CN101942523B - Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof - Google Patents
Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof Download PDFInfo
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Abstract
The invention discloses a liquid-phase chip method for detecting a PCA3 gene and a PSA gene and a diagnostic reagent kit thereof. A primer and a probe are designed for mRNA of the PCA3 gene and/or the PSA gene; a mixture, formed by the covalent bonding of the probe and microsphere, is hybridized with a reverse transcription P CR amplification product, after streptavidin phycoerythrin is added fluorescent signals of different microspheres can be detected to determine whether a sample to be detected contains the PCA3 and PSA genes and the expression condition thereof, and simultaneously, the PCA3/PSA ratio can be obtained. The liquid-phase chip method and the reagent kit have the advantages of high sensitivity, high throughput, quick and accurate detection and the like, and can perform qualitative and quantitative detection on the PCA3 and PSA genes related to different degrees of prostate diseases including prostate cancer and benign prostatic hyperplasia and perform specific diagnosis on the prostate cancer.
Description
Technical field
The present invention relates to in-vitro diagnosis detection technique field, be specifically related to liquid-phase chip detection method and the diagnostic kit thereof of relevant specific gene PCA3, PSA of prostatosis.
Background technology
PCA3 (prostate cancer antigen 3) gene, claim again DD3 (differential display code 3) gene, a kind of function that is the discoveries such as Bussemakers in 1999 is the gene of non-coding RNA, is comprised of 4 exons and 3 introns.The PCA3 gene only is expressed in prostata tissue, and it is expressed extremely low or does not express in normal prostatic and benign prostatic hyperplasia (BPH) tissue, and is high expression level in prostate cancer (PCa) cell.Therefore, the PCA3 gene is considered to the most special gene of prostate cancer at present.
Prostate specific antigen (PSA, prostate specific antigen) although be widely used at present the generaI investigation, diagnosis of prostate cancer, by stages, observation of curative effect and prognosis judge, but the PSA gene does not have tumour-specific, in the benign lesions such as hyperplasia of prostate (BPH) and prostatitis, also can raise, and the PSA gene can not improve the early diagnostic rate of prostate cancer.
Aspiration biopsy of prostatic gland is the gold standard of different prostatosis differential diagnosis, but it is that wound inspection is arranged, and false-negative generation is often arranged.Rectal touch and relevant auxiliary examination such as B ultrasonic, MRI (Magnetic resonance imaging) though etc. be Noninvasive, limitation is arranged.Therefore, the inexorable trend that to seek more special, early stage, noninvasive detection means be prostatosis differential diagnosis.
In the method for the specific gene that the outer widely used molecular Biological Detection prostatosis of Present Domestic are relevant, quantitative fluorescent PCR exists the limitation that detects flux, so all can't really satisfy the needs that clinical diagnosis detects.Traditional solid phase biological chip (Biochip) technology exist repeatable poor, insufficient sensitivity good and the outstanding weakness of complex operation.Therefore need that clinically a kind of detection method is arranged, can be rapidly, specific gene stable, that exactly prostatosis are correlated with carries out joint-detection, liquid-phase chip (xMAP) technology is a kind of so novel detection technique just.
Liquid-phase chip can carry out qualitative and quantitative analysis to a small amount of sample, has high-throughput, easy and simple to handle, good reproducibility, highly sensitive, the outstanding advantages such as linearity range is wide.This system is that main matrix consists of by many microballoons, in the middle of the manufacturing processed of microballoon, two kinds of different redness classification fluorescence have been mixed, ratio according to these two kinds of fluorescence is different, sphere matrix is divided into 100 kinds, 100 kinds of different probe molecules on can mark, can be simultaneously in the sample nearly 100 kinds of different target molecules detect.According to the difference that detects thing, microsphere surface can the various detection of nucleic acids probes of covalent attachment, add fluorescent mark when hybridization carries out.In same reaction system, can add simultaneously different detection microballoons, so just can utilize a small amount of sample to carry out quick, high-throughout detection.After reaction finishes, by micro-fluidic technologies with microballoon defiled Rapid Flow through the liquid-phase chip detector, each microballoon can be arrived by two bundle laser detection simultaneously, red laser excites the redness classification fluorescence on the microballoon, the reaction that each is different makes a distinction and qualitative; Green laser then excites the fluorescent mark that is combined on the sample to be tested to carry out quantitatively.When the good sample to be tested of mark and the probe on the specific microballoon combined, the light that two bundle laser excite all can be detected.At last, by the high speed digital signal processor of computer, the average fluorescent strength on the specific microballoon be can automatic statistical analysis draw, thereby kind and the quantity of thing determined to detect.
The present invention is based on the high-throughput of liquid-phase chip technology, easy and simple to handle, good reproducibility, highly sensitive, the outstanding advantages such as linearity range is wide, the specific gene relevant to prostatosis detects, and can better be used in clinical detection.
Summary of the invention
The technical issues that need to address of the present invention provide detection method and the diagnostic kit of the relevant specific gene of a kind of prostatosis.The method and test kit comprise PCA3 gene and/or PSA gene are detected, can be to prostatosis in various degree, especially prostate cancer carry out special and early diagnosis, by stages, observation of curative effect and prognosis judge, benign prostatic hyperplasia and prostate cancer are carried out differential diagnosis, and other relative diseases are carried out early diagnosis, observation of curative effect and prognosis judge.This detection method and diagnostic kit have the advantages such as highly sensitive, high specific, split hair caccuracy, detection be rapid.
For solving the problems of the technologies described above, in one aspect of the invention, provide a kind of liquid-phase chip method that detects the ratio of prostatosis relevant specific gene PCA3 gene, PSA gene and PCA3 gene/PSA gene, may further comprise the steps:
(1) a kind of carboxyl microballoon Beads of design, covalent attachment is for the designed specific dna probe of mRNA of PCA3 gene or PSA gene on this microballoon; Perhaps design comprises two kinds of carboxyl microballoon Beads that difference is fluorescence-encoded, distinguishes covalent attachment for the designed specific dna probe of mRNA of PCA3 gene and gene PS A on every kind of microballoon; Described PCA3 gene can be separately detects as the specific gene of prostate cancer, the PSA gene can be separately detects as the specific gene of relevant prostatosis, the specific gene that PCA3 and PSA gene can combine as relevant prostatosis carries out joint-detection, can also carry out differential diagnosis to the prostatosis of being correlated with according to the ratio of PCA3 gene/PSA gene simultaneously.PCA3 gene and/or PSA gene also can work the specific gene that is used as relevant prostatosis with other assortment of genes and carry out joint-detection;
(2) for the mRNA of PCA3 gene and/or PSA gene, design respectively the upstream and downstream primer, amplify corresponding product by reverse transcription PCR;
(3) contain the reverse transcription amplification product hybridization of mRNA of the microballoon of specific dna probe and PCA3 gene and/or PSA gene after, add SA-PE (Streptavidin-PE), detect fluorescent signal by Luminex xMAP;
(4) fluorescent signal that detects and the fluorescent signal of reference gene are compared, thereby determine whether have prostatosis relevant gene PCA3 gene and/or PSA gene in the test sample, and the expression situation in sample, the while also can calculate the ratio of PCA3 gene/PSA gene.
Microballoon described in the step of above detection method (1) is that mean diameter is 5.6 μ m, combines the polyphenyl alkene microballoon of different fluorescence dyes, i.e. color-code microballoon (color-coded beads);
In the step (1), the described designed specific dna probe of mRNA for PCA3 gene and/or PSA gene that is covalently bonded on the microballoon comprises following sequence (wherein 5 ' end contains amido modified):
MRNA for the PCA3 gene:
Probe (1): 5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ', shown in SEQ ID NO.1;
Probe (2), (3), (5), (6) same probe (1) are shown in SEQ ID NO.1;
Probe (4): 5 '-AminolinkerC12 CTCACCTCTGTATCATCAG-3 ', shown in SEQ ID NO.2;
Probe (7): 5 '-AminolinkerC12 ATCTCTGTGCTTCCTTTTGT-3 ', shown in SEQ ID NO.3;
The same probe of probe (8) (7) is shown in SEQ ID NO.3
Probe (9): 5 '-AminolinkerC12 CAAATCTGTAATCCCGTTCA-3 ', shown in SEQ ID NO.4;
Probe (10): 5 '-AminolinkerC12 TATGTGTCAAGAGGAGAGCC-3 ', shown in SEQ ID NO.5;
Perhaps include holding or/and the sequence that 3 ' end prolongs to 5 ' of above-mentioned sequence (containing complementary sequence);
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
MRNA for the PSA gene:
Probe A:5 '-AminolinkerC12 CAGAATCACCCGAGCAGGTG-3 ' is shown in SEQ ID NO.6;
Probe B:5 '-AminolinkerC12 GGGGTCAAGAACTCCTCTGG-3 ' is shown in SEQ ID NO.7;
Probe C:5 '-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
Probe D is with PSA probe A, shown in SEQ ID NO.6;
Probe E is with PSA probe B, shown in SEQ ID NO.7;
Perhaps include holding or/and the sequence that 3 ' end prolongs to 5 ' of above-mentioned sequence (containing complementary sequence);
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
In the step (2), described for the designed upstream and downstream primer of the mRNA of PCA3 gene and/or PSA gene, comprise following sequence (wherein upstream primer 5 ' end contains biotin label):
MRNA for the PCA3 gene:
Primer sets (1): upstream 5 '-biotin AAGAAATAGCAAGTGCCGAGAAG-3 ', shown in SEQ ID NO.9;
Downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3 ' is shown in SEQ ID NO.10;
Primer sets (2): upstream 5 '-biotin TGGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.11;
Downstream 5 '-TCTCCCAGGGATCTCTGTGCTT-3 ' is shown in SEQ ID NO.12;
Primer sets (3): upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14;
Primer sets (4): upstream 5 '-biotin AGCCGAGGGAGACCAGGAAG-3 ', shown in SEQ ID NO.15;
Downstream 5 '-CAGCAGATGTGTGGCCTCAGAT-3 ' is shown in SEQ ID NO.16;
Primer sets (5): upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT-3 ', shown in SEQ ID NO.17;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.18;
Primer sets (6): upstream 5 '-biotin AGAAATAGCAAGTGCCGAGAAGC-3 ', shown in SEQ ID NO.19;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.20;
Primer sets (7): upstream 5 '-biotin TATCCACACACACAGGAAGCAC-3 ', shown in SEQ ID NO.21;
Downstream 5 '-CCTCTCATTGGTAATGCTCACTTT-3 ' is shown in SEQ ID NO.22;
Primer sets (8): upstream 5 '-biotin AAGGCTGCTGACTTTACCATCTG-3 ', shown in SEQ ID NO.23;
Downstream 5 '-TCTAATGTCCTTCCCTCACAAGC-3 ' is shown in SEQ ID NO.24;
Primer sets (9): upstream 5 '-biotin CGCTTGTGAGGGAAGGACATTAG-3 ', shown in SEQ ID NO.25;
Downstream 5 '-GTGAAGCCATCAAGATTTTCTCGTC-3 ' is shown in SEQ ID NO.26;
Primer sets (10): upstream 5 '-biotin CAGCAGGACCCAACGCAT-3 ', shown in SEQ ID NO.27;
Downstream 5 '-GAGAGAGGATTGGTAAGCGATGTG-3 ' is shown in SEQ ID NO.28;
Perhaps include holding or/and the sequence that 3 ' end prolongs to 5 ' of above-mentioned sequence (containing complementary sequence);
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
MRNA for the PSA gene:
Primer sets A: upstream 5 '-biotin TTGACCCCAAAGAAACTTCAGTGT-3 ', shown in SEQ ID NO.29;
Downstream 5 '-TGCCCCATGACGTGATACCT-3 ' is shown in SEQ ID NO.30;
Primer sets B: upstream 5 '-biotin TCCCACACCCGCTCTACGAT-3 ', shown in SEQ ID NO.31;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.32
Primer sets C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34
Primer sets D: upstream 5 '-biotin GGCAGCATTGAACCAGAGGAGT-3 ', shown in SEQ ID NO.35;
Downstream 5 '-CGATGGTGTCCTTGATCCACTT-3 ' is shown in SEQ ID NO.36
Primer sets E: upstream 5 '-biotin TCCTCAGGCCAGGTGATGACT-3 ', shown in SEQ ID NO.37;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.38;
Perhaps include holding or/and the sequence that 3 ' end prolongs to 5 ' of above-mentioned sequence (containing complementary sequence);
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
More than corresponding one by one with PCA3 probe (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) of above-mentioned design for the designed primer sets of PCA3 (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), namely the product that amplifies of corresponding primer sets and corresponding probe carry out the hybridization in the step (3);
More than corresponding one by one with PSA probe A, B, C, D, the E of above-mentioned design for the designed primer sets A of PSA, B, C, D, E, namely the product that amplifies of corresponding primer sets and corresponding probe carry out the hybridization in the step (3);
In the step (4), primer and the probe sequence of described reference gene β-actin gene are as follows:
Upstream primer: 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ', shown in SEQ ID NO.39;
Downstream primer: 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
Probe: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
Perhaps include holding or/and the sequence that 3 ' end prolongs to 5 ' of above-mentioned sequence (containing complementary sequence);
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
In the present invention on the other hand, a kind of relevant specific gene PCA3 of prostatosis, diagnostic kit of PSA of detecting is provided, the mixture of microspheres of the mRNA specific probe of the comprised covalent attachment mRNA of PCA3 gene and/or PSA gene, combine the microballoon of β-actin gene probe, for upstream and downstream primer, the upstream and downstream primer of β-actin gene, SA-PE Streptavidin-PE, the quality control product (negative control and positive control) of the mRNA of the mRNA of PCA3 gene and/or PSA.
Quality control product described in the above test kit comprises positive control and negative control, wherein positive control is the plasmid mixed solution (comprising the plasmid that contains β-actin gene) that contains PCA3 gene and/or PSA gene, and the negative control product are not for containing the plasmid of PCA3 gene, PSA gene and β-actin gene; Described mixture of microspheres is according to the needs independent assortment of different test sample.
Another aspect of the present invention, provide a kind of diagnostic kit that detects prostatosis relevant specific gene PCA3, PSA detecting vitro samples, to prostatosis (especially prostate cancer) in various degree carry out special and early diagnosis, by stages, observation of curative effect and the prognosis application in judging, benign prostatic hyperplasia and prostate cancer are carried out application in the differential diagnosis, and to other relative diseases carry out early diagnosis, by stages, observation of curative effect and the prognosis application in judging.
Because the present invention has utilized liquid-phase chip technology, make detection method and test kit have the outstanding advantages such as highly sensitive, high specific, high-throughput, good stability, detection be rapid, accurate, can carry out the quantitative and qualitative analysis detection to PCA3 gene and/or PSA gene, and can calculate the ratio of PCA3 gene/PSA gene, therefore can better be used in clinical detection and diagnosis.
Embodiment
Describe the present invention in detail below in conjunction with specific embodiment, but can not be interpreted as limitation of the present invention.Described embodiment only provides the Production and application method of illustrating nucleic acid primer, probe, detection method, test kit and is not limited by it.Various versions are expected in the scope of the present invention and described claim during this time.
Experiment material:
Primer and probe are synthetic by invitrogen company; Trizol is available from invitrogen company; The total RNA extraction reagent box is available from TaKaRa company; Reverse transcription cDNA synthetic agent box is available from Fermentas company; The microballoon (surperficial carboxyl modified) of multiple PCR reagent kit, different numberings, SA-PE are all available from QIAGEN company; 1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) is available from Pierce company; 2-(N-morpholine)-ethyl sulfonic acid (MES), N-lauroyl propylhomoserin sodium (Sarkosyl), tetramethyl ammonium chloride (TMAC) are all available from sigma company.
The preparation of damping fluid, hybridization solution and other solution:
Coupling buffer, pH4.5 250ml
MES 4.88g；
1.5 * TMAC hybridization solution 250ml
5M TMAC 225ml
20％Sarkosyl 1.88ml
1M Tris-HCl，pH8.0 18.75ml
0.5M EDTA，pH8.0 3.0ml
H
2O 1.37ml；
1.5 * TMAC hybridization solution 250ml
5M TMAC 150ml
20％Sarkosyl 1.25ml
1M Tris-HCl，pH8.0 12.5ml
0.5M EDTA，pH8.0 2.0ml
H
2O 84.25ml；
The TE damping fluid, pH8.0 500ml
1M Tris-HCl，pH8.0 5ml
0.5M EDTA，pH8.0 1ml
H
2O 444ml
The preparation of D-Hanks liquid: KCl 0.4g, NaCI 8.0g, NaHCO
30.35g, Na
2HPO412H
2O 0.15g,
KH
2PO4 0.06g is dissolved in deionized water successively, adds ionized water to 1000ml.15
KPa * 30min sterilization, 4 ℃ of preservations.
DEPC processes the water configuration: deionized water adds DEPC, and to make its concentration be 0.1%, 37.C water-bath 2 hours, 15KPa
* 20min autoclaving.
Embodiment 1: different primers and probe combinations detect the liquid-phase chip method of PCA3 gene in the urine
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PCA3 gene
1. according to following sequence synthetic oligonucleotide probe:
MRNA for the PCA3 gene:
Probe (1) 5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ' is shown in SEQ ID NO.1;
Probe (2), (3), (5), (6) same probe (1) are shown in SEQ ID NO.1;
Probe (4) 5 '-AminolinkerC12 CTCACCTCTGTATCATCAG-3 ' are shown in SEQ ID NO.2; Probe (7) 5 '-AminolinkerC12 ATCTCTGTGCTTCCTTTTGT-3 ' are shown in SEQ ID NO.3;
The same probe of probe (8) (7) is shown in SEQ ID NO.3
Probe (9) 5 '-AminolinkerC12 CAAATCTGTAATCCCGTTCA-3 ' are shown in SEQ ID NO.4; Probe (10) 5 '-AminolinkerC12 TATGTGTCAAGAGGAGAGCC-3 ' are shown in SEQ ID NO.5;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41.
2. will contain amido modified oligonucleotide probe respectively with the 11 kinds of carboxyl microballoons coupling that is numbered 11,13,15,21,25,61,62,63,64,65, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PCA3, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 11 kinds of carboxyl microballoons of 11,13,15,21,25,61,62,63,64,65, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4 get respectively 2.5 * 10
6Microballoon stores in centrifuge tube of suspension to ten;
2.5 10,000g is centrifugal, 1-2min;
2.6 remove supernatant liquor, with 50 μ l 0.1M MES, the coupling buffer of pH4.5, resuspended microballoon, vortex concussion 20 seconds;
2.7 be that the 1mM oligonucleotide probe is used respectively dH with 11 kinds of concentration
2O was with 1: 10 dilution proportion, and making its concentration is 0.1nmol/ μ l;
2.8 every kind of probe adds 2 μ l (concentration is 0.1nmol/ μ l) in the corresponding microballoon of mixing, vortex mixed;
2.9 use dH
2The fresh EDC solution of O preparation 10mg/ml (noting: keep the EDC powder for drying, be convenient to the use of next step EDC);
2.10 add 2.5 μ l 10mg/ml EDC fresh EDC solution divide and be clipped to (25 μ g final concentrations are about 0.5 μ g/ μ l) vortex mixing in 11 kinds of microballoons;
2.11 the room temperature lucifuge is hatched 30min;
2.12 use dH
2The EDC fresh solution of second part of 10mg/ml of O preparation (noting: if EDC powder deliquescence then should abandon, advise that each step coupling process all uses fresh EDC powder);
2.13 respectively add the EDC fresh solution of 2.5 μ l 10mg/ml in 11 kinds of microballoons, the vortex mixing;
2.14 the room temperature lucifuge is hatched 30min;
2.15 respectively add 1ml 0.02%Tween-20 in 11 kinds of coupling microballoons;
2.16 10,000g is centrifugal, 1-2min;
2.17 remove supernatant, use respectively the resuspended 11 kinds of coupling microballoons of 1ml 0.1%SDS, the vibration mixing;
2.18 10,000g is centrifugal, 1-2min;
2.19 remove supernatant, add respectively 100 μ l TE, pH8.0, vortex mixed 20s, resuspended microballoon;
2.20 with the cell counter microballoon of oligonucleotide probe of having counted 11 kinds of couplings;
A. use dH
2O diluted the coupling microballoon with 1: 100;
B. vortex shakes abundant mixing;
C. get 10 μ l to cell counter;
D. count the microballoon total amount of 4 large lattice in angle on the cell counter;
E. microballoon/μ l=(4 large lattice microballoon total amount) * 2.5 * 100 (extension rates).
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PCA3 probe (1) microballoon 11, PCA3 probe (2) microballoon 13, PCA3 probe (3) microballoon 15, PCA3 probe (4) microballoon 21, PCA3 probe (5) microballoon 25, PCA3 probe (6) microballoon 61, PCA3 probe (7) microballoon 62, PCA3 probe (8) microballoon 63, PCA3 probe (9) microballoon 64, PCA3 probe (10) microballoon 65, β-actin probe microballoon 66, equal proportion is mixed, the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
The 1-5 clinical sample is prostate cancer (PCa) patient's urine, and the 6-8 clinical sample is normal people's urine:
1. the experimenter is adopted traditional massage of prostate method, namely the examiner does rectal touch, from prostate gland two side direction median groovees, vertically massages from top to bottom 2 to 3 times, massages median groove once again, and prostatic fluid is clamp-oned urethra.
2. enjoin the experimenter rear collection initial segment urine 20～30ml that urinates immediately, put into immediately ice and cool off.Collect arena in 4 ℃ of centrifugal 10min of 3000r/min, add an amount of precooling D-Hanks liquid washed twice;
3. arena is collected in the 1.5ml Eppendorf pipe, adds immediately sex change liquid (being RNAiso Reagent) the 500 μ l mixings that contain acid guanidinium isothiocyanate and put-70 ℃ of preservations.
4. added the arena sample that contains 500 μ l RNAiso Reagent from-70 ℃ of taking-ups, rear centrifugal wait melting.
5. add 1/5 RNAiso Reagent volume chloroform, the vibration mixing, after room temperature leaves standstill 5 minutes, 4 ℃ of centrifugal 15min of 12000r/min.
6. carefully upper water is moved in the 1.5ml Eppendorf pipe of RNase-free mutually, then add and the isopyknic Virahol of supernatant, put upside down mixing, after room temperature leaves standstill 5 minutes, the centrifugal 10min of 12000r/min.
7. abandon supernatant liquor, with 1ml 75% pre-cooled ethanol washing precipitation 2 times, centrifugal thorough removal ethanol.
8. the RNA that obtains is precipitated and dissolved in an amount of DEPC and processes in the water, the total RNA that extracts in the sample is carried out quantitatively at the uv-spectrophotometric instrument.
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-8 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the first chain is synthetic
1. get the Eppendorf tube of processing once DEPC, place on ice, set up following reaction system;
Total RNA 5～10μg/3μl
Oligo(dT)18Primer(0.5μg/μl) 1μl
RNase-free water forward to 12μl
The mixing reaction solution arrives the pipe end with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge gently;
2. centrifuge tube is in 70 ℃ of incubations 5 minutes, take out rapidly afterwards to place ice to cool off, with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge to the pipe end;
3. centrifuge tube is positioned on ice, is sequentially added into following reaction solution;
5×reaction buffer 4μl
RNase Inhibitor(20U/μl) 1μl
10mM dNTPs Mix 2μl
The mixing reaction solution arrives the pipe end with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge gently;
4. centrifuge tube was in 37 ℃ of incubations 5 minutes;
5. add 1 μ l RevertAidTM Reverse Transcriptase (200U/ μ l), final volume is 20 μ l;
6. centrifuge tube was in 42 ℃ of reactions 60 minutes;
7. centrifuge tube is in 10 minutes termination reactions of 70 ℃ of heating, and taking-up places ice to cool off rapidly afterwards;
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
MRNA for the PCA3 gene:
Primer sets (1) upstream 5 '-biotin AAGAAATAGCAAGTGCCGAGAAG-3 ' is shown in SEQ ID NO.9;
Downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3 ' is shown in SEQ ID NO.10;
Primer sets (2) upstream 5 '-biotin TGGTGGGAAGGACCTGATGATAC-3 ' is shown in SEQ ID NO.11;
Downstream 5 '-TCTCCCAGGGATCTCTGTGCTT-3 ' is shown in SEQ ID NO.12;
Primer sets (3) upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ' is shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14;
Primer sets (4) upstream 5 '-biotin AGCCGAGGGAGACCAGGAAG-3 ' is shown in SEQ ID NO.15;
Downstream 5 '-CAGCAGATGTGTGGCCTCAGAT-3 ' is shown in SEQ ID NO.16;
Primer sets (5) upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT-3 ' is shown in SEQ ID NO.17;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.18;
Primer sets (6) upstream 5 '-biotin AGAAATAGCAAGTGCCGAGAAGC-3 ' is shown in SEQ ID NO.19;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.20;
Primer sets (7) upstream 5 '-biotin TATCCACACACACAGGAAGCAC-3 ' is shown in SEQ ID NO.21;
Downstream 5 '-CCTCTCATTGGTAATGCTCACTTT-3 ' is shown in SEQ ID NO.22;
Primer sets (8) upstream 5 '-biotin AAGGCTGCTGACTTTACCATCTG-3 ' is shown in SEQ ID NO.23;
Downstream 5 '-TCTAATGTCCTTCCCTCACAAGC-3 ' is shown in SEQ ID NO.24;
Primer sets (9) upstream 5 '-biotin CGCTTGTGAGGGAAGGACATTAG-3 ' is shown in SEQ ID NO.25;
Downstream 5 '-GTGAAGCCATCAAGATTTTCTCGTC-3 ' is shown in SEQ ID NO.26;
Primer sets (10) upstream 5 '-biotin CAGCAGGACCCAACGCAT-3 ' is shown in SEQ ID NO.27;
Downstream 5 '-GAGAGAGGATTGGTAAGCGATGTG-3 ' is shown in SEQ ID NO.28;
More than corresponding one by one with PCA3 probe (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) of above-mentioned design for the designed primer sets of PCA3 (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), correspondence is combined as PCA3 primer/probe groups 1,2,3,4,5,6,7,8,9,10 (see Table 1 and table 2) respectively;
β-actin gene:
Upstream primer 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ' is shown in SEQ ID NO.39;
Downstream primer 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
2.2 multi-PRC reaction:
What adopt is the Multiplex PCR test kit of QIAGEN company, and 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 μ l
(contain warm start TaqDNA enzyme, MgCl among 2 * QIAGEN Multiplex PCR Master Mix
2With dNTP Mix.)
Pcr amplification program: 95 ℃ of 15min; 94 ℃ of 30s, 55 ℃ of 90s, 72 ℃ of 90s, 35 circulations; 72 ℃ of 10min; 4 ℃ of insulations.
Four. the hybridization of oligonucleotide probe and PCR product
1. the oligonucleotide probe mixture of microspheres of preparing in the selecting step one;
2. vortex shakes 20s, mixing microballoon;
3. preparation microballoon working fluid is 150 microballoons/μ l with 1.5 * TMAC hybridization solution dilution coupling microballoon to concentration.(annotate: the microballoon working fluid of each reaction needed 33 μ l);
4. vortex shakes 20s, mixing microballoon working fluid;
5. in sample aperture, positive control hole, negative control hole, add respectively 33 μ l microballoon working fluids;
6. add respectively pcr amplification product and the TE solution (pH is 8.0) of 1-8 sample in each sample well, cumulative volume is 17 μ l;
7. inhale up and down with the volley of rifle fire and beat gentle mixing reaction solution;
8. cover the reaction lid and avoid evaporation, under 95-100 ℃, hatch 1-3min, make the oligonucleotide in the sample remove secondary structure;
9. incubation reaction plate 15min under hybridization temperature;
10. prepare fresh detection mixed solution, with 1 * TMAC hybridization solution dilution Streptavidin phycoerythrin solution to 10 μ g/ml (the detection mixed solutions of each reaction needed 25 μ l);
11. add the detection mixed solution of 25 μ l to every hole, inhale up and down with the volley of rifle fire and beat gentle mixing;
12. under hybridization temperature, hatch 5min;
Above-mentioned steps can be programmed its merging by following scheme by the PCR instrument:
95℃，1-3min；
Hybridization temperature, forever;
13. on liquid-phase chip instrument (LiquiChip 200, QIAGEN and Luminex company), keep hybridization temperature, each reacting hole is detected analysis, measure fluorescence MFI value (average fluorescent strength).
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 1:
Table 1
Interpretation of result is as shown in table 2:
Table 2
The PCA3 that different primers/probe groups detects in 1-5 prostate cancer (PCa) Urine in Patients is all positive.
The PCA3 that different primers/probe groups detects in 6-8 normal people's urine is all negative.
Simultaneously, the fluorescence MFI value that the patient presents is compared with the fluorescence MFI value of confidential reference items β-actin gene, can be drawn the expression situation of PCA3 gene.
Embodiment 2: different primers and probe combinations detect the liquid-phase chip method of PSA gene in the urine
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PSA gene
1. according to following sequence synthetic oligonucleotide probe:
MRNA for the PSA gene:
Probe A:5 '-AminolinkerC12 CAGAATCACCCGAGCAGGTG-3 ' is shown in SEQ ID NO.6;
Probe B:5 '-AminolinkerC12 GGGGTCAAGAACTCCTCTGG-3 ' is shown in SEQ ID NO.7;
Probe C:5 '-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
Probe D: with PSA probe A, shown in SEQ ID NO.6;
Probe E: with PSA probe B, shown in SEQ ID NO.7;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
2. will contain amido modified oligonucleotide probe respectively with the 6 kinds of carboxyl microballoons coupling that is numbered 11,13,15,21,25, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PSA, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 6 kinds of carboxyl microballoons of 11,13,15,21,25, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4-2.20 with the 2.4-2.20 among the embodiment 1, just with corresponding 6 pipes that reduce to of 11 pipes, other are constant.
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PSA probe A microballoon 11, PSA probe B microballoon 13, PSA probe C microballoon 15, PSA probe D microballoon 21, PSA probe E microballoon 25, β-actin probe microballoon 66, equal proportion is mixed, the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
The 1-5 clinical sample is prostate cancer (PCa) patient's urine, and the 6-8 clinical sample is normal people's urine, and the preparation method is with the preparation method of sample among the embodiment 1.
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-8 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the synthetic method of the first chain is with the synthetic method of cDNA the first chain among the embodiment 1.
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
MRNA for the PSA gene:
Primer sets A: upstream 5 '-biotin TTGACCCCAAAGAAACTTCAGTGT-3 ', shown in SEQ ID NO.29;
Downstream 5 '-TGCCCCATGACGTGATACCT-3 ' is shown in SEQ ID NO.30;
Primer sets B: upstream 5 '-biotin TCCCACACCCGCTCTACGAT-3 ', shown in SEQ ID NO.31;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.32
Primer sets C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34
Primer sets D: upstream 5 '-biotin GGCAGCATTGAACCAGAGGAGT-3 ', shown in SEQ ID NO.35;
Downstream 5 '-CGATGGTGTCCTTGATCCACTT-3 ' is shown in SEQ ID NO.36
Primer sets E: upstream 5 '-biotin TCCTCAGGCCAGGTGATGACT-3 ', shown in SEQ ID NO.37;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.38
More than corresponding one by one with PSA probe A, B, C, D, the E of above-mentioned design for the designed primer sets A of PSA, B, C, D, E, correspondence is combined as PSA primer/probe groups 1,2,3,4,5 (see Table 3 and table 4) respectively;
β-actin gene:
Upstream 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ' is shown in SEQ ID NO.39;
Downstream 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
2.2 multi-PRC reaction:
Method is with (three, 2.2) multi-PRC reaction method among the embodiment 1.
Four. the hybridization of oligonucleotide probe and PCR product
Method is with the hybridizing method of (four) oligonucleotide probe among the embodiment 1 and PCR product.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 3:
Table 3
Interpretation of result is as shown in table 4:
Table 4
The PSA that different primers/probe groups detects in 1-5 prostate cancer (RCa) Urine in Patients is all positive.
The PSA that different primers/probe groups detects in 6-8 normal people's urine is all negative.
Simultaneously, the fluorescence MFI value that the patient presents is compared with the fluorescence MFI value of confidential reference items β-actin gene, can be drawn the expression situation of PSA gene.
Embodiment 3: the liquid-phase chip method of PCA3, PSA and PCA3/PSA ratio in the joint-detection urine
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PSA gene
1. according to following sequence synthetic oligonucleotide probe:
PCA3:5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ' is shown in SEQ ID NO.1;
PSA:5 '-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
2. will contain amido modified oligonucleotide probe respectively with the 3 kinds of carboxyl microballoons coupling that is numbered 11,13, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PCA3, PSA, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 3 kinds of carboxyl microballoons of 11,13, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4-2.20 with the 2.4-2.20 among the embodiment 1, just with corresponding 3 pipes that reduce to of 11 pipes, other are constant.
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PCA3 probe microballoon 11, PSA probe microballoon 13, β-actin probe microballoon 66, equal proportion is mixed, and the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
1-10 patient's clinical sample is prostate cancer (PCa) patient's urine, and 11-20 patient's clinical sample is benign prostatic hyperplasia (BPH) patient's urine, and the preparation method is with the preparation method of sample among the embodiment 1.
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-20 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the synthetic method of the first chain is with the synthetic method of cDNA the first chain among the embodiment 1.
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
PCA3: upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14
PSA: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34
β-actin gene:
Upstream 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ' is shown in SEQ ID NO.39;
Downstream 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
2.2 multi-PRC reaction:
Method is with (three, 2.2) multi-PRC reaction method among the embodiment 1.
Four. the hybridization of oligonucleotide probe and PCR product
Method is with the hybridizing method of (four) oligonucleotide probe among the embodiment 1 and PCR product.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 5:
Table 5
Interpretation of result is as shown in table 6:
Table 6
The mean value of PCA3/PSA ratio and analyze as shown in table 7:
Table 7
PCA3 and PSA in 1-10 prostate cancer (PCa) the group Urine in Patients are all positive; PCA3 in 11-20 benign prostatic hyperplasia (BPH) the group Urine in Patients is negative, and PSA is positive; Two groups of patient PCA3/PSA mean values differ 21 times.This method makes a distinction patients with prostate cancer and Benign Prostatic Hypertrophy well.
Simultaneously, the patient is presented positive fluorescence MFI value compare with the fluorescence MFI value of confidential reference items β-actin gene, can draw the expression situation of PCA3 gene and PSA gene.
Embodiment 4: different primers and probe combinations detect the Luminex of PCA3 gene in the blood
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PCA3 gene
1. according to following sequence synthetic oligonucleotide probe:
For the PCA3 gene:
Probe (1): 5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ', shown in SEQ ID NO.1;
Probe (3), (5) same probe (1) are shown in SEQ ID NO.1;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
2. will contain amido modified oligonucleotide probe respectively with the 4 kinds of carboxyl microballoons coupling that is numbered 11,13,15, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PCA3, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 4 kinds of carboxyl microballoons of 11,13,15, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4-2.20 with the 2.4-2.20 among the embodiment 1, just with corresponding 4 pipes that reduce to of 11 pipes, other are constant.
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PCA3 (1) probe microballoon 11, PCA3 (3) probe microballoon 13, PCA3 (5) probe microballoon 15, β-actin probe microballoon 66, equal proportion is mixed, the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
The 1-5 clinical sample is prostate cancer (PCa) patient's venous blood, and the 6-8 clinical sample is normal people's venous blood, extracts according to the following steps respectively RNA:
1. lymphocyte extracts: get early before the meal anti-freezing venous blood 5ml of experimenter, 3000r/min is centrifugal behind the mixing, and 10min, gets that pale yellow chromatograph is lymphocyte on the blood cell layer by 4 ℃;
2. get 1 centrifuge tube (through the DEPC water treatment) adding lymphocyte liquid, 150 μ l. and then add 1ml TRIZOL, room temperature is placed 5min, makes its abundant cracking;
3.12 the centrifugal 5min of 000rpm gets supernatant;
4. add 200 μ l chloroforms among every 1ml Trizol, room temperature is placed 15min behind the acute vibration mixing;
5.4 ℃ 12, the centrifugal 15min of 000g;
6. draw the upper strata water, to another centrifuge tube;
7. add 500 μ l Virahol mixings among every 1ml Trizol, room temperature is placed 5-10min;
8.4 ℃ 12, the centrifugal 10min of 000g abandons supernatant, RNA is sunken to the pipe end;
9. by adding 1ml 75% ethanol among every 1ml Trizol, gentle vibration centrifuge tube, suspension precipitation;
10.4 ℃ 8, the centrifugal 5min of 000g abandons supernatant as far as possible;
11. room temperature is dried or vacuum-drying 5-10min;
12. with 50ul RNase-free dH
2O dissolving RNA sample, 55-60 ℃, 5-10min;
13. survey the quantitative RNA concentration of OD value.
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-8 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the synthetic method of the first chain is with the synthetic method of cDNA the first chain among the embodiment 1.
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
For the PCA3 gene:
Primer (1) upstream 5 '-biotin AAGAAATAGCAAGTGCCGAGAAG-3 ' is shown in SEQ ID NO.9;
Downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3 ' is shown in SEQ ID NO.10;
Primer (3) upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ' is shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14;
Primer (5) upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT-3 ' is shown in SEQ ID NO.17;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.18;
More than corresponding one by one with PCA3 gene probe (1), (3), (5) of above-mentioned design for the designed primer sets of PCA3 gene (1), (3), (5), correspondence is combined as PSA primer/probe groups 1,3,5 (see Table 8 and table 9) respectively;
β-actin gene: upstream primer 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ', shown in SEQ ID NO.39; Downstream primer 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
2.2 multi-PRC reaction:
Method is with (three, 2.2) multi-PRC reaction method among the embodiment 1.
Four. the hybridization of oligonucleotide probe and PCR product
Method is with the hybridizing method of (four) oligonucleotide probe among the embodiment 1 and PCR product.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 8:
Table 8
Interpretation of result is as shown in table 9:
Table 9
The PCA3 that different primers/probe groups detects in 1-5 prostate cancer (PCa) blood samples of patients is all positive.
The PCA3 that different primers/probe groups detects in the 6-8 normal human blood is all negative.
Simultaneously, the fluorescence MFI value that the patient presents is compared with the fluorescence MFI value of confidential reference items β-actin gene, can be drawn the expression situation of PCA3 gene.
Embodiment 5: different primers and probe combinations detect the liquid-phase chip method of PSA gene in the blood
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PSA gene
1. according to following sequence synthetic oligonucleotide probe:
For the PSA gene:
Probe A:5 '-AminolinkerC12 CAGAATCACCCGAGCAGGTG-3 ' is shown in SEQ ID NO.6;
Probe C:5 '-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
2. will contain amido modified oligonucleotide probe respectively with the 3 kinds of carboxyl microballoons coupling that is numbered 11,13, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PSA, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 3 kinds of carboxyl microballoons of 11,13, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4-2.20 with the 2.4-2.20 among the embodiment 1, just with corresponding 3 pipes that reduce to of 11 pipes, other are constant.
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PSA probe A microballoon 11, PSA probe C microballoon 13, β-actin probe microballoon 66, equal proportion is mixed, and the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
The 1-5 clinical sample is prostate cancer (PCa) patient's venous blood, and the 6-8 clinical sample is normal people's venous blood, and the preparation method is with the preparation method of (two) sample among the embodiment 4.
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-8 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the synthetic method of the first chain is with the synthetic method of cDNA the first chain among the embodiment 1.
2. multiplex PCR
2.1 according to following sequence synthesized primer thing
For the PSA gene:
Primer A: upstream 5 '-biotin TTGACCCCAAAGAAACTTCAGTGT-3 ', shown in SEQ ID NO.29;
Downstream 5 '-TGCCCCATGACGTGATACCT-3 ' is shown in SEQ ID NO.30;
Primer C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34;
More than corresponding one by one with PSA probe A, the C of above-mentioned design for the designed primer sets A of PSA, C, correspondence is combined as PSA primer/probe groups 1,3 (see Table 10 and table 11) respectively;
β-actin gene:
Upstream primer 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ' is shown in SEQ ID NO.39;
Downstream primer 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40.
2.2 multi-PRC reaction:
Method is with (three, 2.2) multi-PRC reaction method among the embodiment 1.
Four. the hybridization of oligonucleotide probe and PCR product
Method is with the hybridizing method of (four) oligonucleotide probe among the embodiment 1 and PCR product.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 10:
Table 10
Interpretation of result is as shown in table 11:
Table 11
The PSA that different primers/probe groups detects in 1-5 prostate cancer (PCa) blood samples of patients is all positive.
The PSA that different primers/probe groups detects in the 6-8 normal human blood is all negative.
Simultaneously, the fluorescence MFI value that the patient presents is compared with the fluorescence MFI value of confidential reference items β-actin gene, can be drawn the expression situation of PSA gene.
Embodiment 6: the liquid-phase chip method of PCA3 gene, PSA gene and PCA3/PSA ratio in the joint-detection blood
Concrete detection method comprises the steps:
One. detect the preparation of the microballoon mixed solution of PSA gene
1. according to following sequence synthetic oligonucleotide probe:
PCA3:5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ' is shown in SEQ ID NO.1;
PSA:5 '-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
β-actin gene: 5 '-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
2. will contain amido modified oligonucleotide probe respectively with the 3 kinds of carboxyl microballoons coupling that is numbered 11,13, No. 66
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolves respectively the oligonucleotide probe of PCA3, PSA, β-actin, and concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 3 kinds of carboxyl microballoons of 11,13, No. 66 and stores at least 3min of suspensions respectively, produces the microballoon suspension of homogeneous;
2.4-2.20 with the 2.4-2.20 among the embodiment 1, just with corresponding 3 pipes that reduce to of 11 pipes, other are constant.
3. the preparation of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: be respectively PCA3 probe microballoon 11, PSA probe microballoon 13, β-actin probe microballoon 66, equal proportion is mixed, and the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
1-10 patient's clinical sample is prostate cancer (PCa) patient's venous blood, and 11-20 patient's clinical sample is benign prostatic hyperplasia (BPH) patient's venous blood, and the preparation method is with the preparation method of sample among the embodiment 4 (two).
Three. multiple RT-PCR
As follows the mRNA of above-mentioned 1-20 sample carried out the multiple reverse transcription pcr amplification:
1.cDNA the synthetic method of the first chain is with the synthetic method of cDNA the first chain among the embodiment 1.
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
PCA3: upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14
PSA: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34
β-actin gene:
Upstream 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ' is shown in SEQ ID NO.39;
Downstream 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
2.2 multi-PRC reaction:
Method is with (three, 2.2) multi-PRC reaction method among the embodiment 1.
Four. the hybridization of oligonucleotide probe and PCR product
Method is with the hybridizing method of (four) oligonucleotide probe among the embodiment 1 and PCR product.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as shown in table 12:
Table 12
Interpretation of result is as shown in table 13:
Table 13
PCA3/PSA mean value and analysis are such as table 14
Table 14
PCA3 and PSA in 1-10 prostate cancer (PCa) the group blood samples of patients are all positive; PCA3 in 11-20 benign prostatic hyperplasia (BPH) the group blood samples of patients is negative, and PSA is positive; Two groups of patient PCA3/PSA mean values differ 27 times.This method makes a distinction patients with prostate cancer and Benign Prostatic Hypertrophy well.
Simultaneously, the patient is presented positive fluorescence MFI value compare with the fluorescence MFI value of confidential reference items β-actin gene, can draw the expression situation of PCA3 gene and PSA gene.
About after the those set forth of the present invention, those skilled in the art can do various modifications or variation to the present invention more than having read, and these equivalent form of values belong to the scope defined in the application's appended claims equally.
Sequence table
＜110〉Shao's Chinese bush cherry
＜120〉a kind of Luminex and diagnostic kit thereof that detects PCA3 gene, PSA gene
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Claims (3)
1. diagnostic kit that detects PCA3 gene, PSA gene, it is characterized in that, the mixture of microspheres of the mRNA specific probe of the comprised covalent attachment mRNA of PCA3 gene and/or PSA gene and combine the microballoon of β-actin gene probe, for the upstream and downstream primer of the mRNA of the mRNA of PCA3 gene and/or PSA gene and upstream and downstream primer, SA-PE and the quality control product of β-actin gene;
The mRNA specific probe of the mRNA of described PCA3 gene and/or PSA gene adopts following sequence, and wherein 5 ' end contains amido modified:
MRNA for the PCA3 gene:
Probe (1): 5 '-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3 ', shown in SEQ ID NO.1;
Probe (2), (3), (5), (6) same probe (1) are shown in SEQ ID NO.1;
Probe (4): 5 '-Aminol inkerC12CTCACCTCTGTATCATCAG-3 ', shown in SEQ ID NO.2;
Probe (7): 5 '-AminolinkerC12 ATCTCTGTGCTTCCTTTTGT-3 ', shown in SEQ ID NO.3;
The same probe of probe (8) (7) is shown in SEQ ID NO.3;
Probe (9): 5 '-AminolinkerC12 CAAATCTGTAATCCCGTTCA-3 ', shown in SEQ ID NO.4;
Probe (10): 5 '-AminolinkerC12TATGTGTCAAGAGGAGAGCC-3 ' is shown in SEQ ID NO.5;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
MRNA for the PSA gene:
Probe A:5 '-Aminol inkerC12CAGAATCACCCGAGCAGGTG-3 ' is shown in SEQ ID NO.6;
Probe B:5 '-AminolinkerC12GGGGTCAAGAACTCCTCTGG-3 ' is shown in SEQ ID NO.7;
Probe C:5 '-AminolinkerC12CACGCTTTTGTTCCTGATGC-3 ' is shown in SEQ ID NO.8;
Probe D is with probe A, shown in SEQ ID NO.6;
Probe E is with probe B, shown in SEQ ID NO.7;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
Described for the PCA3 gene mRNA and/or the upstream and downstream primer of the mRNA of PSA gene, adopt following sequence, wherein upstream primer 5 ' end contains biotin label;
MRNA for the PCA3 gene:
Primer sets (1): upstream 5 '-biotin AAGAAATAGCAAGTGCCGAGAAG-3 ', shown in SEQ ID NO.9;
Downstream 5 '-GTGTGGCCTCAGATGGTAAAGTC-3 ' is shown in SEQ ID NO.10;
Primer sets (2): upstream 5 '-biotinTGGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.11;
Downstream 5 '-TCTCCCAGGGATCTCTGTGCTT-3 ' is shown in SEQ ID NO.12;
Primer sets (3): upstream 5 '-biotin GGTGGGAAGGACCTGATGATAC-3 ', shown in SEQ ID NO.13;
Downstream 5 '-TAAAGGGGCTGGAAATGTGC-3 ' is shown in SEQ ID NO.14;
Primer sets (4): upstream 5 '-biotin AGCCGAGGGAGACCAGGAAG-3 ', shown in SEQ ID NO.15;
Downstream 5 '-CAGCAGATGTGTGGCCTCAGAT-3 ' is shown in SEQ ID NO.16;
Primer sets (5): upstream 5 '-biotin CCGAGGGAGACCAGGAAGAT-3 ', shown in SEQ ID NO.17;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.18;
Primer sets (6): upstream 5 '-biotin AGAAATAGCAAGTGCCGAGAAGC-3 ', shown in SEQ ID NO.19;
Downstream 5 '-CACAGGGCGAGGCTCATC-3 ' is shown in SEQ ID NO.20;
Primer sets (7): upstream 5 '-biotin TATCCACACACACAGGAAGCAC-3 ', shown in SEQ ID NO.21;
Downstream 5 '-CCTCTCATTGGTAATGCTCACTTT-3 ' is shown in SEQ ID NO.22;
Primer sets (8): upstream 5 '-biotin AAGGCTGCTGACTTTACCATCTG-3 ', shown in SEQ ID NO.23;
Downstream 5 '-TCTAATGTCCTTCCCTCACAAGC-3 ' is shown in SEQ ID NO.24;
Primer sets (9): upstream 5 '-biotin CGCTTGTGAGGGAAGGACATTAG-3 ', shown in SEQ ID NO.25;
Downstream 5 '-GTGAAGCCATCAAGATTTTCTCGTC-3 ' is shown in SEQ ID NO.26;
Primer sets (10): upstream 5 '-biotin CAGCAGGACCCAACGCAT-3 ', shown in SEQ ID NO.27;
Downstream 5 '-GAGAGAGGATTGGTAAGCGATGTG-3 ' is shown in SEQ ID NO.28;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
MRNA for the PSA gene:
Primer sets A: upstream 5 '-biotin TTGACCCCAAAGAAACTTCAGTGT-3 ', shown in SEQ ID NO.29;
Downstream 5 '-TGCCCCATGACGTGATACCT-3 ' is shown in SEQ ID NO.30;
Primer sets B: upstream 5 '-biotin TCCCACACCCGCTCTACGAT-3 ', shown in SEQ ID NO.31;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.32;
Primer sets C: upstream 5 '-biotin TGCACCCCTCATCCTGTCTC-3 ', shown in SEQ ID NO.33;
Downstream 5 '-GCTGTGGCTGACCTGAAATACC-3 ' is shown in SEQ ID NO.34;
Primer sets D: upstream 5 '-biotin GGCAGCATTGAACCAGAGGAGT-3 ', shown in SEQ ID NO.35;
Downstream 5 '-CGATGGTGTCCTTGATCCACTT-3 ' is shown in SEQ ID NO.36;
Primer sets E: upstream 5 '-biotin TCCTCAGGCCAGGTGATGACT-3 ', shown in SEQ ID NO.37;
Downstream 5 '-CGTCCAGCACACAGCATGAACT-3 ' is shown in SEQ ID NO.38;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences;
More than corresponding one by one for the designed primer sets of PCA3 gene (1), (2), (3), (4), (5), (6), (7), (8), (9), (10) and PCA3 probe (1), (2), (3), (4), (5), (6), (7), (8), (9), (10);
More than corresponding one by one for the designed primer sets A of PSA gene, B, C, D, E and PSA probe A, B, C, D, E;
Described β-actin gene, its primer and probe sequence are as follows:
Upstream primer: 5 '-biotin TGGGTCAGAAGGATTCCTATGTG-3 ', shown in SEQ ID NO.39;
Downstream primer: 5 '-GCTGGGGTGTTGAAGGTCTC-3 ' is shown in SEQ ID NO.40;
Probe: 5 '-Aminol inkerC12TCATTGTAGAAGGTGTGGTG-3 ', shown in SEQ ID NO.41;
Perhaps with the complementary base sequences thereof of above-mentioned sequence.
2. the diagnostic kit of detection PCA3 gene as claimed in claim 1, PSA gene is characterized in that described mixture of microspheres is according to the needs independent assortment of different test sample.
3. the diagnostic kit of detection as claimed in claim 1 PCA3 gene, PSA gene, it is characterized in that, described quality control product comprises positive control and negative control, and wherein positive control is the mixed solution that contains PCA3 gene and/or PSA gene plasmid, comprises the plasmid that contains β-actin gene; Negative control is not for containing the plasmid solution of PCA3 gene, PSA gene and β-actin gene.
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| CN 201010509924 CN101942523B (en) | 2010-10-15 | 2010-10-15 | Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof |
| PCT/CN2011/079361 WO2012048607A1 (en) | 2010-10-15 | 2011-09-06 | Method for detecting pca3 and psa genes, and diagnostic kit thereof |
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| CN 201010509924 CN101942523B (en) | 2010-10-15 | 2010-10-15 | Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof |
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| CN101942523B (en) * | 2010-10-15 | 2013-04-03 | 邵棠 | Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof |
| CN103436594A (en) * | 2012-10-16 | 2013-12-11 | 科蒂亚(新乡)生物技术有限公司 | Application of BDNA for detecting PCA3 in urine |
| CN104164474A (en) * | 2013-05-17 | 2014-11-26 | 刘代新 | Fluorescent quantitative PCR method for detecting expression quantities of PCA3 gene and PSA gene in urine and diagnosis kit thereof |
| CN104878077A (en) * | 2014-02-28 | 2015-09-02 | 谭巍 | PCA3 mRNA/ACPP mRNA RT-PCR detection primer and detection kit thereof |
| CN103966352B (en) * | 2014-05-29 | 2015-07-29 | 苏州工业园区为真生物医药科技有限公司 | PCA3, CST1 and CST4 are at the application prepared in prostate cancer marker and test kit thereof |
| CN106290919B (en) * | 2016-08-31 | 2018-03-27 | 天津市泌尿外科研究所 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
| CN108441557A (en) * | 2018-01-31 | 2018-08-24 | 广州瑞博奥生物科技有限公司 | PCA3 and PSA gene detecting kits and its application |
| CN110951871B (en) * | 2019-11-21 | 2024-01-05 | 瑞博奥(广州)生物科技股份有限公司 | PCA3 and PSA RNA detection kit and amplification system |
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| BRPI0921043A2 (en) * | 2008-11-12 | 2018-08-07 | Caris Life Sciences Luxembourg Holdings | methods and systems for using exosomes to determine phenotypes |
| CN101942523B (en) * | 2010-10-15 | 2013-04-03 | 邵棠 | Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof |
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Non-Patent Citations (2)
| Title |
|---|
| 刘光香.前列腺癌患者PCA3 mRNA的表达及其临床意义.《临床泌尿外科杂志》.2008, * |
| 陈占国.PCA3(DD3)基因多态性与前列腺癌风险关系的研究.《中国卫生检验杂志》.2009, * |
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| CN101942523A (en) | 2011-01-12 |
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