JPH10276780A - Detection of psa mrna and detection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly and accurately diagnose the prostatic cancer by amplifying a prostate-specific antigen mRNA in the presence of a labeled sense primer, a labeled antisense primer and a specific DNA polymerase by a reverse transcription PCR method and subsequently detecting the amplified product. SOLUTION: This method for detecting the prostate-specific antigen(PSA) mRNA comprises amplifying a constant domain of the PSA mRNA in the presence of a sense primer and an antisense primer labeled with a label A such as 5'-terminal 2,4-dinitrophenyl(DNP) label and in the presence of rTth DNA polymerase, hybridizing the amplified product with an amplified product- specific probe immobilized on a support, reacting the hybridized and bound product with an antibody specific to the label A and labeled with an enzyme, and subsequently detecting the bound product by a reaction specific to the enzyme. Thereby, the target PSA mRNA as a prostatic cancer marker in a specimen can surely and simply be detected in high sensitivity and in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a prostate-specific antigen (prostate cancer marker) useful as a prostate cancer marker, which is useful for the definitive diagnosis of prostate cancer, early detection and postoperative monitoring, and detection of presence or absence of cancer metastasis in the fields of medicine and clinical examination. PSA) for the detection of mRNA.

[0002]

2. Description of the Related Art Detection of PSA in blood components is known as a useful method for diagnosing prostate cancer. At present, many kits for detecting PSA in blood components using an antigen-antibody reaction are commercially available and widely used in clinical tests for prostate-related diseases. However, in metastatic prostate cancer, PS in blood components
When it is determined that the A antigen is positive, it is often too late, and the need for a detection method and a detection reagent for early detection of prostate cancer is increasing. Currently, definitive diagnosis of prostate cancer is mainly performed using a biopsy,
Biopsy does not cover the entire prostate tissue, which raises the problem of accuracy. Recent studies have begun to suggest that the detection of PSA mRNA in PSA-producing cells contaminating peripheral blood during prostate cancer may lead to more accurate prostate cancer diagnosis and early cancer detection, Genetic engineering techniques are being introduced into this field. To date, Moreno JG
(Cancer Res. 52: 6110-6112, 1992), Robert. A et al. (Urology 45: 597-603, 1995), Katz AE et al. (Cancer 7
5: 1642-1648, 1995) have succeeded in detecting PSA mRNA from cells in blood by a detection method using a reverse transcription polymerase chain reaction (RT-PCR) method. Also, Israel
li et al. describe a new prostate-specific antigen, the prostate membrane antigen (pr
ostate membrane antigen (PSM), and the detection of PSA mRNA has been successfully detected by RT-PCR (Cancer Res. 53: 227-230, 1993; J. Uro).
l. 153: 573-577, 1995). In addition, Hoshi et al.
2: 781-785, 1996) is FNAB (Fine Needle Aspiration)
Biopsy) using lymph node aspiration biopsy cells and detecting the presence of PSA mRNA in cancer cells contaminated by RT-PCR to determine the presence or absence of pelvic lymph node metastasis.
This method increases the accuracy of conventional cytology,
It is reported that it is useful for understanding the presence or absence of micrometastasis.

[0003] Currently, these methods of detecting PSA mRNA generally include (1) detecting PSA mRNA extracted from cell components, tissues, or lymph node aspirated biopsy cells in peripheral blood of prostate cancer patients. (2) PSA
After amplification by PCR (nested PCR if high sensitivity is required) using primers specific to a part of the mRNA sequence, and (3) separating the target amplification product by electrophoresis, This amplification product is detected by Southern blotting using a labeled probe specific to the target amplification product.
The problem here is that PSA belongs to a superfamily of serine proteases called kallikrein, and the nucleotide sequences of mRNA of kallikrein derived from human submandibular gland and that of PSA are very similar (see FIG. 2). ). To solve this problem, kallikrein mRNA from the submandibular gland
It is necessary to set a primer at a site having low homology to the sequence of (1). Furthermore, since genomic contamination may be considered to extract PSA mRNA from peripheral blood cell components, the issue is how to detect PSA mRNA specifically and with high sensitivity from these unintended contaminants. Has become.

[0004]

The conventional detection methods as described above require several days from the acquisition of a sample to the detection thereof, take time, and include techniques requiring skill such as electrophoresis and blotting. In addition, since the presence or absence of detection is finally determined by visual observation of a human, there are disadvantages such as poor objectivity of the data and inability to obtain a stable result.

Accordingly, in the present invention, the process from obtaining a specimen to detection is completed in one day, and can be carried out without special equipment and skill requiring skill as long as the temperature conditions are maintained. An object of the present invention is to provide a PSA mRNA detection kit and a detection method using the reagent, which provide more objective data.

[0006]

Means for Solving the Problems The present inventors have developed PSA mRNA.
In the method for detecting a specific region of PSA mRNA, amplifying a certain region of the PSA mRNA, performing reverse transcription PCR using a primer set and rTth DNA polymerase, and further, a specific `` probe used for immobilization of primer set and amplification product '' Can be operated easily and in a short time,
In addition, they have found a method for detecting PCR mRNA with high specificity and high sensitivity, and have completed the present invention.

[0007] The present invention relates to a primer set comprising a labeled sense primer and an antisense primer, and rTth DNA having reverse transcription activity and polymerase activity.
The present invention relates to a method for detecting PSA mRNA in a sample, comprising amplifying a certain region of prostate specific antigen (PSA) mRNA using a polymerase by reverse transcription PCR, and detecting an amplified product.

In the detection of an amplification product, the amplification product is hybridized and bound to an amplification product-specific probe immobilized on a support, and an antibody that is specific for labeling and labeled with an enzyme is added to the probe. React with the conjugate,
The conjugate can be detected by an enzyme-specific reaction.

[0009] As a primer set for the reverse transcription PCR method, a sense primer: 5'-TGTGCGCAAGTTCACCCTCA-3 '
(SEQ ID NO: 1); and antisense primer: 5 ′
It is preferable to use -CCACGATGGTGTCCTTGATC-3 '(SEQ ID NO: 2).

[0010] As an amplification product-specific probe, 5'-AGA
It is preferable to use ATCACCCGAGCAGG-3 ′ (SEQ ID NO: 3).

As the label of the primer, any label known as a nucleic acid label can be used.
Dinitrophenyl (DNP) labeling is preferred, and detection sensitivity can be increased by linking a plurality of DNPs.

Further, according to the present invention, 5′-TGTGCGCAAGTTCACCCTCA-3 ′, each of which is labeled with DNP at the 5 ′ end.
(SEQ ID NO: 1) sense primer and 5′-CCACGATGG
A reverse transcription PCR reaction reagent comprising an antisense primer of TGTCCTTGATC-3 '(SEQ ID NO: 2), 5'-AGAATCACCCGAGC
A kit for detecting PSA mRNA in a sample, comprising a solid support having an AGG-3 ′ (SEQ ID NO: 3) probe, an alkaline denaturing solution, and a hybridization buffer, is also provided.

The detection of PSA mRNA according to the present invention comprises the following steps: a (a-1 to a-3) and b (b-1 to b-5)
However, it goes without saying that modifications such as additions and changes may be made within the scope of common knowledge of those skilled in the art.

(A) Reverse transcription and PCR amplification reaction (a-1) The sample containing the extracted PSA mRNA is re-dissolved in purified water or 10 μl of an aqueous solution containing an RNase inhibitor for the purpose of preventing RNA degradation.

(A-2) Next, a final concentration of 50 mM bicine, 1
15 mM potassium acetate, 8% (w / w) glycerol and dAT
P, dTTP, dGTP, dCTP, manganese (II) acetate, rTth DNA
The PSA mRNA solution prepared in (a-1) is added to the mixed solution of the polymerase, the sense primer and the antisense primer to make a total of 50 μl.

The pH of the PSA mRNA solution is preferably 8.0 to 8.4,
Most preferably, it is 8.2.

The final concentrations of dA, T, G and CTP are each preferably 0.1-0.5 mM, most preferably 0.3 mM.

[0018] dUTP may be used in place of dTTP, and in this case, the final concentration is preferably 0.1 to 1.5 mM, and most preferably 0.7 mM.

The reason for using dUTP is as follows.
In recent years, a method for preventing carryover contamination using an enzyme called uracil-N-glycosylase (UNG) having an enzymatic activity of recognizing U (uracil) base in DNA and cleaving the bond between U and pentose has been developed. Was. That is, dU
By performing PCR amplification using TP, it is possible to obtain an amplification product in which the thymine originally replaces uracil.
Even if this amplification product enters the newly prepared reaction solution before amplification, pre-incubation with the addition of UNG can decompose this amplification product and prevent contamination by past amplification products. it can. Therefore, to make the contamination prevention method applicable to the system of the present invention, dUTP
, Reverse transcription and PCR amplification reaction conditions can be set.

The final concentration of manganese (II) acetate is 1.0 to 5.0
mM is preferred, and most preferably about 4.0 mM.

The final concentration of rTth DNA polymerase is 0.05 to
It is preferably 0.2 U / μl, most preferably about 0.1 U / μl.

The final concentration of each primer is preferably from 0.1 to 1.0 μM, most preferably about 0.5 μM.

RTth DNA polymerase has both reverse transcription and polymerase activities. However, from reverse transcription
The balance between the efficiency of reverse transcription and PCR amplification in the process of performing a PCR amplification reaction in a single tube largely depends on the sequence of the template nucleic acid and the primer sequence, and depending on the selection of the sequence, RT-PCR Amplification may be incomplete or not amplified at all. Therefore, it is necessary to appropriately select the nucleic acid sequence of the site to be amplified and the sequence of the primer. Therefore, in view of the above, in the present invention, a primer that provides performance comparable to or higher than the amplification sensitivity of the conventional RT-nested PCR in reverse transcription and PCR amplification using rTth DNA polymerase. And a primer selected from a site having relatively low homology to the human submandibular gland-derived kallikrein mRNA sequence.

The primer of the present invention has an amplification site of PS
On the genome encoding A, it is set to span exon 4 (sense side) and exon 5 (antisense side). There is an intron of about 1300 bases between exon 4 and exon 5, but with this setting,
Under the conditions of temperature change in PCR amplification, it is difficult to generate an amplification product derived from the genome.

(A-3) PCR amplification is carried out by applying the following temperature changes to the above solution. 65 ° C 30 minutes 95 ° C 1 minute 95 ° C 25 seconds, 55 ° C 30 seconds: 5 cycles 90 ° C 25 seconds, 55 ° C 30 seconds: 40 cycles 60 ° C 7 minutes

(B) Detection of PCR amplification product on plate (b-1) An equal volume of an alkali denaturing solution is added to the PCR amplification product (50 μl) obtained in (a) and mixed. Fifteen
Minutes, most preferably about 10 minutes. A sodium hydroxide solution of about 0.1 N is used for the alkali denaturing solution.
Further, by adding salmon sperm DNA having a final concentration of preferably 0.1 to 5.0 mg / ml, most preferably 1.0 mg / ml, non-specific adsorption at the next hybridization is prevented, and more accurate hybridization is performed. I do.

(B-2) Next, a microwell on which the above-mentioned probe specific to PSA mRNA has been previously immobilized is prepared.

The probe of the present invention is set in a region containing the boundary between exon 4 and exon 5 so that amplification products other than those intended to be amplified due to contamination of genomic DNA are not captured. .

The PSA mRNA has a sequence very similar to that of human submandibular gland kallikrein mRNA.
A site with relatively low homology in sequence between NA and human submandibular gland kallikrein mRNA is used.If an amplification product derived from human submandibular gland kallikrein mRNA is generated, it is not captured and false positive Design.

Next, 100 μl of a hybridization buffer is added to the microwell on which the probe is immobilized. The hybridization buffer was urea, sodium chloride, sodium dihydrogen phosphate, ethylenediaminetetraacetic acid, polyoxyethylene sorbitan monolaurate (Twe
en20), a solution containing polyethylene alcohol, the pH of which is adjusted to 7.0 by adding sodium hydroxide. Further, before the hybridization, an appropriate amount of hydrochloric acid or sulfuric acid is added to the present hybridization buffer, and the mixture is adjusted to be neutral (pH 6.5 to 7.5) when 25 μl of the denatured amplification product is added.

The concentration of urea in the hybridization buffer is preferably 4 to 8M, most preferably 6M.
It is about.

The polyethylene glycol preferably has a molecular weight of about 6000, and the concentration is preferably 2 to 6% (w / v), most preferably about 3%.

In addition, the concentration of sodium chloride is about 0.3 M, sodium dihydrogen phosphate is about 20 mM, the concentration of ethylenediaminetetraacetic acid is about 2 mM, and the concentration of polyoxyethylene sorbitan monolaurate is about 1% (w / v). Is preferred.

(B-3) 25 μl of the alkali-denatured PCR product prepared in (b-1) is added to the well containing the hybridization buffer prepared in (b-2), and gently mixed. Then at 35-40 ° C, preferably 37 ° C
For 30 to 90 minutes, and most preferably about 60 minutes to perform hybridization.

(B-4) Next, 0.5 × PBS (Ph) containing 0.1% (w / v) polyoxyethylene sorbitan monolaurate
osphate-buffered saline: 8 g / l sodium chloride, 0.2
g / l potassium chloride, 1.15 g / l sodium monohydrogen phosphate (anhydrous) and 0.2 g / l sodium dihydrogen phosphate (anhydrous)), and then wash well with anti-dinitrophenyl (DNP) rabbit polyclonal antibody / A solution containing a peroxidase label is added and left at 37 ° C. for 30 minutes.

(B-5) The wells were washed in the same manner as in (b-4), and the peroxidase activity was measured using 3,3 ′, 5,5′-tetramethylbenzidine (100 μl of the substrate solution) to develop a color reaction. Measured as Color development is performed for 15 minutes, stopped with an acidic solution (100 μl of stop solution), and the absorbance is measured at a wavelength of 450 nm. A case where the absorbance is 0.3 or more is determined to be positive. In addition, a blank is prepared by preparing a well in addition to detection, adding 100 μl of a substrate solution and a stop solution, setting the absorbance thereof to 0, and performing other measurements.

[0037]

【Example】

Embodiment 1 FIG. Prostate cancer cell line derived from lymph node metastasis (LNCaP)
Detection of PSA mRNA from LNCaP cells is known as a cancer cell line of human prostate cancer (Cancer Res 43: 1809-1818, 1983) and used as a cell producing PSA. This culture 10 ⁶ cells than AGPC method (acid guanidinium thiocyanate-phenol-
PSA mRNA was extracted by a modified method (using a coprecipitant) of the chloroform method and Experimental Medicine 15: 99-102, 1991) to prepare a dilution series of PSA mRNA. Since one cell is considered to be producing a plurality of PSA mRNA, in a cell-number conversion, to produce a 10 ² to 10 ^-3 or equivalent dilution series. Using this dilution series, detection of PSA mRNA was attempted by the method of the present invention.

The extracted LNCap PSA mRNA was redissolved in 10 μl of an aqueous solution containing an RNase inhibitor (manufactured by Pharmacia Biotech) so that the final concentration was 250 U / ml, and the final concentration was 50 mM bicine, 115 mM potassium acetate, 8
% (W / w) glycerol and 0.3 mM dATP, dTTP, dG
TP and dCTP, 4 mM manganese (II) acetate, 0.1 U / μl r
Tth DNA polymerase (PerkinElmer), 0.5 each
A mixed solution of μM sense primer (SEQ ID NO: 1) and antisense primer (SEQ ID NO: 2) was added to make a total of 50 μl, and the pH was adjusted to 8.2. Primers were synthesized using a model 8750 manufactured by Milligene (the same applies to the following probes).

PCR amplification was performed by applying the following temperature changes to the above solution. 65 ° C 30 minutes 95 ° C 1 minute 95 ° C 25 seconds, 55 ° C 30 seconds: 5 cycles 90 ° C 25 seconds, 55 ° C 30 seconds: 40 cycles 60 ° C 7 minutes

An equal volume of an alkali denaturing solution (0.1N NaOH) was added to the PCR amplification product (50 μl), mixed, and allowed to stand for 10 minutes. Further, non-specific adsorption at the time of hybridization was prevented by adding salmon sperm DNA (manufactured by Sigma) at a final concentration of 1.0 mg / ml. Next, a probe (SEQ ID NO: 3) specific for PSA mRNA was previously immobilized on a microwell.
100 μl of hybridization buffer (6 M urea, 0.
3 M NaCl, 20 mM NaH ₂ PO ₄ , 2 mM EDTA, 1% (w / v) Twee
n20 and 3% polyethylene alcohol (molecular weight 600
0), pH 7.0). Before the hybridization, an appropriate amount of sulfuric acid was added to the hybridization buffer, and neutralized (pH 6.5 to 7.
5)

25 μl of the alkaline-denatured PCR product was added to the wells containing the above hybridization buffer, and mixed gently. Then, the mixture was left at 37 ° C. for 60 minutes to perform hybridization.

Next, the wells were thoroughly washed with 0.5 × PBS containing 0.1% (w / v) polyoxyethylene sorbitan monolaurate, and then containing an anti-DNP rabbit polyclonal antibody / peroxidase label (manufactured by Dako Japan). The solution was added and left at 37 ° C. for 30 minutes. After washing the wells in the same manner as above, the peroxidase activity was measured as a color reaction using 3,3 ′, 5,5′-tetramethylbenzidine (100 μl of the substrate solution). Color development was performed for 15 minutes, stopped with an acidic solution (100 μl of stop solution), and the absorbance was measured at a wavelength of 450 nm. Absorbance meter is model MTP-32 manufactured by Corona
It was used. A case where the absorbance was 0.3 or more was determined to be positive. The blank was prepared by adding a well in addition to the detection, and adding 100 μl of a substrate solution and a stop solution in the same manner. The results are shown in Table 1 below.

[0043]

[Table 1]

As shown in Table 1 above, when the cutoff value was tentatively set to 0.3, it was possible to measure up to 10 ^-2 cells in terms of cell number. FIG. 3 is a schematic view of a plate well showing the results of Table 1.

Embodiment 2 FIG. PSA mRNA from various cancer lineage cells
To date, detection of PSA mRNA from cancer cell lines other than various prostate cancers using existing PSA mRNA detection technology has been studied. Mitchell R et al. Report ovarian cancer (BG-1), lung cancer (SK
-MES-1), a small amount of PSA mRNA was detected in lineage cells derived from myeloma (HL-60) (Canc
er Res. 55: 2640-2644, 1995). Also Nosratollah
Z. Report that PSA mRNA was also detected in breast cancer in women (Clin Chem 42: 361-366, 1996). Thus, the present inventors extracted RNA from about 10 ⁶ types of urological cancer cell lines excluding LNCaP cells by a modified AGPC method in the same manner as in Example 1 and then carried out PSA mRNA Attempted detection. The results are shown in Table 2 below.

[0046]

[Table 2]

The 15 types of cells shown in Table 2 were obtained from the following sources or as described in the following literature. PUC3: Invest. Urol., 17: 16-23, 1979 DU145: Int. J. Cancer, 21: 274-281, 1978 R3 and R4: obtained from the Department of Urology, Tohoku University School of Medicine TOS1 to 3 and 3LN: obtained from the School of Urology, Tohoku University NEC8 and 14: manufactured by RIKEN ACHN: manufactured by Dainippon Pharmaceutical Co., Ltd. KK47: Obtained from Urology, Tohoku University School of Medicine T24: Int. J. Cancer, 5: 310-319, 1970 J82: Obtained from Urology, Tohoku University School of Medicine YTS-1: Obtained from Urology, Tohoku University School of Medicine From the results shown in Table 2 above, TOS3, NEC8 and NE
It is determined that PSA mRNA was detected in C14 cells.

Embodiment 3 FIG. Example 1. Detection of PSA mRNA from cancer cells mixed in lymphocytes of human peripheral blood. Nucleated component was collected from blood of a patient with prostate disease.
In the same manner as described above, RNA was extracted by the modified AGPC method, and PSA mRNA was detected by the method of the present invention.
PSA mRNA was detected in the example and its clinical stage was D2 for prostate cancer (results not shown).

Embodiment 4 FIG. Detection of PSA mRNA from cancer cells remaining in human serum We also tried to detect PSA mRNA from nucleated cells remaining in serum. 500 to 1000 μl of serum from a prostate disease patient was collected and centrifuged at 12,000 × g at 4 ° C. for 20 minutes in a refrigerated high-speed centrifuge to collect remaining cells. As in Example 1, RNA was extracted from the cells by a modified AGPC method, and then PSA mRNA was detected by the method of the present invention. As a result, PSA mRNA was extracted and detected from nucleated cells, which are considered to remain in the serum, although the value of the absorbance was low from the serum. As shown in Table 3, 3 out of 72 cases were clearly judged to be PCR mRNA positive. Therefore, it was suggested that PSA mRNA could be easily detected without using human peripheral blood lymphocytes, which are difficult to preserve or pretreat from whole blood.

[0050]

[Table 3]

Embodiment 5 FIG. PSA mRNA from cancer cells contaminated in lymph node aspirated biopsy cells FNAB aspirated cells and lymph node tissue from pelvic lymph nodes of prostate and bladder cancer patients were degraded with proteolytic enzymes Thereafter, as in Example 1, RNA was extracted by a modified AGPC method, and then PSA mRNA was extracted by the method of the present invention.
Was detected. As a result, PSA mR was found in lymph node aspirated biopsy cells from patients with relapsed prostate cancer or bone metastases.
NA was detected (Samples 4 and 5 in Table 4), and lymph node aspirated biopsy cells and lymph node tissue from bladder cancer patients and lymph node aspirated biopsy cells from prostate cancer patients without metastasis and lymph node tissue PSA mRNA was not detected.

[0052]

[Table 4]

[0053]

According to the present invention, the reverse transcription and the PCR amplification are performed separately, and the gene is more easily and sensitively compared with the conventional method in which the detection is attempted by the nested PCR method. It is possible to detect PSA mRNA that reduces the risk of contamination that requires the most attention in the test. In addition, unlike the conventional method in which the amplification product is electrophoresed and then transferred to a membrane and subjected to Southern hybridization, detection is completed in a short time (one day) because hybridization is performed on a plate, operability is good, and the number of samples is small. It can handle cases where there is a lot of data, and is excellent in objectivity of data.

Such effects of the present invention are particularly effective when early detection of prostate cancer, presence or absence of cancer metastasis, distinction from prostatic hypertrophy, postoperative follow-up, and discrimination from cancers other than prostate cancer. It is advantageous.

The method of labeling the primer of the present invention, the sensitization method by devising a reagent at the time of hybridization, and the improvement of specificity are useful for the detection of other cancer markers similar in characteristics to PSA mRNA detection and the detection of viral genes. Is also applicable, and its application range is extremely wide.

[0056]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Genomic DNA sequence TGTGCGCAAG TTCACCCTCA 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: Genomic DNA sequence CCACGATGGT GTCCTTGATC 20

SEQ ID NO: 3 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA sequence AGAATCACCC GAGCAGG 17

[Brief description of the drawings]

FIG. 1 is a diagram showing a structure and a part of a base sequence of PSA mRNA and showing a set position of a primer and a probe used in the present invention.

FIG. 2 is a diagram showing the setting positions of primers and probes used in the present invention on the base sequence of PSA mRNA. The homology with the PSA mRNA is also shown by listing the human submandibular gland kallikrein mRNA.

FIG. 3 is a schematic plate diagram showing the detection results of PSA mRNA from LNCaP cells detected on a plate by the detection method of the present invention. The numbers shown are the PSA mRNA used
Is expressed in terms of the number of cells (cells).

Claims (6)

[Claims] Amplifying a certain region of prostate specific antigen (PSA) mRNA by reverse transcription PCR using a primer set consisting of a sense primer and an antisense primer labeled with a label A and rTth DNA polymerase;
And the PSA in the sample consists of detecting the amplification product
A method for detecting mRNA. 2. In the detection of an amplification product, the amplification product is hybridized and bound to an amplification product-specific probe immobilized on a support, and an antibody specific for label A and labeled with an enzyme. The detection method according to claim 1, comprising reacting the conjugate with the conjugate and detecting the conjugate by a reaction specific to the enzyme. 3. The primer set of the reverse transcription PCR method, wherein the sense primer is 5'-TGTGCGCAAGTTCACCCTCA-3 '(SEQ ID NO: 1) and the antisense primer is 5'
-CCACGATGGTGTCCTTGATC-3 '(SEQ ID NO: 2) and the amplification product-specific probe is 5'-AGAATCACCCGAGCA
The detection method according to claim 1 or 2, which is GG-3 '(SEQ ID NO: 3). 4. The detection method according to claim 1, wherein the label A is a 2,4-dinitrophenyl (DNP) label at the 5 ′ end of the sense primer and the antisense primer. 5. The detection method according to claim 4, wherein a plurality of DNPs are linked. 6. (a) 5′-TGTGCGCAAGTTCACCCTCA-3 ′ (SEQ ID NO: 1) sense primer and 5′-CCACGATGGTGTC
It consists of an antisense primer of CTTGATC-3 '(SEQ ID NO: 2), provided that the 5' end of each primer is
(B) a solid support having a 5′-AGAATCACCCGAGCAGG-3 ′ (SEQ ID NO: 3) probe, (c) an alkali denaturing solution, and (d) a A kit for detecting PSA mRNA in a sample, comprising a hybridization buffer.