detected, a primer labeled with a radioisotope, and an amplified gene can be obtained by carrying out a PCR reaction using the primer.
The PCR reaction was carried out in the coexistence of a known amount of an artificial gene (hereinafter abbreviated as “competitive gene”) designed to give an amplified gene having a chain length slightly different from that of the CR reaction. To do. Then, the reaction solution is subjected to electrophoresis to separate the amplified gene obtained from the gene to be detected and the amplified gene obtained from the competitive gene, and the radioactivity of each band is measured. Such a measurement is performed while changing the addition amount of the competing gene, and the concentration of the gene to be detected is determined by assuming that the concentrations of both bands are equal when the intensities of both bands are equal.

On the other hand, measuring the amplified gene by enzyme immunoassay has become popular in recent years [JP-A-5-1.
No. 76800; Proceedings of the National Academy of Sciences of USA (Proc. Natl. Acad. Sci. USA), Vol. 86, 24
Page 23 (1989); Clinical Chemistry (Clin.Che
m.), 37, 422 (1991); 37, 1626 (199).
1 year); Analytical Biochemistry (Anal. B
iochem.), 205, p. 1 (1992); and ibid. 208.
Vol. 110, p. 1993).

All of these methods are methods for performing qualitative analysis of amplified genes, and are described in, for example, JP-A-5-17680.
The method described in Japanese Patent No. 0 will be described below. First, 1 corresponding to the nucleotide sequence of the gene to be detected
PCR is performed by selecting a pair of primers. In order to measure the amplified gene by enzyme immunoassay, these primers are labeled with a ligand in advance. The obtained PCR reaction solution is provided on a solid phase on which a receptor for the ligand used for labeling one of the primers is immobilized, and the amplified gene is captured on the solid phase. After removing the free labeled primer, the conjugate of the receptor for the ligand used for labeling the other of the primers with the detection label E is reacted, and the activity of E is measured. As described above, in the enzyme immunoassay method for an amplified gene, a primer for PCR or a probe for detecting an amplified gene is labeled with a ligand, and the enzyme immunoassay is performed through these labels.

[0006]

As described above, the "competitive PCR method" is the only relatively reliable method for quantitatively measuring an amplified gene. In order to separate and quantify the amplified gene obtained from the gene and the amplified gene obtained from the competitive gene according to the difference in their chain lengths, electrophoresis and radioactivity measurement are required. Electrophoresis requires complicated operations and long measurement time, and is not suitable for measurement and automation of many samples. Furthermore, radioactivity measurement requires special facilities for measurement and waste disposal. On the other hand, although the enzyme immunoassay method is suitable for the measurement and automation of a large number of samples, all the amplified gene measurement methods to which the method is applied are for qualitative analysis.

Therefore, an object of the present invention is to provide a method which is simple in operation, suitable for measuring and automating a large number of samples, and quantitatively measuring an amplified gene with high accuracy.

[0008]

According to the present invention, the above object is to provide a primer P1 labeled with a ligand A.
PCR of the gene to be detected using a primer pair consisting of primer P2 labeled with ligand B
Amplification step, reacting the amplified gene with a solid phase on which receptor Ra for ligand A is immobilized, reacting receptor Rb for ligand B labeled with detection label E on the solid phase, and detecting In the method for quantifying a gene, which comprises a step of measuring the amount of the labeled substance E for use in the PCR reaction solution, a predetermined amount of an internal standard gene is allowed to coexist, and the internal standard gene is labeled with a primer C, a primer P3 and a ligand B. PCR was carried out at the same time by using a primer pair consisting of the primer P4 labeled by the above, and a part of the resulting reaction solution was solid phase on which the receptor Ra for ligand A was immobilized, and another part was a receptor for ligand C. The solid phase on which Rc is immobilized is reacted with each solid phase, and each solid phase is labeled with the detection label E. By providing a method for quantifying a gene, which comprises reacting the receptor Rb with respect to the prepared ligand B, measuring a signal corresponding to each of the detection labels E on each solid phase, and determining a signal ratio between the two. To be achieved.

The primer, solid phase and receptor used in the present invention may be abbreviated herein as follows. Primer P1 labeled with ligand A: AP
1 Primer P2 labeled with ligand B: BP
2 Primer P3 labeled with ligand C: C · P
3 Primer P4 labeled with ligand B: BP
4 Solid phase on which receptor Ra for ligand A is immobilized: Ra on solid phase on which Ra is immobilized Receptor on ligand C on solid phase: Rc on solid phase: Rc immobilized solid phase Receptor for ligand B labeled with detection label E Rb: Rb ・ E

The steps of the gene quantification method of the present invention are shown in FIG. The step will be described in more detail. (1) In the method of the present invention, an internal standard gene is first selected. A predetermined amount of internal standard gene is allowed to coexist in a PCR reaction solution containing the target gene, and ligand-specific primers (A • P1 and B • P2) specific for the target gene are detected, as well as the internal standard gene. Specific ligand-labeled primers (C / P3 and B / P4)
) Is added and PCR is performed. As a result, an amplified gene having ligands A and B at both ends was detected from the detection target gene, and ligands C and B were detected from the internal standard gene.
Amplified genes having the two ends are produced respectively.

(2) Part of the obtained solution after the PCR reaction is reacted with Ra-immobilized solid phase, and another part is reacted with Rc-immobilized solid phase. By such a reaction, the amplified gene obtained from the gene to be detected is bound on the Ra-immobilized solid phase via the binding of Ra and the ligand A, and the amplified gene is bound on the Rc-immobilized solid phase via the binding of Rc and the ligand C. The amplified gene obtained from the internal standard gene is captured. The solid phase on which the amplified gene is captured is washed to remove unbound free primers and amplified gene.

(3) On the solid phase on which the amplified gene obtained from the gene to be detected or the internal standard gene is captured, Rb.
React E. As a result, a solid phase to which the detection marker E corresponding to the concentration of the gene to be detected or the amplified gene obtained from the internal standard gene is bound can be obtained. Free Rb
Wash each solid phase to remove E.

(4) The activity of the label E for detection on each solid phase is measured. The signal intensities S and So are obtained for the Ra-immobilized solid phase and the Rc-immobilized solid phase, respectively. Next, the concentration of the gene to be detected is calculated using the signal intensity ratio S / So.

The internal standard gene used in the method of the present invention is not particularly limited as long as it is different from the gene to be detected, but the sample used for the measurement in the present invention is a multi-species extracted from a living body such as human or animal or plant. Internal control genes must be distinguished from those that often contain different types of genes. For that purpose, it is preferable to use an artificial gene having a low probability of naturally occurring as the internal standard gene. The internal standard gene is
It can be synthesized according to a usual gene synthesis method, but it is also possible to use a commercially available artificial gene. For example, various plasmid vectors sold by Promega (pGEM series, pSP series, pBR series, pCAT series, etc.) or lambda vectors (Lambda GEM series, Lamb)
(dagt series, etc.) is preferable because it has high purity, is stable, and is easy to handle.

Since the internal standard gene is used for correcting the tube-to-tube variation in the PCR reaction, it is necessary to use it at a concentration that can sufficiently reflect the influence of the tube-to-tube variation factor. For that purpose, it is preferable to select the amount of the internal standard gene used so that the signal intensity So of the Rc-immobilized solid phase is near the center of the detectable region of the signal intensity measurement system. If So is close to the lower limit or upper limit of detection of the measurement system, it is not preferable because the influence of the inter-tube variation factor cannot be sufficiently reflected.

Primer pair P1 and P2, or P3
And P4 are selected from among the nucleotide sequences of the gene to be detected or the internal standard gene by searching for a sequence specific to the gene. Further, in order to avoid generation of complementary strands between the primers, it is necessary that the 3'sides of both primers have no complementarity and that a palindrome is not formed in the primers. Furthermore, regarding the base sequences of the primers P3 and P4, it is preferable to select and use the artificial internal standard gene sequence having a natural existence probability as low as possible. The length of the primer is not particularly limited, but usually 10 to 40 bases,
It is preferably 15 to 30 bases.

Of the base sequence of the gene to be detected, the region of the base sequence sandwiched between the pair of primers is the region amplified by PCR. The chain length of this PCR amplification region is
Although not particularly limited, it is usually 50 bases to 5000 bases,
It is preferable to select the primer pair so that it is preferably 100 to 500 bases. Furthermore, in order that the PCR efficiencies of the gene to be detected and the internal standard gene respond in parallel to an unexpected change in PCR conditions, it is preferable that the chain lengths of the PCR amplification regions of the two do not differ greatly. . That is, the PCR amplification region chain length of the internal standard gene is 1/3 of the PCR amplification region chain length of the detected gene.
It is preferably ˜3 times, more preferably ½ to 2 times.

In the method of the present invention, the ligand used for labeling the primer is a relatively low molecular weight that does not inhibit the reactivity of the primer when the primer is labeled with the ligand, and a specific protein (antibody, Receptor, other binding protein) means a compound capable of binding. For example, a compound capable of binding to a specific protein (avidin) such as biotin; or digoxigenin, fluorescein isothiocyanate (hereinafter referred to as FIT
Abbreviated as C. ), Haptens and the like capable of binding to antibodies such as trinitrophenol and dinitrophenol.

As a method of binding the ligand molecule to the primer, a method of first introducing a functional group such as an amino group into the 5'end of the primer and reacting the modified ligand molecule so as to be able to bind to the functional group is Commonly used (eg translation by Saito, PCR experiment manual, HB
See J Publishing, 1991, p. 89). Apart from this, there is also a method of incorporating a nucleotide triphosphate labeled with a ligand molecule when a primer is synthesized by a DNA synthesizer. The former method introduces ligands only at the 5'end, whereas the latter method introduces several ligands into the primer strand.

The receptor in the method of the present invention means a protein capable of binding to the above-mentioned ligand. For example,
Avidin for biotin, anti-digoxigenin antibody for digoxigenin, anti-FIT for FITC
C antibody is the receptor for each ligand. When various haptens are used as ligands, antibodies against them can be used as receptors.
In the method of the present invention, three types of receptors, Ra, Rb and Rc, are used. Ra and Rc are used after being immobilized on a solid phase, and Rb is used after being bound to the detection label E.

As the solid phase used in the method of the present invention, the same solid phase as used in the known enzyme immunoassay can be used. Specifically, microtiter plates, tubes, beads, microparticles,
Further, a small diameter tube described in JP-A 1-212347 is used. Two types of solid phase are used: receptor Ra-immobilized solid phase and Rc-immobilized solid phase. Immobilization of the receptor on the solid phase is performed by a conventional method. Most commonly, a receptor is adsorbed to a solid phase made of plastic by physical adsorption, and then so-called blocking is performed with bovine serum albumin or the like. It is also possible to immobilize the receptor by covalent bonding to a solid phase having a functional group such as an amino group.

The detection label used in the method of the present invention is the same as that used in the known enzyme immunoassay, for example, enzymes such as urease, alkaline phosphatase and peroxidase. Further, instead of the enzyme, fluorescent molecules such as FITC, Rhodamine D, and erythrosine isothiocyanate; chemiluminescent molecules such as acridinium esters and luminols; iodine 12
Radioisotopes such as 5 are used.

When an enzyme is used as the detection label, the enzyme activity is measured by the same means as used in the usual enzyme immunoassay. That is, an absorptiometer is used if the substrate for the enzyme is chromogenic, a fluorimeter is used if it is fluorescent, and a luminometer is used if it is chemiluminescent. Further, as in the system disclosed in JP-A No. 1-212347, when the activity of urease trapped on the inner wall of the small diameter tube is detected as the pH change of the substrate solution in the small diameter tube, it is used as a detector. A pH electrode or a pH sensitive field effect transistor may be used. When a fluorescent molecule, a chemiluminescent molecule or a radioisotope is used as a label for detection, the label captured on the solid phase is directly used in a fluorometer, a luminometer, or a radioactivity. Each is detected by the counter.

Next, the PCR reaction conditions in the method of the present invention will be described. As a polymerase for gene amplification, thermostable Taq DNA polymerase is usually used.

As the substrate for the polymerase, deoxynucleoside 5'-triphosphate (dNTP);
That is, adenine-5'-triphosphate, guanine-5'-
Triphosphate, cytosine-5'-triphosphate, and thymine-
5'-triphosphate.

The composition of a standard PCR feed solution is as follows. Gene to be detected Predetermined amount of internal standard gene 0.5 to 500 mM KCl 10 to 200 mM Tris-MCl buffer (pH 8.
3) 0.25-15 mM MgCl ₂ 0.01-0.5 wt% gelatin 20-200 μM each dNTP 0.01-1 μM each ligand-labeled primer 1-3 unit Taq DNA polymerase

In PCR, first, the gene in solution is heated to 92 ° C. to 96 ° C. for 10 seconds to 3 minutes, and the gene in solution is converted into single-stranded D
Heat denature to NA. Then at 55 ℃ ~ 72 ℃ 10 seconds ~
The ligand labeled primer is hybridized to the single-stranded DNA by annealing for 3 minutes. Further, the primer extension reaction is performed by heating at 70 ° C. to 73 ° C. for 30 seconds to 3 minutes. This thermal denaturation, annealing, and primer extension reaction are sequentially performed multiple times to obtain
A gene in the R amplification region is amplified. The number of PCR cycles is usually 10 to 50 times, preferably 20 to 35 times. Among the PCR conditions described above, in particular, the amount of the internal standard gene, the concentration of the ligand labeled primer, and the P
The number of CR cycles usually needs to be decided by thorough examination.

The reaction solution after completion of PCR may be directly used for enzyme immunoassay, but unreacted A / P1
Since the remaining C and P3 prevent the amplified gene from reacting with the solid phase, it is desirable to remove unreacted primers by ethanol precipitation or simple column separation and then subject to enzyme immunoassay.

Next, the conditions for enzyme immunoassay will be described.
First, the solution after the PCR reaction is left as it is, or the solution obtained by separating and removing the unreacted primer is divided into two, and one is reacted with the Ra-immobilized solid phase and the other with the Rc-immobilized solid phase. At this time, the solution may be diluted with an appropriate buffer solution, and animal serum components such as bovine serum albumin may be added to the buffer solution to prevent nonspecific adsorption. The reaction between the solution and the solid phase after the PCR reaction is usually 0 to 50 ° C, preferably 25 to 40 ° C.
It is done in the range of. The reaction time is 10 minutes to overnight, preferably 10 minutes to 2 hours. After such reaction, the solid phase is thoroughly washed with a washing buffer.

Next, the two solid phases after the washing are reacted with a solution of Rb.E. Also in this case, in order to prevent non-specific adsorption, animal serum and further a surfactant may be added if necessary. The reaction temperature is 0 to 50 ° C, preferably 2
It is in the range of 5 to 40 ° C. Reaction time is 10 minutes to 1
Overnight, preferably in the range of 10 minutes to 2 hours. After the reaction, the solid phase is washed with a washing buffer. Finally, in order to measure the activity of the enzyme captured on the solid phase, a substrate solution is added, and the decomposition rate of the substrate or the amount of decomposition of the substrate within a predetermined time is measured according to the detection method as described above.

In order to actually quantify a gene by the method of the present invention, it is necessary to first prepare a calibration curve. In order to create a calibration curve, instead of the detection target gene, a primer pair (P1 and P2) for the detection target gene
The measurement is performed using a known concentration of "external standard gene" that is amplified by. As this external standard gene, a gene to be detected may be purified in advance and the concentration of which has been determined by other means, but it is generally difficult,
Usually created artificially by recombinant technology. Since the external standard gene is used in place of the detection target gene, it has a base sequence that is PCR-amplified by the primer pair for the detection target gene as described above, that is, at least the region to which the primer hybridizes. The nucleotide sequence must be the same as that of the gene to be detected. Furthermore, it is desirable that the chain length of the region amplified by P1 and P2 is almost the same as the chain length of the PCR amplification region of the internal standard gene. That is,
The chain length of the PCR amplification region of the external standard gene is preferably 1/3 to 3 times, more preferably 1/2 to 2 times that of the internal standard gene.

When the gene to be detected is a gene for various human cytokines or various growth factors, a pAW type plasmid used as a competitive gene in the above-mentioned "competitive PCR method" should be adopted as the external standard gene. Is possible. The plasmid is obtained by connecting the sequences of the primer portions of various cytokine or growth factor genes with a linker sequence, and can be isolated and purified to determine the concentration. Using a known amount of external standard gene and a predetermined amount of internal standard gene, the signal intensity for each was measured according to the quantification method of the present invention, and S /
Find So. S / So was measured for several concentrations of the external standard gene, and the external standard gene concentration and S / So were measured.
A calibration curve can be obtained by determining the relationship with.

The gene to be detected in the method of the present invention is DNA directly. However, the gene to be detected is RNA
In this case, the RNA can be quantified according to the quantification method of the present invention (hereinafter, RN to be detected).
A is abbreviated as “RNA to be detected”. ). That is, an internal standard gene (hereinafter abbreviated as “internal standard RNA”) is selected for RNA to be detected, and RNA to be detected and internal standard RNA are allowed to coexist in a reverse transcriptase solution,
Both are simultaneously reverse transcribed to produce their respective cDNAs. Hereinafter, the cDNA obtained according to the quantification method of the present invention
Quantify.

Generally, reagents used for PCR are expensive, and a sample containing a gene to be detected is often valuable. Therefore, it is desirable to carry out PCR with as little solution as possible. In the enzyme immunoassay device disclosed in the above-mentioned Japanese Patent Laid-Open No. 1-212347, since the inside of the tip of the thin tube is used as an immunoreaction field and an enzyme reaction field, a required sample volume of 1 μl or less is sufficient. It is suitable as an enzyme immunoassay device for the above method.

[0035]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Example 1 IL-1β mRNA in peripheral blood mononuclear cells of healthy subjects and rheumatic patients and synovial cells of rheumatic patients was quantified as follows.

1. Preparation of labeled primer pAW109RNA used as a competitive gene in the above-mentioned “competitive PCR method” as an external standard gene (RNA) corresponding to human IL-1β mRNA
[Gene Amp; Proceedings of the National Academy of Sciences of USA (Proc. Natl. Acad. Sci. USA), 86, 9717
See page (1989)]. The following pair was selected as a primer for the cDNA obtained by reverse transcribing each of the external standard RNA and human IL-1β mRNA. Sense primer P1: 5'AAACAGATGAAG
TGCTCCTTCCAGG 3'antisense primer P2: 5'TGGAGAACA
The chain length amplified by PCR with CCACTTGTTGCTCCCA 3'primer pair P1 and P2 was 388 bases for IL-1β cDNA, pAW10
There are 306 bases in 9 cDNAs.

The internal standard RNA is pRSET which is an artificial expression vector having a T7 promoter region.
A DNA [Invitrogen Corp .; Gene (Gen
e), 124, 83 (1993)]. The following pair was selected as a primer for cDNA obtained by reverse transcription of the internal standard RNA. Sense primer P3: 5'ACCACAACGGTT
TCCCTCTA 3'antisense primer P4: 5'GATCAAGCT
The chain length of pRSET A cDNA amplified by PCR with the TCGAATTTCCAT 3'primer pair P3 and P4 is 210 bases.

The synthesis of these primers was carried out by an automatic synthesizer (Model 380B; manufactured by Applied Biosystems Inc.).

Then, the synthesized primer P1 was labeled with digoxigenin, the primers P2 and P4 were labeled with biotin, and the primer P3 was labeled with FITC as follows. First, an amino group was introduced at the 5'end of each primer. The reaction was carried out by binding Aminolink-2 (manufactured by Applied Biosystems Inc.) with the above-mentioned automatic DNA synthesizer. Then, the aminated primer P1 was treated with digoxigenin-3-o-methylcarbonyl-ε-aminocaproic acid N-hydroxysuccinimide ester (Digoxigenin-3-o-methylca).
rbonyl- ε-aminocaproic acid-N-hydroxysuccinimide
ester; Boehringer Mannheim Biochemica) and aminated primers P2 and P4 with biotin labeling kit (Biotin Labeling Kit; Clontech)
And the aminated primer P3 is 5 (6)-
Carboxyfluorescein N-hydroxysuccinimide ester (5 (6) -carboxyfluorescein-N-hydroxysuc
cinimide ester; manufactured by Boehringer Mannheim Biochemica), respectively, to give a primer P1 labeled with digoxigenin, a primer P2 and a primer P4 labeled with biotin, and FITC.
Primers P3 labeled with were obtained respectively. Each labeled primer was purified by liquid chromatography.

2. Preparation of Pipette Tip Type Solid Phase The pipette tip used as the solid phase is shown in FIG. The pipette tip is a polypropylene molded product with a total length of 48 m.
m. The inner and outer diameters of the most tip are 0.55m
m and 1.00 mm (FIG. 4). The anti-digoxigenin antibody or the anti-FITC antibody was immobilized on the inner wall of the tip shown by the broken line 11 in FIG. Ie 50 μg / ml
Fab part of anti-digoxigenin antibody of Boehringer Man
nheim Biochemica) or PBS (pH 7.) containing 100 μg / ml of anti-FITC antibody (DAKO).
4) Put 1 μl inside the tip of the chip and place in a moisturizing box at 37
Incubated for 2 hours at ° C. The tip of the tip was then placed in a blocking solution (1% bovine serum albumin and 1%
After washing with PBS containing 0% sucrose), the tip 15 mm of the chip is immersed in the same blocking solution,
The mixture was left standing at 4 ° C for 16 hours. The chip after blocking was vacuum dried and stored at 8 ° C. in a polyethylene bag.

3. Preparation of avidin-urease conjugate First, a maleimide group was introduced into avidin. 6.4 mg of streptavidin (5532LY; manufactured by Gibco) 0.1M PBE (EDTA-containing PBS; pH 7.0)
To 2.5 ml of the solution, 8.7 mg of EMCS [N- (ω-
Maleimidocaproyloxy) succinimide] DMF
Add 0.87 ml of (dimethylformamide) solution and add 3
The reaction was allowed for 0 minutes. The reaction was stopped by adding 1 M glycine, and the maleimidized streptavidin was added to PD10.
Separation was performed using a column (Pharmacia). On the other hand, in order to regenerate the SH group of urease, 50.2 mg of urease (U-0376; manufactured by Sigma) in phosphate buffer (p
H6.5) 2.1 ml to 0.42 ml 0.1 M D
TT (dithiothreitol) was added and reacted at 25 ° C for 30 minutes, and the product was separated on a PD10 column. 8.0
1 nmol maleimidized streptavidin and an equimolar amount of reduced urease were added at 0.
Coupling was performed in 6.36 ml of 1M phosphate buffer (pH 6.5), and β-mercaptoethanol was added to stop the reaction. After the reaction, the solution is Sephacryl S-
Fractionation was performed with 400 HR (Pharmacia), and the fraction with high conjugate activity was fractionated and stored at 4 ° C. In addition, the conjugation activity of each fraction was determined by biotin-labeled sheep Ig.
It was determined by binding the product to a microplate on which G (manufactured by EY Lab) was immobilized and measuring its urease activity.

4. Extraction of mRNA from clinical specimens Peripheral blood mononuclear cells (5 specimens), peripheral blood mononuclear cells (10 specimens) of rheumatic patients, and synovial cells (5 specimens) collected from knee joints of rheumatic patients were used as clinical specimens. 1 × 10 ⁷ pieces were used. Isolation of mRNA from these cells is Quick
Prep Micro mRNA Purification Kit (Pharmacia)
The purified mRNA was purified using 400 μl of buffer [1 mM EDTA-containing 10 mM Tris-HCl.
(PH 7.5)] as a solution.

5. Preparation of internal standard RNA As described above, the internal standard RNA was pRSET.
What transcribed A DNA into RNA was used. The transfer was performed as follows. First, the plasmid pRSET A
The linear DNA was obtained by digesting the DNA with a restriction enzyme Sca I (Takara Shuzo Co., Ltd.), extracting with phenol, and then performing ethanol precipitation. The linear DNA was dissolved in distilled water to obtain 20 μl of a 200 ng / μl solution. Next, 1 μg of the linear DNA was used as a template and transferred with T7 polymerase (Takara Shuzo Co., Ltd.) using 4 kinds of ribonucleoside-5′-triphosphates as substrates, followed by phenol extraction,
Then, ethanol precipitation was performed to obtain RNA. The resulting RNA was dissolved in 100 μl of RNase inhibitor-distilled water to obtain a stock solution of the internal standard RNA, which was diluted 25,000 times to prepare “pRS for internal standard”.
ET RNA solution ”.

6. Reverse transcription reaction A reverse transcription reaction solution was prepared by mixing the following. Buffer for 10 × PCR 90.0 μl 100 mM DTT 90.0 μl 1.25 mM dNTP 144.0 μl 40 u / μl RNase inhibitor 26.3 μl 32 u / μl Reverse Transcriptase 1.9 μl 10 μM biotin-P4 30.0 μl 20 μM oligo-dT18 150. 0 μl “pRSET A RNA solution for internal standard” 30.0 μl distilled water 217.8 μl total 780.0 μl In the above composition, the 10 × PCR buffer is 50
0 mM KCl, 200 mM Tris-HCl (pH
8.4) means 25 mM MgCl ₂ , 1 mg / ml bovine serum albumin in distilled water. Further, oligo-dT18 was used as a primer for reverse transcription of clinical sample-derived mRNA or pAW109 RNA having a poly A sequence.

When preparing a calibration curve, 0, 10 ² , 10 ³ , 10 ⁴ , 1 was added to 26 μl of the reverse transcription reaction solution having the above composition.
TE buffer containing 10 ⁵ or 10 ⁶ copies of pAW109 RNA (10 mM Tris-HCl (pH 7.
6) Reverse transcription was carried out by adding 4 μl each of 1 mM EDTA). In the measurement of clinical specimens, reverse transcription reaction was carried out by adding 4 μl each of TE buffer containing mRNA corresponding to 1 × 10 ⁵ clinical specimen cells to 26 μl of the reverse transcription reaction solution having the above composition. Reverse transcription reaction is 1 at 43 ℃
After heat denaturation at 95 ° C. for 10 minutes,
Quenched to 10 ° C.

7. PCR The composition of the PCR solution is as follows. Buffer for 10 × PCR 90.0 μl 1.25 mM dNTP 144.0 μl 5u / μl Taq DNA polymerase 6.0 μl 10 μM digoxigenin-P1 50.0 μl 10 μM biotin-P2 50.0 μl 10 μM FITC-P3 20.0 μl P 10 μM biotin 10.0 μl distilled water 530.0 μl total 900.0 μl In the above composition, the 10 × PCR buffer was the same as that used in the reverse transcription reaction.

15 μl of the solution obtained by the reverse transcription reaction was added to 45 μl of the PCR solution, and heat denatured at 93 ° C. for 5 minutes. Then, annealing was performed at 55 ° C. for 45 seconds and primer extension reaction was performed at 72 ° C. for 2 minutes. Then at 93 ° C for 1 minute,
29 cycles of PCR at 55 ° C. for 45 seconds and 72 ° C. for 2 minutes were performed. The solution after PCR was applied to a CHROMA SPIN column (manufactured by Clontech) to remove unreacted primers.

8. Detection of amplified gene by enzyme immunoassay 1 μl of the PCR solution treated with the CHROMA SPIN column was placed inside the tip of the anti-FITC antibody-immobilized pipette tip, and another 1 μl was placed inside the tip of the anti-digoxigenin antibody-immobilized pipette tip. Inhale each, 10 at room temperature
After leaving it for a minute, it was washed with PBS (pH 7.8). Then, 2 μl of the avidin-urease conjugate solution was sucked into each pipette tip and allowed to stand at room temperature for 5 minutes.
It was washed with a solution containing 10 mM ammonium chloride and 154 mM sodium chloride. The activity of urease trapped on the inner wall of the tip of the pipette tip after the reaction was measured by inserting the pipette tip into the cell shown in FIG.

The cell is a substrate solution of urease (155 mM
Urea, 10 mM ammonium chloride and 154 mM
It consists of a flow cell 1 having an inlet 2 and an outlet 3 for salt. pH-sensitive field effect transistor (hereinafter referred to as "p
H-FET "is abbreviated. ) 6 is embedded in the tip of the sensor body 5, which is inserted and fixed from the bottom of the cell. A sensor coupler 4 is attached to the tip of the sensor body. This acts as a guide for coupling the pipette tip 8 inserted from the upper part of the cell and the pH-FET. Reference numeral 7 is a liquid junction type reference electrode. Enlarged views and dimensions from the top and sides of the coupled state of the pH-FET and pipette tip are shown in FIGS. 3 and 4, respectively. The pipette tip was inserted into the cell while the flow of the substrate solution was stopped, and the pH-FET was coupled to the cell, and the pH change due to the decomposition of urea inside the tip of the chip was measured as the change rate of the source potential of the pH-FET. That is, the source potential from 10 seconds to 20 seconds after the pipette tip was inserted was read at 0.1 second intervals, and the changing speed for 10 seconds was calculated by the least squares method. The output may become unstable for 10 seconds immediately after the chip is inserted, so data was not collected. After reading the potential, the chip was taken out and the substrate solution was fed to wash the cell. In addition, 9 in FIG.
The pH sensitive part on the pH-FET element, and 10 in FIGS. 3 and 4 show the flow path of the substrate solution. In addition, the italicized numbers in FIG. 4 exemplify the size (unit: mm).

9. Preparation of calibration curve for IL-1β mRNA As mentioned above, a fixed amount of pRSET A RN for internal standard was used.
A and 0 to 10 ⁶ copies of external standard pAW109 RN
The solution containing A was reverse-transcribed and PCR, each PCR product was detected by the above-mentioned enzyme immunoassay, and the signal intensity ratio was plotted against the copy number of pAW109 RNA for external standard. The results are shown in FIG. It was This figure shows that it is possible to quantify 10 ² to 10 ⁶ copies of pAW109 RNA by this method.

10. IL-1β mRNA in clinical samples
Next, a fixed amount of pRSET A RN for internal standard is also used.
A solution containing A and mRNA corresponding to cells from 10 ⁵ clinical specimens was reverse-transcribed and PCR, and the results of measuring the PCR products of pRSET A RNA and IL-1β by the above-mentioned enzyme immunoassay and the signal intensity ratio of both Is shown in Table 1. Next, IL-1β calculated using the signal ratio and the calibration curve of FIG.
Table 1 shows the mRNA copy number, and further, the IL-1β mRNA expression number per clinical specimen cell. In addition,
N, RAB and RAS in Table 1 represent peripheral blood mononuclear cells of healthy subjects, peripheral blood mononuclear cells of rheumatic patients and synovial cells of rheumatic patients, respectively.

[0053]

[Table 1]

Summarizing the results in Table 1, the average value ± standard deviation of the number of IL-1β mRNA expressed in each cell was 0.021 ± 0.01 in peripheral blood mononuclear cells of healthy subjects.
3. 0.049 ± in peripheral blood mononuclear cells of patients with rheumatism
0.068, 0.146 in rheumatoid patient synovial cells
It was ± 0.133. This trend is consistent with the trend reported previously.

[0055]

EFFECTS OF THE INVENTION A method for quantitatively measuring an amplified gene with high accuracy is provided, which is simple in operation, suitable for measurement and automation of a large number of samples.

[Brief description of drawings]

FIG. 1 shows steps of the quantification method of the present invention.

FIG. 2 shows an example of a cell for measuring the activity of urease trapped on the inner wall of the tip of a solid phase pipette tip.

FIG. 3 shows an enlarged view from the top and dimensions of the coupled state of the pH-FET and pipette tip.

FIG. 4 shows an enlarged side view and dimensions of the coupled pH-FET and pipette tip.

FIG. 5 is a calibration curve for IL-1β mRNA.

[Explanation of symbols]

 1: Flow Cell 2: Inlet of Urease Substrate Solution 3: Outlet of Urease Substrate Solution 4: Sensor Coupler 5: Sensor Body 6: pH Sensitive Field Effect Transistor (pH-FET) 7: Liquid Junction Reference Electrode 8: Pipette Chip 9: pH sensitive portion 10: Substrate solution flow channel 11: Receptor-immobilized portion

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hitoshi Tsuruta, Kuraray Co., Ltd., 2045, Satsuki, Kurashiki, Okayama Prefecture (72) Toshihiko Namba, 1621 Sakata, Kurashiki, Okayama, Kuraray Co., Ltd.

Claims (4)

[Claims] 1. A step of amplifying a gene to be detected by polymerase chain reaction using a primer pair consisting of a primer P1 labeled with a ligand A and a primer P2 labeled with a ligand B; The step of reacting the amplified gene with the immobilized solid phase, the step of reacting the receptor Rb for the ligand B labeled with the detection label E with the solid phase, and the amount of the detection label E are measured In the method for quantifying a gene, the polymerase chain reaction reaction solution is allowed to coexist with a predetermined amount of an internal standard gene, and the internal standard gene is converted from a primer P3 labeled with a ligand C and a primer P4 labeled with a ligand B. With the primer pair Sometimes performing a polymerase chain reaction, the ligand part of the obtained reaction liquid A
To the solid phase having the receptor Ra immobilized thereto and the other part thereof to the solid phase having the receptor Rc immobilized to the ligand C respectively, and the respective solid phases are labeled with the detection label E. Receptor Rb for B is reacted to give a detection label E on each solid phase
A method for quantifying a gene, which comprises measuring a signal corresponding to and determining a signal ratio between them. 2. The method for quantifying a gene according to claim 1, wherein the label for detection is urease. 3. The pH-sensitive field-effect transistor is used as the means for measuring the detection label.
A method for quantifying the described gene. 4. The method for quantifying a gene according to claim 1, wherein the solid phase is a thin tube.