WO2012048607A1 - Method for detecting pca3 and psa genes, and diagnostic kit thereof - Google Patents

Abstract

Disclosed is a method for detecting PCA3 and PSA genes, and diagnostic kits thereof. This method comprises designing primers and probes for the mRNAs of the PCA3 and/or PSA genes, bonding covalently the probes with microbeads to form a probe-microbead mixture, hybriding with the products of reverse transcription PRC amplification, then adding streptavidin-phycoerythrin; fluorescence signals from the various microbeads can be detected so as to determine whether the samples to be tested contain PCA3 and PSA genes, or not, and determine the expression level thereof.

Description

[Name of invention established by ISA according to Rule 37.2] Method for detecting PCA3 gene, PSA gene and diagnostic kit thereof Technical Field

The invention relates to the technical field of in vitro diagnostic detection, in particular to a liquid phase chip detection method for a specific gene PCA3, PSA related to prostate diseases and a diagnostic kit thereof.

Background Art

 PCA3 (prostate cancer antigen 3) gene, also known as DD3 ( Differential display code 3 ) The gene, a gene found to be non-coding RNA discovered by Bussemakers et al in 1999, consists of four exons and three introns. The PCA3 gene is expressed only in prostate tissue, and its expression is extremely low or not expressed in normal prostate and benign prostatic hyperplasia (BPH) tissues, but is highly expressed in prostate cancer (PCa) cells. Therefore, the PCA3 gene is currently considered to be the most specific gene for prostate cancer.

 Prostate specific antigen (PSA, prostate specific Although the antigen has been widely used in the screening, diagnosis, staging, efficacy observation and prognosis of prostate cancer, the PSA gene does not have tumor specificity. Benign lesions such as benign prostatic hyperplasia (BPH) and prostatitis can also be elevated, and the PSA gene does not improve the early diagnosis rate of prostate cancer.

Prostate biopsy is the gold standard for differential diagnosis of different prostate diseases, but it is an invasive examination and often has a false negative. Digital rectal examination and related auxiliary examinations such as B-ultrasound, MR I (magnetic resonance imaging) Although non-invasive, it has limitations. Therefore, finding more specific, early, non-invasive detection methods is an inevitable trend in the differential diagnosis of prostate diseases.

 Molecular biological tests widely used at home and abroad for prostate diseases Among the related specific gene methods, the quantitative quantitative PCR has the limitation of detecting flux, so it can not really meet the needs of clinical diagnosis and detection. Traditional solid-phase biochip technology (Biochip) technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, and cumbersome operation. Therefore, there is a need for a clinical detection method that can rapidly, stably and accurately detect the specific genes related to prostate diseases. The liquid phase chip (xMAP) technology is such a new detection technology. .

 Liquid phase chip, also known as liquid chip, It can perform qualitative and quantitative detection on a small number of samples, and has outstanding advantages such as high throughput, easy operation, good repeatability, high sensitivity and wide linear range. The system is composed of many microspheres as the main matrix. In the manufacturing process of the microspheres, two different red-classified fluorescences are incorporated. According to the ratio of the two kinds of fluorescence, the spherical matrix is divided into 100 kinds, and 100 different probe molecules can be labeled. Can simultaneously up to 100 in one sample Different target molecules are detected. Depending on the analyte, the surface of the microspheres can be covalently bound to a variety of nucleic acid detection probes, and fluorescent labels are added as the hybridization reaction proceeds. Different detection microspheres can be added simultaneously in the same reaction system, so that a small amount of sample can be used for rapid, high-throughput detection. After the reaction, the microspheres are arranged into a single column and flow through the liquid phase chip detector by microfluidic technology. Each microsphere can be detected by two lasers at the same time. The red laser excites the red classified fluorescence on the microsphere, and distinguishes the different reactions to be qualitative; The green laser excites the fluorescent label bound to the sample to be tested for quantification. When the labeled sample to be tested is combined with the probe on a specific microsphere, the light excited by the two lasers can be detected. At last , Through the computer's high-speed digital signal processor, the average fluorescence intensity on a particular microsphere can be automatically statistically analyzed to determine the type and amount of the analyte.

The invention is based on the high-throughput, easy-to-operate, reproducible, high-sensitivity, wide linear range and the like of the liquid-phase chip technology, and specific genes related to prostate diseases. Testing can be better applied in clinical testing.

Technical Problem

 The technical problem to be solved by the present invention is to provide a detection method and a diagnostic kit for a specific gene related to prostate diseases. . The method and kit comprise detecting the PCA3 gene and/or the PSA gene, which can be Different degrees of prostate disease, especially prostate cancer, for specific and early diagnosis, staging, efficacy observation and prognosis, differential diagnosis of benign prostatic hyperplasia and prostate cancer, and early diagnosis, efficacy observation and prognosis of other related diseases . The detection method and the diagnostic kit have the advantages of high sensitivity, high specificity, high accuracy, and rapid detection.

Technical Solution

 In order to solve the above technical problem, in an aspect of the present invention, a detection is provided A liquid phase chip method for the ratio of the specific genes PCA3 gene, PSA gene and PCA3 gene/PSA gene associated with prostate disease, comprising the following steps:

(1) comprising a fluorescently encoded carboxyl (-COOH) microsphere, Beads, which covalently binds to a specific nucleic acid probe designed for mRNA of the PCA3 gene or the PSA gene; or the design comprises two different fluorescent codes The carboxylated microspheres Beads, each of which is covalently bound to a specific nucleic acid probe designed for the mRNA of the PCA3 gene and the gene PSA; the PCA3 gene can be detected as a specific gene for prostate cancer alone, the PSA gene It can be used as a specific gene for related prostate diseases, and PCA3 and PSA genes can be combined to detect specific genes related to prostate diseases, and the relevant prostate diseases can be identified based on the ratio of PCA3 gene/PSA gene. diagnosis. PCA3 gene and / or PSA gene can also be combined with other genes for joint detection of specific genes related to prostate diseases;

 (2) Targeting the PCA3 gene and / or PSA gene mRNA, respectively, designed upstream and downstream primers, one of which contains biotin (Biotin) label; the corresponding product is amplified by reverse transcription PCR;

 (3) Microspheres containing specific nucleic acid probes and PCA3 gene and/or PSA After hybridization of the reverse transcription amplification product of the mRNA of the gene, streptavidin-PE is added, and the fluorescent signal is detected by liquid phase chip method xMAP;

(4) comparing the detected fluorescent signal with the fluorescent signal of the reference gene to determine whether there is a prostate disease-related gene PCA3 gene and/or PSA gene in the test sample, and its expression in the sample, The ratio of the PCA3 gene/PSA gene can also be calculated.

The microspheres described in the above step (1) of the detection method are polyphenylene microspheres having an average diameter of 5.6 μm combined with different fluorescent dyes, that is, color-coded beads;

 In the step (1), the PCA3 gene and/or the PSA gene covalently bound to the microspheres Specific nucleic acid probes designed for mRNA, including the following sequences (wherein the 5' end contains an amino group modification):

 mRNA for the PCA3 gene:

 Probe (1) 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as shown in SEQ ID NO.

 Probes (2), (3), (5), (6) are the same as probe (1), as shown in SEQ ID NO.

 Probe (4) 5 ' - AminolinkerC12 CTCACCTCTGTATCATCAG -3 ' , as shown in SEQ ID NO. 2;

Probe (7) 5 ' - AminolinkerC12 ATCTCTGTGCTTCCTTTTGT -3 ' as shown in SEQ ID NO. 3;

 Probe (8) is the same as probe (7), as shown in SEQ ID NO.

 Probe (9) 5 ' - AminolinkerC12 CAAATCTGTAATCCCGTTCA -3 ' , as shown in SEQ ID NO. 4;

Probe (10) 5 ' - AminolinkerC12 TATGTGTCAAGAGGAGAGCC -3 ' as shown in SEQ ID NO. 5;

 Or a sequence comprising the above sequence (including a complementary sequence) extending to the 5' end or/and the 3' end;

 Or a sequence having greater than 85% homology to the above sequence (comprising a complementary sequence);

 Or a base complementary sequence to the above sequence;

 Or using any one or more of the above sequences;

 mRNA for the PSA gene:

 Probe A: 5 ' - AminolinkerC12 CAGAATCACCCGAGCAGGTG -3 ' , as shown in SEQ ID NO.

Probe B: 5 ' - AminolinkerC12 GGGGTCAAGAACTCCTCTGG -3 ' , as shown in SEQ ID NO.

Probe C: 5 ' - AminolinkerC12 CACGCTTTTGTTCCTGATGC -3 ' , as shown in SEQ ID NO.

 Probe D is the same as PSA probe A, as shown in SEQ ID NO.

 Probe E is the same as PSA probe B, as shown in SEQ ID NO.

 Or a sequence comprising the above sequence (including a complementary sequence) extending to the 5' end or/and the 3' end;

 Or a sequence having greater than 85% homology to the above sequence (comprising a complementary sequence);

 Or a base complementary sequence to the above sequence;

 Alternatively, any one or a combination of more than one of the above sequences may be used.

 In step (2), the PCA3 gene and/or the PSA gene are described. Upstream and downstream primers designed for mRNA, including the following sequences (where the upstream primer contains a biotin tag at the 5' end):

 mRNA for the PCA3 gene:

 Primer set (1) upstream 5 ' -biotin AAGAAATAGCAAGTGCCGAGAAG -3 ' , as shown in SEQ ID NO. 9; downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3', as shown in SEQ ID NO.

 Primer set (2) upstream 5 '-biotin TGGTGGGAAGGACCTGATGATAC-3', such as SEQ ID NO. 11; downstream 5'-TCTCCCAGGGATCTCTGTGCTT-3', as shown in SEQ ID NO.

 Primer set (3) upstream 5 ' -biotin GGTGGGAAGGACCTGATGATAC -3 ' , as shown in SEQ ID NO. 13; downstream 5'-TAAAGGGGCTGGAAATGTGC-3', as shown in SEQ ID NO.

 Primer set (4) upstream 5 '-biotin AGCCGAGGGAGACCAGGAAG -3 ' , as SEQ ID NO. 15; downstream 5 ' - CAGCAGATGTGTGGCCTCAGAT-3' as shown in SEQ ID NO.

 Primer set (5) upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT -3 ' , as SEQ ID NO. 17; downstream 5 ' - CACAGGGCGAGGCTCATC-3' as shown in SEQ ID NO.

 Primer set (6) upstream 5 ' -biotin AGAAATAGCAAGTGCCGAGAAGC -3 ' , as shown in SEQ ID NO. 19; downstream 5'-CACAGGGCGAGGCTCATC-3', as shown in SEQ ID NO.

 Primer set (7) upstream 5 ' -biotin TATCCACACACACAGGAAGCAC -3 ' , as shown in SEQ ID NO. 21; downstream 5 ' - CCTCTCATTGGTAATGCTCACTTT-3', as SEQ ID NO. Shown

 Primer set (8) upstream 5 ' -biotin AAGGCTGCTGACTTTACCATCTG -3 ' , as shown in SEQ ID NO. 23; downstream 5 ' - TCTAATGTCCTTCCCTCACAAGC-3', as shown in SEQ ID NO.

 Primer set (9) upstream 5 ' -biotin CGCTTGTGAGGGAAGGACATTAG -3 ' , as shown in SEQ ID NO. 25; downstream 5 ' - GTGAAGCCATCAAGATTTTCTCGTC-3', as SEQ ID NO. Shown

 Primer set (10) upstream 5 '-biotin CAGCAGGACCCAACGCAT -3 ' , as SEQ ID NO. 27; downstream 5'-GAGAGAGGATTGGTAAGCGATGTG-3' as shown in SEQ ID NO.

 Or a sequence comprising the above sequence (including a complementary sequence) extending to the 5' end or/and the 3' end;

 Or a sequence having greater than 85% homology to the above sequence (comprising a complementary sequence);

 Or a base complementary sequence to the above sequence;

 Or using any one or more of the above sequences;

 mRNA for the PSA gene:

 Primer set A: upstream 5 ' -biotin TTGACCCCAAAGAAACTTCAGTGT -3 ' , as shown in SEQ ID NO. 29; downstream 5 ' - TGCCCCATGACGTGATACCT-3', as shown in SEQ ID NO.

 Primer set B: upstream 5 ' -biotin TCCCACACCCGCTCTACGAT-3', as SEQ ID NO.31; downstream 5 ' - CGTCCAGCACACAGCATGAACT-3' as shown in SEQ ID NO.

 Primer group C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33; downstream 5 ' - GCTGTGGCTGACCTGAAATACC-3' as shown in SEQ ID NO.

 Primer set D: upstream 5 ' -biotin GGCAGCATTGAACCAGAGGAGT -3 ' , as shown in SEQ ID NO. 35; downstream 5 ' - CGATGGTGTCCTTGATCCACTT-3', as shown in SEQ ID NO.

 Primer set E: upstream 5 ' -biotin TCCTCAGGCCAGGTGATGACT -3 ' , as SEQ ID NO. 37; downstream 5'-CGTCCAGCACACAGCATGAACT-3', as shown in SEQ ID NO.

 Or a sequence comprising the above sequence (including a complementary sequence) extending to the 5' end or/and the 3' end;

 Or a sequence having greater than 85% homology to the above sequence (comprising a complementary sequence);

 Or a base complementary sequence to the above sequence;

 Alternatively, any one or a combination of more than one of the above sequences may be used.

 The above primer set designed for PCA3 (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) and PCA3 probes designed as described above (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) one-to-one correspondence, that is, the corresponding primer set amplification The product is hybridized with the corresponding probe in step (3) ;

 The above primer sets A, B, C, D, E designed for PSA and the PSA probe A designed above , B, C, D, E correspond one-to-one, that is, the product amplified by the corresponding primer set and the corresponding probe are hybridized in step (3);

 In the step (4), the primers and probe sequences of the internal reference gene β-actin gene are as follows:

 Upstream primer 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG -3' , as SEQ ID NO. 39;

 Downstream primer 5'-GCTGGGGTGTTGAAGGTCTC -3', as SEQ ID NO. 40 Shown

 Probe 5 ' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' , as shown in SEQ ID NO. 41;

 Or a sequence comprising the above sequence (including a complementary sequence) extending to the 5' end or/and the 3' end;

 Or a sequence having greater than 85% homology to the above sequence (comprising a complementary sequence);

 Or a base complementary sequence to the above sequence;

 Alternatively, any one or a combination of more than one of the above sequences may be used.

In another aspect of the present invention, a diagnostic kit for detecting a prostate cancer-associated specific gene PCA3, PSA comprising mRNA encoding a PCA3 gene and/or a PSA gene is provided. Microsphere mixture of specific probes, microspheres bound to β-actin gene probe, mRNA against PCA3 gene and/or mRNA of PSA Upstream and downstream primers, upstream and downstream primers of β-actin gene, streptavidin-PE, control substance (negative control and positive control).

 The controls described in the above kits contain a positive control and a negative control, wherein the positive control contains the PCA3 gene and / Or a plasmid mix of the PSA gene (including the plasmid containing the β-actin gene), and the negative control does not contain the PCA3 gene, PSA gene. And a plasmid of the β-actin gene; the microsphere mixture is freely combined according to the needs of different test samples.

 Another aspect of the invention provides A diagnostic kit for detecting specific genes related to prostate diseases, PCA3 and PSA, for detecting in vitro samples, for specific and early diagnosis, staging, efficacy observation and prognosis of different degrees of prostate diseases (especially prostate cancer), The application of differential diagnosis of benign prostatic hyperplasia and prostate cancer, as well as early diagnosis, staging, efficacy observation and prognosis judgment of other related diseases.

Advantageous Effects

 Since the present invention utilizes liquid phase chip technology, the detection method and kit have high sensitivity, high specificity, high throughput, Good stability, rapid detection, accurate and other advantages, can qualitatively and quantitatively detect PCA3 gene and / or PSA gene, and can calculate the ratio of PCA3 gene / PSA gene, so Better application in clinical testing and diagnosis.

Description of Drawings

Best Mode

Mode for Invention

The invention is described in detail below with reference to specific embodiments, but is not to be construed as limiting. The examples described are merely illustrative of the nucleic acid primers, probes, detection methods, kits, and methods of application and are not limited thereto. Various variations are contemplated as being within the scope of the invention and the scope of the invention.

 Experimental Materials:

 Primers and probes were synthesized by Invitrogen; Trizol was purchased from Invitrogen; Total RNA extraction kit from TaKaRa The reverse transcription cDNA synthesis kit was purchased from Fermentas; the multiplex PCR kit, different numbered microspheres (surface carboxyl modification), streptavidin-phycoerythrin were purchased from QIAGEN; 1-ethyl-(3) -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Pierce; 2-(N-morpholine)-ethanesulfonic acid (MES), N-lauroyl Sarkosyl and tetramethylammonium chloride (TMAC) were purchased from sigma.

 Preparation of buffers, hybrids and other solutions:

 Coupling buffer, pH 4.5 250 ml

 MES 4.88g ;

 1.5 × TMAC Hybrid Solution 250 ml

 5M TMAC 225 ml

 20% Sarkosyl 1.88 ml

 1M Tris-HCl, pH 8.0 18.75 ml

 0.5M EDTA, pH 8.0 3.0 ml

H ₂ O 1.37 ml ;

 1.5 × TMAC Hybrid Solution 250 ml

 5M TMAC 150 ml

 20% Sarkosyl 1.25 ml

 1M Tris-HCl, pH 8.0 12.5 ml

 0.5M EDTA, pH 8.0 2.0 ml

H ₂ O 84.25 ml ;

 TE buffer, pH 8.0 500 ml

 1M Tris-HCl, pH 8.0 5 ml

 0.5M EDTA, pH 8.0 1 ml

H ₂ O 444 ml

Preparation of D-Hanks solution: KCl 0.4g, NaCI 8.0g, NaHC0 ₃ 0.35 g, Na ₂ HP04 · 12H ₂ 0 0.15 g, KH ₂ P04 0.06 g, dissolved in 1000 ml dH ₂ O. Sterilize at 15 KPa x 30 min and store at 4 °C.

 Example 1 : Liquid phase chip method for detecting PCA3 gene in urine by different primers and probe combinations

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PCA3 gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 mRNA for the PCA3 gene:

 Probe (1) 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as shown in SEQ ID NO.

 Probes (2), (3), (5), (6) are the same as probe (1), as shown in SEQ ID NO.

 Probe (4) 5 ' - AminolinkerC12 CTCACCTCTGTATCATCAG -3 ' , as shown in SEQ ID NO. 2; probe (7) 5 ' - AminolinkerC12 ATCTCTGTGCTTCCTTTTGT -3 ' , as SEQ ID NO. 3;

 Probe (8) is the same as probe (7), as shown in SEQ ID NO.

 Probe (9) 5 ' - AminolinkerC12 CAAATCTGTAATCCCGTTCA -3 ' , as shown in SEQ ID NO. 4; probe (10) 5 ' - Aminolinker C12 TATGTGTCAAGAGGAGAGCC -3 ' , as shown in SEQ ID NO. 5;

 --actin gene: 5' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' is shown as SEQ ID NO.

2. The amino-modified oligonucleotide probes were coupled to 11 carboxyl microspheres numbered 11, 13, 15, 21, 25, 61, 62, 63, 64, 65, 66, respectively.

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 Dissolving the oligonucleotide probes of PCA3 and β-actin with d H ₂ O at a concentration of 1 mM (1 nmol/μl);

 2.3 The full speed vortex numbers are 11, 13 , 15 , 21 , 25 , 61 , 62 , Storage of 11 kinds of carboxyl microspheres of 63, 64, 65, 66 for at least 3 min, resulting in a uniform suspension of microspheres;

2.4 Take 2.5×10 ⁶ microspheres to store the suspension into eleven centrifuge tubes;

 2.5 10,000g, centrifuged, 1-2min;

 2.6 Remove the supernatant with 50μl 0.1M MES, pH 4.5 coupling buffer, resuspend the microspheres, vortex for 20 seconds;

2.7 eleven concentrations of 1 mM oligonucleotide probes were diluted with d H ₂ O at a ratio of 1:10 to a concentration of 0.1 nmol / μl;

 2.8 each probe was added 2 μl (concentration 0.1nmol / μl) to the corresponding microspheres mixed, vortex mixed;

2.9 Prepare 10mg/ml fresh EDC solution with d H ₂ O (Note: keep the EDC powder dry to facilitate the use of EDC in the next step);

 2.10 Add 2.5μl 10mg/ml The fresh EDC solution of EDC was vortexed into eleven kinds of microspheres (25 μg final concentration: about 0.5 μg/μl);

 2.11 Incubate at room temperature for 30 min in the dark;

2.12 Prepare a second 10mg/ml EDC fresh solution with d H ₂ O (Note: EDC powder should be discarded if deliquescent, it is recommended to use fresh EDC powder for each step of the coupling process);

 2.13 Add 0.1 μl of 10 mg/ml EDC fresh solution to each of the eleven microspheres, and vortex and mix;

 2.14 Incubate at room temperature for 30 min in the dark;

 2.15 Add 1 ml of 0.02% Tween-20 to each of the eleven coupled microspheres;

 2.16 10,000g, centrifuged, 1-2min;

 2.17 Remove the supernatant, resuspend eleven coupled microspheres with 1 ml of 0.1% SDS, and mix by shaking;

 2.18 10,000g, centrifuged, 1-2min;

 2.19 Remove the supernatant, add 100 μl TE, pH 8.0, vortex for 20 s, and resuspend the microspheres;

 2.20 Counting eleven microspheres coupled with an oligonucleotide probe using a cell counter;

a. The coupled microspheres were diluted 1:100 with d H ₂ O;

 b. Vortex and shake thoroughly;

 c. Take 10μl onto the cell counter;

 d. The total number of microspheres in the four corners of the number cell counter;

 e. Microspheres/μl=(4 large microspheres total)×2.5×100 (dilution factor).

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PCA3 probe (1) microsphere 11, PCA3 Probe (2) Microsphere 13, PCA3 Probe (3) Microsphere 15, PCA3 Probe (4) Microsphere 21, PCA3 Probe (5) Microsphere 25, PCA3 Probe (6) Microsphere 61, PCA3 Probe (7) microsphere 62, PCA3 probe (8) microsphere 63, PCA3 probe (9) microsphere 64, PCA3 probe (10) microsphere 65, β-actin probe microsphere 66, In a proportional mixture, the final concentration of each microsphere is 1500/μl, and it is stored at 2-8 °C in the dark.

 two. Sample preparation

 The clinical sample No. 1-5 is the urine of patients with prostate cancer (PCa), and the clinical sample of No. 6-8 is the urine of normal people:

 1. The subject adopts the traditional prostate massage method, that is, the examiner performs a digital rectal examination, from the sides of the prostate to the central groove, massages from the top to the bottom 2 to 3 times, and then massages the central ditch once to squeeze the prostatic fluid into the urethra.

 2. The subject immediately urinates and collects 20 to 30 ml of the initial urine, which is immediately placed in ice for cooling. At 4 ° C 3000 r /min was centrifuged for 10 min to collect urine sediment, and an appropriate amount of pre-cooled D-Hanks solution was washed twice;

 3. Collect urinary sediment in RNase-free centrifuge tubes Immediately, 500 μl of a denaturing solution containing guanidinium isothiocyanate (ie, RNAiso Reagent) was added and stored at 70 ° C.

 4. Remove 500 μl RNAiso Reagent from a 70 ° C The urine sediment specimen is centrifuged after being melted.

 5. Add 1/5 RNAiso Reagent volume of chloroform, mix by shaking, and let stand for 5 minutes at room temperature. Centrifuge for 15 min at 12000 r /min 4 °C.

 6. Carefully move the upper aqueous phase into the RNase-free centrifuge In the tube, an equal volume of isopropanol was added to the supernatant, and the mixture was inverted. The mixture was allowed to stand at room temperature for 5 minutes, and then centrifuged at 12000 r/min for 10 minutes.

 7. Discard the supernatant and wash the pellet twice with 1 ml of 75% pre-chilled ethanol. Centrifuge thoroughly to remove the ethanol.

8. Dissolve the RNA sample with 50 ul (RNase-free) dH2O at 55-60 ° C for 5-10 min; measure the OD value to quantify the RNA concentration.

 three. Multiple RT-PCR

 Multiple reverse transcription PCR amplification of mRNAs from samples 1-8 above was performed as follows:

 1. Synthesis of cDNA first strand

 1 Take an RNase-free centrifuge tube and place on ice to establish the following reaction system;

 Total RNA 5 ~ 10μg / 3μl

 Oligo(dT)18 Primer (0.5μg/μl) 1μl

 RNase-free water forward to 12μl

 Mix the reaction solution gently, and centrifuge the centrifuge to collect the reaction solution on the tube wall to the bottom of the tube;

 2 Centrifuge the tube at 70 ° C for 5 minutes, then quickly remove it and place it in ice for cooling. Use a refrigerated centrifuge slightly.

 Centrifuge the reaction liquid on the wall of the tube to the bottom of the tube;

 3 Place the centrifuge tube on ice and add the following reaction solutions in order;

 5×reaction buffer 4μl

 RNase Inhibitor (20U/μl) 1μl

 10mM dNTPs Mix 2μl

 Mix the reaction solution gently, and centrifuge the centrifuge to collect the reaction solution on the tube wall to the bottom of the tube;

 4 centrifuge tube at 37 ° C for 5 minutes;

 5 Add 1μl RevertAidTM Reverse Transcriptase (200 U / μl), the final volume is 20 μl;

 6 centrifuge tube at 42 ° C for 60 minutes;

 7 The tube is heated at 70 ° C for 10 minutes to terminate the reaction, then quickly taken out and placed in ice to cool;

 2. Multiplex PCR

 2.1 Synthesize primers according to the following sequence:

 mRNA for the PCA3 gene:

 Primer set (1) upstream 5 ' -biotin AAGAAATAGCAAGTGCCGAGAAG -3 ' , as shown in SEQ ID NO. 9; downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3', as shown in SEQ ID NO.

 Primer set (2) upstream 5 '-biotin TGGTGGGAAGGACCTGATGATAC-3', such as SEQ ID NO. 11; downstream 5'-TCTCCCAGGGATCTCTGTGCTT-3', as shown in SEQ ID NO.

 Primer set (3) upstream 5 ' -biotin GGTGGGAAGGACCTGATGATAC -3 ' , as shown in SEQ ID NO. 13; downstream 5'-TAAAGGGGCTGGAAATGTGC-3', as shown in SEQ ID NO.

 Primer set (4) upstream 5 '-biotin AGCCGAGGGAGACCAGGAAG -3 ' , as SEQ ID NO. 15; downstream 5 ' - CAGCAGATGTGTGGCCTCAGAT-3' as shown in SEQ ID NO.

 Primer set (5) upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT -3 ' , as SEQ ID NO. 17; downstream 5 ' - CACAGGGCGAGGCTCATC-3' as shown in SEQ ID NO.

 Primer set (6) upstream 5 ' -biotin AGAAATAGCAAGTGCCGAGAAGC -3 ' , as shown in SEQ ID NO. 19; downstream 5'-CACAGGGCGAGGCTCATC-3', as shown in SEQ ID NO.

 Primer set (7) upstream 5 ' -biotin TATCCACACACACAGGAAGCAC -3 ' , as shown in SEQ ID NO. 21; downstream 5 ' - CCTCTCATTGGTAATGCTCACTTT-3', as SEQ ID NO. Shown

 Primer set (8) upstream 5 ' -biotin AAGGCTGCTGACTTTACCATCTG -3 ' , as shown in SEQ ID NO. 23; downstream 5 ' - TCTAATGTCCTTCCCTCACAAGC-3', as shown in SEQ ID NO.

 Primer set (9) upstream 5 ' -biotin CGCTTGTGAGGGAAGGACATTAG -3 ' , as shown in SEQ ID NO. 25; downstream 5 ' - GTGAAGCCATCAAGATTTTCTCGTC-3', as SEQ ID NO. Shown

 Primer set (10) upstream 5 '-biotin CAGCAGGACCCAACGCAT -3 ' , as SEQ ID NO. 27; downstream 5'-GAGAGAGGATTGGTAAGCGATGTG-3' as shown in SEQ ID NO.

 The above primer set designed for PCA3 (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) and PCA3 probes designed as described above (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) one-to-one correspondence, respectively corresponding to PCA3 primer/probe set 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (see Tables 1 and 2);

 --actin gene : upstream primer 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG-3', as shown in SEQ ID NO. 39; downstream primer 5'-GCTGGGGTGTTGAAGGTCTC-3' , as shown in SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 QIAGEN's Multiplex PCR kit is used, the system is as follows

 Template cDNA 10μl

 2 ×QIAGEN Multiplex PCR Master Mix 25μl

 10 ×Primer mix 5μl

 RNase-free water 10μl

 Final volume is 50 μl

(The 2×QIAGEN Multiplex PCR Master Mix contains hot-start Taq DNA, MgCl ₂ and dNTP Mix.)

 PCR amplification procedure: 95 ° C for 15 min; 94 ° C for 30 s, 55 ° C for 90 s, 72 ° C for 90 s, 35 cycles; 72 ° C 10 min; 4 ° C insulation.

 four. Hybridization of oligonucleotide probes and PCR products

 1 . Selecting the oligonucleotide probe microsphere mixture prepared in the first step;

 2 . Vortex for 20s, mix the microspheres;

 3 . The microsphere working solution was prepared, and the coupled microspheres were diluted with a 1.5×TMAC hybridization solution to a concentration of 150 microspheres/μl. (Note: 33μl of microsphere working solution is required for each reaction);

 4 . Vortex for 20s, mix the microsphere working solution;

 5 . Add 33 μl of microsphere working solution to the sample well, positive control well and negative control well respectively;

 6. Add PCR amplification product of sample No. 1-8 and TE solution (pH 8.0) to each sample well, the total volume is 17 μl;

 7. Dip up and down with a lance and gently mix the reaction solution;

 8. Cover the reaction cover to avoid evaporation, incubate at 95-100 ° C for 1-3 min, so that the oligonucleotides in the sample remove the secondary structure;

 9. Incubate the reaction plate at the hybridization temperature for 15 min;

 10. Prepare a fresh test mixture, and dilute the streptavidin phycoerythrin solution to 10 μg/ml with 1×TMAC hybridization solution (25 μl of detection mixture per reaction);

 11. Add 25 μl of the test mixture to each well, pipe up and down with a lance, and mix gently;

 12. Incubate for 5 min at the hybridization temperature;

 The above steps can be combined by the PCR program according to the following scheme:

 95 ° C, 1-3 min;

 Hybridization temperature

 13. In the liquid chip device (LiquiChip 200 or Luminex 200 On the above, the hybridization temperature was maintained, and each reaction well was subjected to detection and analysis, and the fluorescence MFI value (average fluorescence intensity) was measured.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 1:

 Table 1

 Sample/primer  PCA3 Primer/Probe Set 1  PCA3 Primer / Probe Set 2  PCA3 Primer / Probe Set 3  PCA3 Primer / Probe Set 4  PCA3 Primer/Probe Set 5  PCA3 Primer / Probe Set 6  PCA3 Primer/Probe Set 7  PCA3 Primer / Probe Set 8  PCA3 Primer/Probe Set 9  PCA3 Primer/Probe Set 10 --actin
Reference gene  1  3172  2786  3618  3293  2851  2374  3035  3496  2915  3371  3562  2  3064  2591  2805  2947  2639  2208  3187  2630  3346  2558  2975  3  2913  2862  3249  2758  2410  2637  3423  3579  2582  2729  3708  4  3485  3170  3762  2924  3286  2413  2651  2768  3607  3146  3437  5  2726  2398  2954  3081  2543  2829  3098  3124  2895  2630  3219  6  127  95  117  104  126  108  131  122  105  113  2804  7  103  124  109  145  92  129  110  146  115  137  3621  8  112  130  118  133  121  101  142  138  123  135  2853  Positive control  3059  2637  3371  2830  2725  2542  2736  3207  3063  2954  3682  Negative control  107  83  125  96  71  68  134  102  119  93  116

 The results are analyzed as shown in Table 2:

 Table 2

 Sample/primer  PCA3 Primer/Probe Set 1  PCA3 Primer / Probe Set 2  PCA3 Primer / Probe Set 3  PCA3 Primer / Probe Set 4  PCA3 Primer/Probe Set 5  PCA3 Primer / Probe Set 6  PCA3 Primer/Probe Set 7  PCA3 Primer / Probe Set 8  PCA3 Primer/Probe Set 9  PCA3 Primer/Probe Set 10  1  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  2  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  3  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  4  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  5  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  Positive  6  negative  negative  negative  negative  negative  negative  negative  negative  negative  negative  7  negative  negative  negative  negative  negative  negative  negative  negative  negative  negative  8  negative  negative  negative  negative  negative  negative  negative  negative  negative  negative

 Different primers/probe groups detect PCA3 in the urine of patients with 1-5 prostate cancer (PCa) Positive.

 Different primers/probe groups were tested. PCA3 was negative in urine of 6-8 normal subjects.

At the same time, the expression of the PCA3 gene can be obtained by comparing the fluorescence MFI value exhibited by the patient with the fluorescent MFI value of the β-actin gene of the internal reference.

 Example 2: Liquid phase chip method for detecting PSA gene in urine by using different primers and probe combinations

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PSA gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 mRNA for the PSA gene:

 Probe A: 5 ' - AminolinkerC12 CAGAATCACCCGAGCAGGTG -3 ' , as shown in SEQ ID NO.

Probe B: 5 ' - AminolinkerC12 GGGGTCAAGAACTCCTCTGG -3 ' , as shown in SEQ ID NO.

Probe C: 5 ' - AminolinkerC12 CACGCTTTTGTTCCTGATGC -3 ' , as shown in SEQ ID NO.

 Probe D: same as PSA probe A, as shown in SEQ ID NO.

 Probe E: same as PSA probe B, as shown in SEQ ID NO.

 --actin gene: 5' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' as shown in SEQ ID NO. 41;

 2 . Coupling an amino-modified oligonucleotide probe to six carboxyl microspheres numbered 11, 13, 15, 21, 25, and 66, respectively

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 Dissolve the oligonucleotide probes of PSA and β-actin with d H ₂ O at a concentration of 1 mM (1 nmol/μl);

 2.3 6 of the full speed vortex numbers 11 , 13 , 15 , 21 , 25 , 66 The carboxy microspheres are stored in suspension for at least 3 minutes to produce a uniform suspension of microspheres;

 2.4-2.20 Same as 2.4-2.20 in Example 1, except that the 11 tubes are correspondingly reduced to 6 tubes, and the others are unchanged.

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PSA probe A microsphere 11, PSA probe B microsphere 13 , PSA probe C microsphere 15, PSA probe D microsphere 21, PSA probe E microsphere 25, β-actin probe microsphere 66, mixed in equal proportion, the final concentration of various microspheres is 1500 /μl, 2-8 °C protected from light.

 two. Sample preparation

 Clinical sample No. 1-5 is the urine of patients with prostate cancer (PCa). The clinical sample No. 6-8 is the urine of a normal person, and the preparation method is the same as the preparation method of the sample in Example 1.

 three. Multiple RT-PCR

 Multiple reverse transcription PCR amplification of mRNAs from samples 1-8 above was performed as follows:

 1. The method for synthesizing the first strand of cDNA is the same as the method for synthesizing the first strand of cDNA in Example 1.

 2. Multiplex PCR

 2.1 Synthesize primers according to the following sequence:

 mRNA for the PSA gene:

 Primer set A: upstream 5 ' -biotin TTGACCCCAAAGAAACTTCAGTGT -3 ' , as shown in SEQ ID NO. 29; downstream 5 ' - TGCCCCATGACGTGATACCT-3', as shown in SEQ ID NO.

 Primer set B: upstream 5 ' -biotin TCCCACACCCGCTCTACGAT-3', as SEQ ID NO.31; downstream 5 ' - CGTCCAGCACACAGCATGAACT-3' as shown in SEQ ID NO.

 Primer group C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33; downstream 5 ' - GCTGTGGCTGACCTGAAATACC-3' as shown in SEQ ID NO.

 Primer set D: upstream 5 ' -biotin GGCAGCATTGAACCAGAGGAGT -3 ' , as shown in SEQ ID NO. 35; downstream 5 ' - CGATGGTGTCCTTGATCCACTT-3', as shown in SEQ ID NO.

 Primer set E: upstream 5 ' -biotin TCCTCAGGCCAGGTGATGACT -3 ' , as SEQ ID NO. 37; downstream 5 ' - CGTCCAGCACACAGCATGAACT-3' as shown in SEQ ID NO.

 The above primer sets A, B, C, D, E designed for PSA and the PSA probe A designed above , B, C, D, E correspond one-to-one, respectively corresponding to PSA primer / probe group 1, 2, 3, 4, 5 (see Table 3 and Table 4);

 --actin gene : upstream 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG-3', as shown in SEQ ID NO. 39; downstream 5'-GCTGGGGTGTTGAAGGTCTC-3', such as SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 The method was the same as in Example 1 (III, 2.2) multiplex PCR reaction method.

 four. Hybridization of oligonucleotide probes and PCR products

 The method is the same as the method of hybridization of the (IV) oligonucleotide probe and the PCR product of Example 1.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 3:

 table 3

 Sample/primer  PSA Primer / Probe Set 1  PSA Primer / Probe Set 2  PSA Primer / Probe Set 3  PSA Primer / Probe Set 4  PSA Primer / Probe Set 5  --actin reference gene  1  3162  2675  3857  2417  3069  3246  2  2574  2916  2939  2783  2581  3095  3  3381  2052  3216  3128  2243  2578  4  2730  2248  3425  2135  2457  2830  5  3597  3129  3641  2871  2062  3314  6  136  121  108  112  105  2681  7  114  128  132  97  132  2759  8  129  140  117  134  91  3128  Positive control  2958  2596  3103  2364  2694  2732  Negative control  92  65  118  86  73  89

 The results are analyzed as shown in Table 4:

 Table 4

 Sample/primer  PSA Primer / Probe Set 1  PSA Primer / Probe Set 2  PSA Primer / Probe Set 3  PSA Primer / Probe Set 4  PSA Primer / Probe Set 5  1  Positive  Positive  Positive  Positive  Positive  2  Positive  Positive  Positive  Positive  Positive  3  Positive  Positive  Positive  Positive  Positive  4  Positive  Positive  Positive  Positive  Positive  5  Positive  Positive  Positive  Positive  Positive  6  negative  negative  negative  negative  negative  7  negative  negative  negative  negative  negative  8  negative  negative  negative  negative  negative

 Different primers/probe groups detect PSA in urine of patients with 1-5 prostate cancer (PCa) Positive.

 Different primers/probe sets were detected. Normally, 6-8 normal people had negative PSA in urine.

At the same time, the expression of the PSA gene can be obtained by comparing the fluorescence MFI value exhibited by the patient with the fluorescent MFI value of the β-actin gene of the internal reference.

 Example 3: Liquid phase chip method for combined detection of PCA3, PSA and PCA3/PSA ratios in urine

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PSA gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 PCA3 : 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as shown in SEQ ID NO.

 PSA : 5 ' - AminolinkerC12 CACGCTTTTGTTCCTGATGC -3 ' , as shown in SEQ ID NO.

 --actin gene: 5' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' as shown in SEQ ID NO. 41;

 2 . Coupling an amino-modified oligonucleotide probe to three carboxy microspheres numbered 11, 13, and 66, respectively

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 with dH 20O separately dissolved PCA3, PSA, β-actin oligonucleotide probe, the concentration was 1 mM (1nmol / μl);

 2.3 Full-speed vortex numbers 11 , 13 , 66 3 The carboxy microspheres are stored in suspension for at least 3 minutes to produce a uniform suspension of microspheres;

 2.4-2.20 Same as 2.4-2.20 in Example 1, except that the 11 tubes are correspondingly reduced to 3 tubes, and the others are unchanged.

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PCA3 probe microsphere 11, PSA probe microsphere 13, respectively , β-actin probe microspheres 66, mixed in equal proportions, the final concentration of various microspheres is 1500 / μl, 2-8 ° C protected from light.

 two. Sample preparation

 1-10 The clinical sample of the patient is the urine of patients with prostate cancer (PCa), and the clinical sample of patients with type 11-20 is the urine of patients with benign prostatic hyperplasia (BPH). The preparation method is the same as the preparation method of the sample in Example 1.

 three. Multiple RT-PCR

 Multiple reverse RT-PCR amplification of mRNA from samples 1-20 above was performed as follows:

 1. The method for synthesizing the first strand of cDNA is the same as the method for synthesizing the first strand of cDNA in Example 1.

 2. Multiplex PCR

 2.1 Synthesize primers according to the following sequence:

 PCA3: upstream 5 ' -biotin GGTGGGAAGGACCTGATGATAC -3 ' , as shown in SEQ ID NO. 13; downstream 5 ' - TAAAGGGGCTGGAAATGTGC-3' , as shown in SEQ ID NO.

 PSA: upstream 5 ' -biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33; downstream 5 ' - GCTGTGGCTGACCTGAAATACC-3' as shown in SEQ ID NO.

 --actin gene : upstream 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG-3', as shown in SEQ ID NO. 39; downstream 5'-GCTGGGGTGTTGAAGGTCTC-3', such as SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 The method was the same as in Example 1 (III, 2.2) multiplex PCR reaction method.

 four. Hybridization of oligonucleotide probes and PCR products

 The method is the same as the method of hybridization of the (IV) oligonucleotide probe and the PCR product of Example 1.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 5:

 table 5

 Patient / gene  PCA3  PSA  PCA3/PSA ratio  --actin reference gene  1  3157  3628  0.87  3356  2  2283  3879  0.59  2804  3  3406  2936  1.16  3173  4  3295  3542  0.93  3695  5  2574  3960  0.65  3482  6  2928  2714  1.08  2641  7  3062  3731  0.82  3328  8  2419  3269  0.74  3057  9  3147  2997  1.05  3563  10  2631  3085  0.85  2714  11  136  3472  0.039  3245  12  125  4196  0.030  2980  13  150  3658  0.041  3526  14  168  2717  0.062  3091  15  139  3964  0.035  2673  16  143  3309  0.043  3748  17  114  4325  0.026  3162  18  157  3081  0.051  2937  19  132  2876  0.046  3419  20  128  3713  0.034  3854  Positive control  2856  3307  3295  Negative control  98  104  81

 The results are analyzed as shown in Table 6:

 Table 6

 Patient / gene  PCA3  PSA  1  Positive  Positive  2  Positive  Positive  3  Positive  Positive  4  Positive  Positive  5  Positive  Positive  6  Positive  Positive  7  Positive  Positive  8  Positive  Positive  9  Positive  Positive  10  Positive  Positive  11  negative  Positive  12  negative  Positive  13  negative  Positive  14  negative  Positive  15  negative  Positive  16  negative  Positive  17  negative  Positive  18  negative  Positive  19  negative  Positive  20  negative  Positive

 The average and analysis of the PCA3/PSA ratios are shown in Table 7:

 Table 7

 Patient/analysis  Average of PCA3/PSA  The difference between the two groups of PCA3/PSA average  No. 1-10 prostate cancer (PCa) group  0.874  21 times  11-20 benign prostatic hyperplasia (BPH) group  0.0407

 Patients with prostate cancer (PCa) 1-10 were positive for PCA3 and PSA in urine; 11-20 PCA3 in the urine of patients with benign prostatic hyperplasia (BPH) Negative, PSA was positive; the mean PCA3/PSA difference between the two groups was 21 times. This method is a good way to distinguish patients with prostate cancer from patients with benign prostatic hyperplasia.

 At the same time, the patient will be presented The positive fluorescent MFI value is compared with the fluorescent MFI value of the internal reference β-actin gene, and the expression status of PCA3 gene and PSA gene can be obtained.

 Example 4: Liquid phase chip method for detecting PCA3 gene in blood by different primers and probe combinations

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PCA3 gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 For the PCA3 gene:

 Probe (1) 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as shown in SEQ ID NO.

 Probe (3), (5) is the same as probe (1), as shown in SEQ ID NO.

 --actin gene: 5 ' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3', as shown in SEQ ID NO.

 2 . Oligonucleotide probes containing amino modifications are assigned to numbers 4, 13, 15, and 66, respectively. Carboxyl microsphere coupling

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 Dissolving the oligonucleotide probes of PCA3 and β-actin with d H ₂ O at a concentration of 1 mM (1 nmol/μl);

 2.3 The full speed vortex numbers are 11, 13, 15 respectively. , the four kinds of carboxyl microspheres of 66 are stored in suspension for at least 3 minutes to produce a uniform suspension of microspheres;

 2.4-2.20 Same as 2.4-2.20 in Example 1, except that the 11 tubes are correspondingly reduced to 4 tubes, and the others are unchanged.

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PCA3(1) probe microsphere 11, PCA3 (3) The probe microspheres 13, PCA3 (5) probe microspheres 15, and β-actin probe microspheres 66 were mixed in equal proportions, and the final concentration of each microsphere was 1500 / μl, and stored at 2-8 ° C in the dark.

 two. Sample preparation

 The clinical sample of No. 1-5 is venous blood of patients with prostate cancer (PCa), and the clinical sample of No. 6-8 is venous blood of normal people. Extract RNA by following the steps below:

 1. Lymphocyte extraction: Take 5 ml of anticoagulated venous blood before breakfast, and centrifuge at 3000 r/min for 10 minutes. Min , 4 °C, taking the light yellow layer on the blood cell layer is the lymphocyte;

 2 . Take 1 centrifuge tube (treated with DEPC water) and add 150 μl of lymphocyte solution. Then add 1 ml. TRIZOL, placed at room temperature for 5 min, allowing it to fully lyse;

 3. Centrifuge at 12,000 rpm for 5 min, and take the supernatant;

 4. Add 200 μl per 1 ml Trizol Chloroform, mixed with shaking and allowed to stand at room temperature for 15 min;

 5. Centrifuge at 12 °C for 12 min at 4 °C;

 6. Pipette the upper aqueous phase into another centrifuge tube;

 7. Add 500 μl of isopropanol per 1 ml of Trizol and mix at room temperature for 5-10 min;

 8. Centrifuge at 4 °C 12,000g for 10min, discard the supernatant, and sink the RNA at the bottom of the tube;

 9. Add 1 ml 75% per 1 ml Trizol Ethanol, gently shake the centrifuge tube, and suspend the precipitate;

 10. Centrifuge at 8 °C 8 ,000g for 5min, discard the supernatant as much as possible;

 11. Dry at room temperature or vacuum dry for 5-10min;

12. Dissolve the RNA sample with 50 ul of RNase-free dH ₂ O, 55-60 ° C, 5-10 min;

 13. Measure the OD value to quantify the RNA concentration.

 three. Multiple RT-PCR

 Multiple reverse transcription PCR amplification of mRNAs from samples 1-8 above was performed as follows:

 1. The method for synthesizing the first strand of cDNA is the same as the method for synthesizing the first strand of cDNA in Example 1.

 2. Multiplex PCR

 2.1 Synthesize primers according to the following sequence:

 For the PCA3 gene:

 Primer (1) upstream 5 ' -biotin AAGAAATAGCAAGTGCCGAGAAG -3 ' , as shown in SEQ ID NO. 9; downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3', as shown in SEQ ID NO.

 Primer (3) upstream 5 ' -biotin GGTGGGAAGGACCTGATGATAC -3 ' , as shown in SEQ ID NO. 13; downstream 5'-TAAAGGGGCTGGAAATGTGC-3', as shown in SEQ ID NO.

 Primer (5) upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT -3 ' , as SEQ ID NO. 17; downstream 5 ' - CACAGGGCGAGGCTCATC-3' as shown in SEQ ID NO.

 The above primer set (1), (3), (5) designed for the PCA3 gene and the PCA3 gene probe designed above (1) , (3), (5) one-to-one correspondence, respectively corresponding to PSA primer / probe set 1, 3, 5 (see Table 8 and Table 9);

 --actin gene : upstream primer 5' -biotin TGGGTCAGAAGGATTCCTATGTG -3' , as shown in SEQ ID NO. 39; downstream primer 5'-GCTGGGGTGTTGAAGGTCTC -3', as shown in SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 The method was the same as in Example 1 (III, 2.2) multiplex PCR reaction method.

 four. Hybridization of oligonucleotide probes and PCR products

 The method is the same as the method of hybridization of the (IV) oligonucleotide probe and the PCR product of Example 1.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 8:

 Table 8

 Sample/primer  PCA3 Primer/Probe Set 1  PCA3 Primer/Probe Set 3  PCA3 Primer/Probe Set 5  --actin reference gene  1  2358  2739  2016  3174  2  2071  1862  1790  2869  3  1986  2415  1683  3402  4  2247  2928  2342  3185  5  1793  2074  1961  3528  6  87  115  122  2816  7  109  98  106  3043  8  95  103  82  3291  Positive control  2105  2631  2057  2937  Negative control  89  102  76  94

 The results are analyzed as shown in Table 9:

 Table 9

 Sample/primer  PCA3 Primer/Probe Set 1  PCA3 Primer/Probe Set 3  PCA3 Primer/Probe Set 5  1  Positive  Positive  Positive  2  Positive  Positive  Positive  3  Positive  Positive  Positive  4  Positive  Positive  Positive  5  Positive  Positive  Positive  6  negative  negative  negative  7  negative  negative  negative  8  negative  negative  negative

 Different primers/probe groups detect PCA3 in the blood of patients with 1-5 prostate cancer (PCa) Positive.

 Different primers/probe sets were found to be negative for PCA3 in the blood of normal humans 6-8.

At the same time, the expression of the PCA3 gene can be obtained by comparing the fluorescence MFI value exhibited by the patient with the fluorescent MFI value of the β-actin gene of the internal reference.

 Example 5: Liquid phase chip method for detecting PSA gene in blood by different primers and probe combinations

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PSA gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 For the PSA gene:

 Probe A: 5 ' - AminolinkerC12 CAGAATCACCCGAGCAGGTG -3 ' , as shown in SEQ ID NO. 6; Probe C: 5 ' - Aminolinker C12 CACGCTTTTGTTCCTGATGC -3 ' , as SEQ ID NO. 8;

 --actin gene: 5' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' as shown in SEQ ID NO. 41;

 2 . Coupling an amino-modified oligonucleotide probe to three carboxy microspheres numbered 11, 13, and 66, respectively

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 Dissolve the oligonucleotide probes of PSA and β-actin with d H ₂ O at a concentration of 1 mM (1 nmol/μl);

 2.3 Full-speed vortex numbers 11 , 13 , 66 3 The carboxy microspheres are stored in suspension for at least 3 minutes to produce a uniform suspension of microspheres;

 2.4-2.20 Same as 2.4-2.20 in Example 1, except that the 11 tubes are correspondingly reduced to 3 tubes, and the others are unchanged.

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PSA probe A microsphere 11, PSA probe C microsphere 13, respectively , β-actin probe microspheres 66, mixed in equal proportions, the final concentration of various microspheres is 1500 / μl, 2-8 ° C protected from light.

 two. Sample preparation

 Clinical sample No. 1-5 is venous blood in patients with prostate cancer (PCa), 6-8 The clinical sample is normal human venous blood, and the preparation method is the same as the preparation method of the sample in (2) in Example 4.

 three. Multiple RT-PCR

 Multiple reverse transcription PCR amplification of mRNAs from samples 1-8 above was performed as follows:

 1. The method for synthesizing the first strand of cDNA is the same as the method for synthesizing the first strand of cDNA in Example 1.

 2. Multiplex PCR

 2.1 Synthesis of primers according to the following sequence

 For the PSA gene:

 Primer A: upstream 5 ' -biotin TTGACCCCAAAGAAACTTCAGTGT -3 ' , as shown in SEQ ID NO. 29; downstream 5 ' - TGCCCCATGACGTGATACCT-3', as shown in SEQ ID NO.

 Primer C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33; downstream 5 ' - GCTGTGGCTGACCTGAAATACC-3' as shown in SEQ ID NO.

 The primer sets A and C designed for PSA are corresponding to the PSA probes A and C of the above design, and the corresponding combinations are respectively PSA primer/probe set 1, 3 (see Table 10 and Table 11);

 --actin gene : upstream primer 5' -biotin TGGGTCAGAAGGATTCCTATGTG -3' , as shown in SEQ ID NO. 39; downstream primer 5'-GCTGGGGTGTTGAAGGTCTC -3', as shown in SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 The method was the same as in Example 1 (III, 2.2) multiplex PCR reaction method.

 four. Hybridization of oligonucleotide probes and PCR products

 The method is the same as the method of hybridization of the (IV) oligonucleotide probe and the PCR product of Example 1.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 10:

 Table 10

 Sample/primer  PSA Primer / Probe Set 1  PSA Primer / Probe Set 3  --actin reference gene  1  2341  2192  3058  2  1863  3085  2976  3  2529  2317  2632  4  2074  2648  2429  5  2756  2493  3215  6  93  85  2504  7  107  111  3168  8  82  79  2791  Positive control  2138  2301  2587  Negative control  63  78  90

 The results are analyzed as shown in Table 11:

 Table 11

 Sample/primer  PSA Primer / Probe Set 1  PSA Primer / Probe Set 3  1  Positive  Positive  2  Positive  Positive  3  Positive  Positive  4  Positive  Positive  5  Positive  Positive  6  negative  negative  7  negative  negative  8  negative  negative

 Different primers/probe groups tested positive for PSA in the blood of patients with 1-5 prostate cancer (PCa).

 Different Primers/Probe Groups The PSA in the blood of normal people 6-8 was negative.

At the same time, the expression of the PSA gene can be obtained by comparing the fluorescence MFI value exhibited by the patient with the fluorescent MFI value of the β-actin gene of the internal reference.

 Example 6: Combined detection of PCA3 gene, PSA gene and PCA3/PSA ratio in blood Liquid phase chip method

 The specific detection method includes the following steps:

 One. Preparation of microsphere mixture for detecting PSA gene

 1. Synthesize oligonucleotide probes according to the following sequence:

 PCA3 : 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as shown in SEQ ID NO.

 PSA : 5 ' - AminolinkerC12 CACGCTTTTGTTCCTGATGC -3 ' , as shown in SEQ ID NO.

 --actin gene: 5' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG -3' as shown in SEQ ID NO. 41;

 2 . Coupling an amino-modified oligonucleotide probe to three carboxy microspheres numbered 11, 13, and 66, respectively

 2.1 Take out a small portion of fresh dry powdered EDC stored at -20 ° C to equilibrate to room temperature;

2.2 Dissolve PCA3, PSA, β-actin oligonucleotide probes with dH2O at a concentration of 1 mM (1 nmol/μl);

 2.3 Full-speed vortex numbers 11 , 13 , 66 3 The carboxy microspheres are stored in suspension for at least 3 minutes to produce a uniform suspension of microspheres;

 2.4-2.20 Same as 2.4-2.20 in Example 1, except that the 11 tubes are correspondingly reduced to 3 tubes, and the others are unchanged.

 3. Preparation of microsphere mixture

 The above microspheres coupled with an oligonucleotide probe are as follows: PCA3 probe microsphere 11, PSA probe microsphere 13, respectively , β-actin probe microspheres 66, mixed in equal proportions, the final concentration of various microspheres is 1500 / μl, 2-8 ° C protected from light.

 two. Sample preparation

 1-10 The clinical sample of the patient is venous blood of patients with prostate cancer (PCa), and the clinical sample of patients with type 11-20 is venous blood of patients with benign prostatic hyperplasia (BPH). The preparation method is the same as that of the example 4 (2) The preparation method of the middle sample.

 three. Multiple RT-PCR

 Multiple reverse RT-PCR amplification of mRNA from samples 1-20 above was performed as follows:

 1. The method for synthesizing the first strand of cDNA is the same as the method for synthesizing the first strand of cDNA in Example 1.

 2. Multiplex PCR

 2.1 Synthesize primers according to the following sequence:

 PCA3: upstream 5 ' -biotin GGTGGGAAGGACCTGATGATAC -3 ' , as shown in SEQ ID NO. 13; downstream 5 ' - TAAAGGGGCTGGAAATGTGC-3' , as shown in SEQ ID NO.

 PSA: upstream 5 ' -biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33; downstream 5 ' - GCTGTGGCTGACCTGAAATACC-3' as shown in SEQ ID NO.

 --actin gene : upstream 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG-3', as shown in SEQ ID NO. 39; downstream 5'-GCTGGGGTGTTGAAGGTCTC-3', such as SEQ ID NO. 40;

 2.2 Multiplex PCR reactions:

 The method was the same as in Example 1 (III, 2.2) multiplex PCR reaction method.

 four. Hybridization of oligonucleotide probes and PCR products

 The method is the same as the method of hybridization of the (IV) oligonucleotide probe and the PCR product of Example 1.

 Fives. Test results and analysis

 The test results (fluorescence MFI values) are shown in Table 12:

 Table 12

 Patient/gene  PCA3  PSA  PCA3/PSA ratio  --actin reference gene  1  2536  2728  0.93  2942  2  1784  2465  0.72  3107  3  2251  2524  0.89  2695  4  2879  2830  1.02  2813  5  2125  2186  0.97  3374  6  1963  2431  0.81  2598  7  2742  2397  1.14  3150  8  2413  2052  1.18  2681  9  2097  3218  0.65  2349  10  2265  2371  0.96  2706  11  109  2963  0.037  3485  12  94  3318  0.028  3029  13  135  2647  0.051  2467  14  112  3092  0.036  3218  15  107  3651  0.029  2743  16  97  2506  0.039  2931  17  104  3275  0.032  3459  18  96  3824  0.025  2870  19  123  2937  0.042  2563  20  101  3582  0.028  3194  Positive control  2918  2769  3026  Negative control  85  97  74

 The results are analyzed as shown in Table 13:

 Table 13

 Patient/gene  PCA3  PSA  1  Positive  Positive  2  Positive  Positive  3  Positive  Positive  4  Positive  Positive  5  Positive  Positive  6  Positive  Positive  7  Positive  Positive  8  Positive  Positive  9  Positive  Positive  10  Positive  Positive  11  negative  Positive  12  negative  Positive  13  negative  Positive  14  negative  Positive  15  negative  Positive  16  negative  Positive  17  negative  Positive  18  negative  Positive  19  negative  Positive  20  negative  Positive

 PCA3/PSA average and analysis are shown in Table 14.

 Table 14

 Patient/analysis  Average of PCA3/PSA  The difference between the two groups of PCA3/PSA average  No. 1-10 prostate cancer (PCa) group  0.927  27 times  11-20 benign prostatic hyperplasia (BPH) group  0.0347

 Patients with prostate cancer (PCa) 1-10 were positive for PCA3 and PSA in the blood; 11-20 PCA3 in the blood of patients with benign prostatic hyperplasia (BPH) Negative, PSA was positive; the mean PCA3/PSA difference between the two groups was 27 times. This method is a good way to distinguish patients with prostate cancer from patients with benign prostatic hyperplasia.

 At the same time, the patient will be presented The positive fluorescent MFI value is compared with the fluorescent MFI value of the internal reference β-actin gene, and the expression status of PCA3 gene and PSA gene can be obtained.

Various modifications or variations of the present invention, including various variations of primers or probes, and various variations of primer or probe labels, may be made by those skilled in the art after reading the above description of the present invention. The valence form is also subject to the scope defined in the claims appended hereto.

Industrial Applicability

 Because the invention utilizes The liquid phase chip technology enables the detection method and kit to have the advantages of high sensitivity, high specificity, high throughput, good stability, rapid detection and accuracy, and can be used for the PCA3 gene and/or PSA gene. Qualitative and quantitative tests can be performed, and the ratio of PCA3 gene/PSA gene can be calculated, so that it can be better applied in clinical detection and diagnosis.

Sequence List Text

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Claims (1)

A liquid phase chip method for detecting a PCA3 gene and a PSA gene, which comprises the steps of: (1) Designing a carboxyl microsphere, Beads, which covalently binds to a specific nucleic acid probe designed for mRNA of the PCA3 gene or PSA gene; or design a carboxyl microsphere Beads containing two different fluorescent codes, each Specific nucleic acid probes designed to covalently bind to mRNAs of the PCA3 gene and the PSA gene, respectively; (2) The upstream and downstream primers are designed for the mRNA of the PCA3 gene and/or the PSA gene, and the corresponding products are amplified by reverse transcription PCR; (3) The microspheres containing the specific nucleic acid probe are hybridized with the reverse transcription amplification product of the mRNA of the PCA3 gene and/or the PSA gene, and after adding streptavidin-PE, the liquid phase chip is added. The xMAP method detects a fluorescent signal; (4) comparing the detected fluorescent signal with the fluorescent signal of the reference gene to determine whether there is a prostate disease-related gene PCA3 gene and/or PSA gene in the test sample, and its expression in the sample, and also The ratio of the PCA3 gene/PSA gene was calculated. 2. The liquid phase chip method for detecting a PCA3 gene and a PSA gene according to claim 1, wherein in the step (1), the microspheres are polyphenylenes having an average diameter of 5.6 μm and combining different fluorescent dyes. Microspheres, ie color-coded microspheres Color-coded Beads. 3. The liquid phase chip method for detecting a PCA3 gene and a PSA gene according to claim 1, wherein in the step (1), the mRNA for the PCA3 gene and the mRNA of the PSA gene covalently bound to the microspheres The specific nucleic acid probe designed includes the following sequence, wherein the 5' end contains an amino modification: mRNA for the PCA3 gene: Probe (1): 5 ' - AminolinkerC12 ATTTCTCACCTCTGTATCATC -3 ' , as SEQ ID NO. 1; Probes (2), (3), (5), (6) are the same as probe (1), as shown in SEQ ID NO. Probe (4): 5 ' - AminolinkerC12 CTCACCTCTGTATCATCAG -3 ' , as SEQ ID NO.2; Probe (7): 5 ' - AminolinkerC12 ATCTCTGTGCTTCCTTTTGT -3 ' , as SEQ ID NO.3; Probe (8) is the same as probe (7), as shown in SEQ ID NO. Probe (9): 5 ' - AminolinkerC12 CAAATCTGTAATCCCGTTCA -3 ' , as SEQ ID NO.4; Probe (10): 5 '-Aminolinker C12TATGTGTCAAGAGGAGAGCC -3 ', as SEQ ID As shown in NO.5; Or a sequence comprising the above sequence or its complementary sequence extending to the 5' end or/and the 3' end; Or a sequence having greater than 85% homology to the above sequence or its complement; Or a base complementary sequence to the above sequence; Or using any one or more of the above sequences; mRNA for the PSA gene: Probe A: 5 ' - AminolinkerC12 CAGAATCACCCGAGCAGGTG -3 ' , as SEQ ID NO.6; Probe B: 5 ' - AminolinkerC12 GGGGTCAAGAACTCCTCTGG -3 ' , as SEQ ID NO.7; Probe C: 5 ' - AminolinkerC12 CACGCTTTTGTTCCTGATGC -3 ' , as SEQ ID NO.8; Probe D is the same as probe A, as shown in SEQ ID NO. Probe E is the same as probe B, as shown in SEQ ID NO. Or a sequence comprising the above sequence or its complementary sequence extending to the 5' end or/and the 3' end; Or a sequence having greater than 85% homology to the above sequence or its complement; Or a base complementary sequence to the above sequence; Alternatively, any one or a combination of more than one of the above sequences may be used. The liquid phase chip method for detecting a PCA3 gene and a PSA gene according to claim 1, wherein In the step (2), the upstream and downstream primers designed for the mRNA of the PCA3 gene and the mRNA of the PSA gene include the following sequence, wherein the upstream primer has a biotin tag at the 5' end; mRNA for the PCA3 gene: Primer set (1): upstream 5 '-biotinAAGAAATAGCAAGTGCCGAGAAG -3 ' , as SEQ ID As shown in NO.9; Downstream 5'-GTGTGGCCTCAGATGGTAAAGTC-3', as shown in SEQ ID NO. Primer set (2): upstream 5'-biotinTGGTGGGAAGGACCTGATGATAC-3', as SEQ ID NO. Shown Downstream 5'-TCTCCCAGGGATCTCTGTGCTT-3', as shown in SEQ ID NO. Primer set (3): upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC -3 ' , as SEQ ID Shown as NO.13; Downstream 5'-TAAAGGGGCTGGAAATGTGC-3', as shown in SEQ ID NO. Primer set (4): upstream 5 ' -biotin AGCCGAGGGAGACCAGGAAG -3 ' , as SEQ ID As shown in NO.15; Downstream 5'-CAGCAGATGTGTGGCCTCAGAT-3', as shown in SEQ ID NO. Primer set (5): upstream 5 ' -biotin CCGAGGGAGACCAGGAAGAT -3 ' , as SEQ ID Shown as NO.17; Downstream 5'-CACAGGGCGAGGCTCATC-3', as shown in SEQ ID NO. Primer set (6): upstream 5 '-biotinAGAAATAGCAAGTGCCGAGAAGC -3 ' , as SEQ ID Shown as NO.19; Downstream 5'-CACAGGGCGAGGCTCATC-3', as shown in SEQ ID NO. Primer set (7): upstream 5 ' -biotin TATCCACACACACAGGAAGCAC -3 ' , as SEQ ID No.21; Downstream 5'-CCTCTCATTGGTAATGCTCACTTT-3', as SEQ ID Shown as NO.22; Primer set (8): upstream 5 ' -biotin AAGGCTGCTGACTTTACCATCTG -3 ' , as SEQ ID Shown as NO.23; Downstream 5'-TCTAATGTCCTTCCCTCACAAGC-3', as shown in SEQ ID NO. Primer set (9): upstream 5 ' -biotin CGCTTGTGAGGGAAGGACATTAG -3 ' , as SEQ ID As shown in NO.25; Downstream 5'-GTGAAGCCATCAAGATTTTCTCGTC-3', as SEQ ID No.26; Primer set (10): upstream 5 ' -biotin CAGCAGGACCCAACGCAT -3 ' , as SEQ ID Shown as NO.27; Downstream 5'-GAGAGAGGATTGGTAAGCGATGTG-3', as SEQ ID Shown as NO.28; Or a sequence comprising the above sequence or its complementary sequence extending to the 5' end or/and the 3' end; Or a sequence having greater than 85% homology to the above sequence or its complement; Or a base complementary sequence to the above sequence; Or using any one or more of the above sequences; mRNA for the PSA gene: Primer set A: upstream 5 ' -biotin TTGACCCCAAAGAAACTTCAGTGT -3 ' , as SEQ ID Shown as NO.29; Downstream 5'-TGCCCCATGACGTGATACCT-3', as shown in SEQ ID NO. Primer set B: upstream 5 '-biotin TCCCACACCCGCTCTACGAT-3', as SEQ ID NO. 31 Shown Downstream 5'-CGTCCAGCACACAGCATGAACT-3', as shown in SEQ ID NO. Primer set C: upstream 5 ' -biotin TGCACCCCTCATCCTGTCTC -3 ' , as SEQ ID NO. 33 Shown Downstream 5'-GCTGTGGCTGACCTGAAATACC-3', as shown in SEQ ID NO. Primer set D: upstream 5 ' -biotin GGCAGCATTGAACCAGAGGAGT -3 ' , as SEQ ID Shown as NO.35; Downstream 5'-CGATGGTGTCCTTGATCCACTT-3', as shown in SEQ ID NO. Primer set E: upstream 5 ' -biotin TCCTCAGGCCAGGTGATGACT -3 ' , as SEQ ID Shown as NO.37; Downstream 5'-CGTCCAGCACACAGCATGAACT-3', as shown in SEQ ID NO. Or a sequence comprising the above sequence or its complementary sequence extending to the 5' end or/and the 3' end; Or a sequence having greater than 85% homology to the above sequence or its complement; Or a base complementary sequence to the above sequence; Or using any one or more of the above sequences; The above primer set designed for the PCA3 gene (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) and the PCA3 probe of claim 3. (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) one-to-one correspondence; Primer sets A, B, C, D, E designed for the PSA gene and PSA probe A in claim 3 , B, C, D, E correspond one by one. 5. The liquid phase chip method for detecting a PCA3 gene and a PSA gene according to claim 1, wherein in the step (4), the internal reference gene is a β-actin gene, and the primers and probe sequences thereof are as follows: Upstream primer: 5 ' -biotin TGGGTCAGAAGGATTCCTATGTG-3', as SEQ ID NO. 39 Shown Downstream primer: 5'-GCTGGGGTGTTGAAGGTCTC-3', as SEQ ID NO. 40 Shown Probe: 5 ' - AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3', as SEQ ID Shown as NO.41; Or a sequence comprising the above sequence or its complementary sequence extending to the 5' end or/and the 3' end; Or a sequence having greater than 85% homology to the above sequence or its complement; Or a base complementary sequence to the above sequence; Alternatively, any one or a combination of more than one of the above sequences may be used. 6. A diagnostic kit for detecting a PCA3 gene, a PSA gene, comprising the mRNA-specific probe comprising the mRNA and/or PSA gene covalently bound to the PCA3 gene according to any one of claims 1 to 5 Microsphere mixture and microspheres incorporating β-actin gene probe, upstream and downstream primers for mRNA and/or PSA gene mRNA and/or β-actin gene according to any one of claims 1 to 5 Upstream and downstream primers, streptavidin-phycoerythrin and control products. 7. Such as The diagnostic kit for detecting a PCA3 gene or a PSA gene according to claim 6, wherein the microsphere mixture is freely combined according to the needs of different test samples. 8. Such as The diagnostic kit for detecting a PCA3 gene and a PSA gene according to claim 6, wherein the control substance comprises a positive control and a negative control, wherein the positive control comprises a PCA3 gene. And/or a mixture of PSA gene plasmids, including plasmids containing the β-actin gene; negative control does not contain the PCA3 gene, PSA gene And a plasmid solution of the β-actin gene. 9. A diagnostic kit for detecting PCA3 gene and PSA gene according to claim 6, for detecting in vitro samples, for specific and early diagnosis, staging, curative effect observation and prognosis of prostate cancer, for benign prostatic hyperplasia and prostate The application of cancer in differential diagnosis and the application of early diagnosis, therapeutic observation and prognosis for other related diseases.