CN110734978B - Application of DLX1, HOXC6 and PCA3 in preparation of prostate cancer markers and kit thereof - Google Patents

Abstract

The invention discloses application of DLX1, HOXC6 and PCA3 in preparation of a prostate cancer marker and a kit thereof. The kit uses a multiple qRT-PCR technology to detect DLX1/HOXC6/PCA3 mRNA in a urine sample simultaneously, and simultaneously uses PSA mRNA as a reference to establish a Logistic regression model as a prostate cancer early diagnosis marker. The kit achieves extremely high coincidence rate to the clinical pathological results of the prostate cancer through the joint detection of a plurality of markers.

Description

Application of DLX1, HOXC6 and PCA3 in preparation of prostate cancer markers and kit thereof

Technical Field

The invention relates to an application of DLX1, HOXC6 and PCA3 in preparation of a prostate cancer marker and a kit thereof, belonging to the technical field of biological medicine.

Background

Prostate Cancer (PCa) is a malignant tumor which seriously threatens male health, and occupies the second place of the global tumor incidence and the sixth place of mortality, and the incidence of PCa in China increases by 4.7% every year on average, which is the fastest-increasing malignant tumor of men. Data from Shanghai disease control centers show that prostate cancer incidence has been the leading cause of male genitourinary malignancies since 2002.

Prostate specific antigen (Prostate Specific Antigen, PSA) is the most widely used early diagnostic marker for prostate cancer worldwide, and elevated PSA suggests that patients may have prostate cancer, but elevated PSA is often caused by other prostate diseases. Taking the PSA gray area (4-10 ng/ml) as an example, these patients have a positive rate of penetration biopsy of only 10-25%. More than 75% of the daily dearth is counted as prostate needle biopsies, which can cause many complications to the patient, such as hematuria, sepsis, infection, etc., and can cause significant pain to the patient. Although PSA has a wide range of applications, there are also the above-mentioned major problems, and new detection targets are needed to compensate for the defects.

Although the correlation between the expression of some genes in urine and prostate cancer has been found in the prior art, there is still a need in the art to further research related genes which can be practically applied to diagnosis of prostate cancer and develop detection reagents with higher detection accuracy, and prostate cancer may be commonly affected by various gene expressions, so that multiple gene expression detection can be jointly performed for different stages of tumorigenesis and development, and the detection rate of tumors can be obviously improved.

Therefore, it is desirable to provide a kit for the joint detection of a plurality of prostate-associated gene expressions, which can detect a plurality of prostate-cancer-associated gene expressions in a joint manner, and can significantly improve the detection rate of tumors.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a combined detection kit for the expression of a plurality of prostate-related genes and application thereof, which can detect the expression of the prostate-related genes in a combined way, can obviously improve the detection rate of tumors, has ingenious design and convenient use, and is suitable for large-scale popularization and application.

In order to achieve the above purpose, the invention provides an application of DLX1, HOXC6 and PCA3 in preparing prostate cancer markers and a kit thereof. The kit can detect the expression of the prostate related genes in a combined way, can obviously improve the detection rate of tumors, has ingenious design and convenient use, and is suitable for large-scale popularization and application.

The combined application of DLX1, HOXC6 and PCA3 in the preparation of a prostate cancer marker, wherein the nucleotide sequence of DLX1 is shown as SEQ ID NO.1, the nucleotide sequence of HOXC6 is shown as SEQ ID NO.2, and the nucleotide sequence of PCA3 is shown as SEQ ID NO. 3.

Preferably, the determination formula for the combined application of DLX1, HOXC6 and PCA3 is p=1/[ 1+exp (-z) ], z=1.741×a+0.03×b+0.489×c-4.652, wherein a is a DLX1 score, B is a HOXC6 score, C is a PCA3 score, DLX1 score=dlx1 copy number/PSA copy number; HOXC6 score = HOXC6 copy number/PSA copy number; PCA3 score = PCA3 copy number/PSA copy number.

A kit for jointly detecting prostate cancer by DLX1, HOXC6 and PCA3, which comprises a quantitative detection reagent for DLX1mRNA, HOXC6mRNA and PCA3 mRNA; the DLX1mRNA quantitative detection reagent comprises detection primers shown in SEQ ID NO.9 and SEQ ID NO.10 and a TaqMAN probe with a nucleotide sequence shown in SEQ ID NO. 11; the HOXC6mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.12 and SEQ ID NO.13 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 14; the PCA3 mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.15 and SEQ ID NO.16 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 17.

Preferably, the kit further comprises a PSA mRNA quantitative detection reagent, wherein the PSA mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.18 and SEQ ID NO.19 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 20.

Preferably, the kit also comprises a one-step RT-PCR reaction solution, comprising a SuperScript IV one-step RT-PCR system and Mg ²⁺ And water.

Preferably, the kit further comprises a standard substance, a negative quality control substance and a positive quality control substance; the standard substance comprises a DLX1 gene recombinant plasmid shown as SEQ ID NO.5, a HOXC6 gene recombinant plasmid shown as SEQ ID NO.6, a PCA3 gene recombinant plasmid shown as SEQ ID NO.7 and a PSA gene recombinant plasmid shown as SEQ ID NO. 8; the positive quality control product is cDNA after reverse transcription of total LNcap RNA of a prostate cancer cell strain, and the negative quality control product is cDNA after reverse transcription of total RWPE-1 RNA of a normal prostate epithelial cell strain.

The invention has the beneficial effects that:

the kit for jointly detecting the expression of the multiple prostate-related genes and the application thereof can jointly detect the expression of the prostate-related genes, can obviously improve the detection rate of tumors, has ingenious design and convenient use, and is suitable for large-scale popularization and application.

Drawings

FIG. 1 is a graph of amplification of a standard sample in the examples;

FIG. 2 is a standard graph in an embodiment;

FIG. 3 is a graph of PCA3 detection ROC;

FIG. 4 is a graph of DLX1+PCA3 detection ROC;

FIG. 5 is a graph of HOXC6+PCA3 detection ROC;

FIG. 6 is a graph of DLX1+HOXC6 detection ROC;

FIG. 7 is a graph of a DLX1+HOXC6+PCA3 joint detection ROC.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

A kit for preparing a marker of prostate cancer of DLX1, HOXC6 and PCA3, comprising quantitative detection reagents for DLX1mRNA, HOXC6mRNA and PCA3 mRNA; the DLX1mRNA quantitative detection reagent comprises detection primers shown in SEQ ID NO.9 and SEQ ID NO.10 and a TaqMAN probe with a nucleotide sequence shown in SEQ ID NO. 11; the HOXC6mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.12 and SEQ ID NO.13 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 14; the PCA3 mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.15 and SEQ ID NO.16 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 17. Because the reaction system of the kit is a quadruple fluorescent RT-PCR reaction system, the probe aiming at each gene is connected with different fluorescent groups. DLX1 probe labeled FAM, HOXC6 probe labeled VIC, PCA3 probe labeled ROX, PSA probe labeled Cy5.

Since PSA is a prostate-specific antigen, PSA gene expression in urine is derived from the prostate, and thus PSA gene (SEQ ID NO. 4) in urine can be used as an internal gene for the amount of prostate-derived RNA. The kit also comprises a PSA mRNA quantitative detection reagent, wherein the PSA mRNA quantitative detection reagent comprises a detection primer shown as SEQ ID NO.18 and SEQ ID NO.19 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 20.

The kit also comprises one-step RT-PCR reaction liquid, and a SuperScript IV one-step RT-PCR system, mg ²⁺ And water. The kit also comprises a standard substance, a negative quality control substance and a positive quality control substance. The standard substances comprise DLX1 gene recombinant plasmid shown as SEQ ID NO.5, HOXC6 gene recombinant plasmid shown as SEQ ID NO.6, PCA3 gene recombinant plasmid shown as SEQ ID NO.7 and PSA gene recombinant plasmid shown as SEQ ID NO. 8; the yang-property controlling substance is the prostateThe cDNA after reverse transcription of the LNcap total RNA of the adenocarcinoma cell line is the cDNA after reverse transcription of the RWPE-1 total RNA of the normal prostatic epithelial cell line as a negative quality control product.

The invention also provides a method for carrying out the combined detection of the expression of the plurality of prostate-related genes by using the combined detection kit for the expression of the plurality of prostate-related genes, which comprises the following steps: (1) RNA extraction of the sample to be tested using Trizol reagent. (2) And detecting the extracted RNA by using the combined detection kit for the expression of the multiple prostate-related genes to obtain the expression levels of DLX1, HOXC6 and PCA3, thereby evaluating the risk of the prostate cancer.

Example 1:

design and screening of primers and probes

12 groups of forward primers, reverse primers and fluorescent probes are designed according to DLX1, HOXC6, PCA3 and PSA cDNA sequences (SEQ ID NO.1-SEQ ID NO. 4), PCR amplification is carried out by using recombinant plasmids prepared by DLX1, HOXC6, PCA3 and PSA mRNA sequences as templates, sequencing analysis is carried out on amplified products, the reliability of the designed 12 groups of primers and probes is verified, and primers and probes with low amplification efficiency and unspecific amplified products are eliminated. After primary screening, 8 groups of forward primers, reverse primers and fluorescent probes are reserved. And then extracting total RNA of the urine sample, reversely transcribing the total RNA into cDNA, and respectively carrying out PCR amplification by using the 6 groups of forward primers, reverse primers and fluorescent probes after the primary screening, wherein the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 10 seconds, annealing/extension at 60℃for 30 seconds, 45 cycles, fluorescence signal detection at the end of extension of each cycle, screening according to the principle of minimum Ct value and maximum fluorescence signal amplification. Through the test process, the forward primer, the reverse primer and the probe sequences (see SEQ ID NO.9-SEQ ID NO. 20) obtained through screening and optimization are obtained.

Example 2, detection System optimization

The purpose is as follows: the concentration of a primer probe (see SEQ ID NO.9-SEQ ID NO. 20) in the reaction system, the selection of an enzyme reaction solution, dNTPs and Mg2+ ion concentration are optimized, so that the PCR reaction system is optimized, and the detection sensitivity and specificity are improved.

1. Primer probe proportional screening

The RT-PCR reactions of four genes are carried out in one reaction tube at the same time, and the PCR effect of other genes is inevitably influenced by each primer probe, so that the concentration of each primer probe is necessary to be screened. We set the amount and ratio of initial primer probes to 0.1uM:0.05uM. The other three primers were kept unchanged at each optimization, and cross-validation was performed sequentially by changing the concentration of only one probe.

The results of the crossover experiments showed that the amounts and ratios of DLX1, HOXC6, PCA3 and PSA primers and probes were 0.3uM, respectively: 0.15uM,0.2uM:0.1uM,0.2uM:0.1uM,0.06uM: a more desirable multiplex amplification effect can be achieved at 0.03. Mu.M.

2. Selection of real-time quantitative PCR reaction premix

For convenient clinical application, the use of one-step RT-PCR reagents is a good choice. The same sample is used for parallel comparison under the same reaction conditions by using a one-step RT-PCR reagent of a plurality of factories for comparing the difference of the fluorescence quantitative reaction premix produced by different factories. The results show that: the SuperScript IV one-step RT-PCR system has the optimal effect, so the SuperScript IV one-step RT-PCR system is selected as a premix for fluorescent quantitative reaction.

3、Mg ²⁺ Ion concentration optimization

Optimizing the concentration of Mg2+ ions in the system, and adding 0mM,1mM,2mM and 5mM of Mg into the SuperScript IV one-step RT-PCR system ²⁺ Ions, using the same sample, were compared in parallel under the same reaction conditions. The results show that: 1mM additional Mg was added ²⁺ The ions have optimal amplification efficiency.

4. dNTP concentration optimization

dNTP concentration in the system is optimized, dNTPs are carried by the SuperScript IV one-step RT-PCR system, 0mM,0.1mM,0.2mM and 0.5mM of dNTPs are additionally added, and parallel comparison is carried out by using the same sample and the same reaction condition. The results show that: the optimal amplification efficiency can be achieved without the addition of dNTPs.

5. Optimization of annealing temperature

According to the Tm value of the primer, the annealing temperature was sequentially increased by 58 ℃, 59 ℃,60 ℃, 61 ℃, 62 ℃, and the annealing temperature was optimized, and the optimal annealing temperature was selected for the subsequent test.

The optimum annealing temperature is preferably 60℃in combination with the efficiency of the PCR amplification reaction and the specificity of the amplification.

6. Optimization of annealing time

And under the condition that the annealing temperature is 60 ℃, the annealing time is respectively set to be 30s, 31s, 32s, 33s and 34s, the annealing time is optimized, and the experimental comparison shows that the finally determined optimal annealing time is 30 seconds.

Example 3 sensitivity experiment

DLX1, HOXC6, PCA3 and PSA cDNA recombinant plasmids were designed according to SEQ ID No.5-SEQ ID No.8 and subjected to gradient dilution respectively, and the concentrations of the recombinant plasmids were 100,000 copies/microliter, 10,000 copies/microliter, 1,000 copies/microliter, 100 copies/microliter, 10 copies/microliter and 1 copy/microliter respectively at 6 dilution points.

PCR detection was performed using the primer probe obtained in example 1 and the reaction conditions in example 2: 95 ℃ for 3 minutes; 95 ℃,10 seconds, 60 ℃,30 seconds, 45 cycles. The amplification curve is shown in FIG. 1, and then a standard curve is drawn according to the detection result, and the result is shown in FIG. 2. The results show that this multiplex fluorescent RT-PCR consisting of DXL1, HOXC6, PCA3 and PSA has very high sensitivity.

Example 4 kit for detection of prostate cancer

Providing a kit comprising: primer probe mixed solution: the primer pair of the 0.3uM DLX1 gene (SEQ ID NO.9, SEQ ID NO. 10), the detection probe of the 0.15uM DLX1 gene (SEQ ID NO. 11), the primer pair of the 0.2uM HOXC6 gene (SEQ ID NO.12, SEQ ID NO. 13), the detection probe of the 0.1uM HOXC6 gene (SEQ ID NO. 14), the primer pair of the 0.2uM PCA3 gene (SEQ ID NO.15, SEQ ID NO. 16), the detection probe of the 0.1uM PCA3 gene (SEQ ID NO. 17), the primer pair of the 0.06uM PSA gene (SEQ ID NO.18, SEQ ID NO. 19), the detection probe of the 0.03uM PSA gene (SEQ ID NO. 20).

PCR reaction solution: superScript IV one-step RT-PCR system, 1mM Mg ²⁺

Controlling the nature of yang: cDNA after reverse transcription of LNcap total RNA of a prostate cancer cell strain;

negative control: cDNA after reverse transcription of RWPE-1 total RNA of normal prostate epithelial cell strain.

Example 5 clinical sample experiment

206 cases of urine of patients who were about to undergo prostate puncture were collected from urology surgery in a drummer hospital affiliated with the university of south Beijing, and used for implementation. Meanwhile, a standard curve was drawn according to example 4, and the expression amounts of the sample DLX1, HOXC6, PCA3 and PSA genes were absolutely quantified, and a DLX1 score, a HOXC6 score and a PCA3 score were calculated, respectively. Wherein DLX1 score = DLX1 copy number/PSA copy number; HOXC6 score = HOXC6 copy number/PSA copy number; PCA3 score = PCA3 copy number/PSA copy number. The prostate puncture results were used as gold standards (83 cases of prostate cancer and 123 cases of non-prostate cancer in 206 cases of samples), and the Logistic regression model was used to calculate the joint judgment formulas of the genes. In this example, the determination formula for the joint application of DLX1 and HOXC6 is p=1/[ 1+exp (-z) ], z=1.19a+0.137 b-2.192. The judgment formula of the combined application of DLX1 and PCA3 is p=1/[ 1+exp (-z) ], and z=1.783×A+0.527×C-4.694. The combined application of HOXC6 and PCA3 has a judgment formula of p=1/[ 1+exp (-z) ], and z=0.136×b+0.242×c-2.991. The judgment formula of the combined application of DLX1, HOXC6 and PCA3 is p=1/[ 1+exp (-z) ], and z=1.741 x A+0.03 x B+0.489 x C-4.652, wherein A is a DLX1 score, B is a HOXC6 score, C is a PCA3 score, and p values are used for respectively drawing a multi-gene combined detection ROC curve of DLX1+ HOXC6, DLX1+ PCA3, HOXC6+ PCA3 and DLX1+ HOXC6+ PCA 3. The results are shown in fig. 3-7, and the detection performance of the combined detection system is as follows: dlx1+hoxc6+pca3> dlx1+hoxc6> pca3+hoxc6> dlx1+pca3> pca3, AUC values are respectively: 0.8607, 0.7443, 0.7087, 0.7066, 0.6314. According to the ROC curve, when the sensitivity is about 90%, the data of the specificity, the negative predictive value, the positive predictive value and the like of each detection system are compared, and the following table 1 is summarized:

cut-off

AUC

sensitivity of

Specificity (specificity)

NPV

PPV

PCA3 scoring

40

0.6314

84％

23％

87％

21％

p-value (DLX 1+ PCA 3)

0.32

0.7066

90％

35％

89％

36％

p-value (PCA3+HOXC6)

0.24

0.7087

91％

38％

90％

34％

p-value (DLX1+HOXC 6)

0.31

0.7443

90％

42％

90％

41％

p-value (DLX1+HOXC6+PCA3)

0.29

0.8607

94％

83％

93％

78％

TABLE 1

The result shows that the sensitivity of the kit for detecting clinical samples can reach 94 percent (78/83), the specificity (102/123) and the total coincidence rate can reach 87 percent, and compared with other molecular markers, the kit has more excellent detection capability and can obviously improve the detection rate of the prostate cancer.

In conclusion, the kit for jointly detecting the expression of the multiple prostate-related genes and the application thereof can jointly detect the expression of the prostate-related genes, can obviously improve the detection rate of tumors, has ingenious design and convenient use, and is suitable for large-scale popularization and application.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Sequence listing

<110> Hangzhou Yiding biotechnology Co Ltd

Application of <120> DLX1, HOXC6 and PCA3 in preparation of prostate cancer markers and kit thereof

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ccctcccctt tgtttgattt tttttccagt ataaagttaa aatgcttagc cttgtactga 1260

ggctgtatac agccacagcc tctccccatc cctccagcct tatctgtcat caccatcaac 1320

ccctcccatg cacctaaaca aaatctaact tgtaattcct tgaacatgtc aggcatacat 1380

tattccttct gcctgagaag ctcttccttg tctcttaaat ctagaatgat gtaaagtttt 1440

gaataagttg actatcttac ttcatgcaaa gaagggacac atatgagatt catcatcaca 1500

tgagacagca aatactaaaa gtgtaatttg attataagag tttagataaa tatatgaaat 1560

gcaagagcca cagagggaat gtttatgggg cacgtttgta agcctgggat gtgaagcaaa 1620

ggcagggaac ctcatagtat cttatataat atacttcatt tctctatctc tatcacaata 1680

tccaacaagc ttttcacaga attcatgcag tgcaaatccc caaaggtaac ctttatccat 1740

ttcatggtga gtgcgcttta gaattttggc aaatcatact ggtcacttat ctcaactttg 1800

agatgtgttt gtccttgtag ttaattgaaa gaaatagggc actcttgtga gccactttag 1860

ggttcactcc tggcaataaa gaatttacaa agagctactc aggaccagtt gttaagagct 1920

ctgtgtgtgt gtgtgtgtgt gtgagtgtac atgccaaagt gtgcctctct ctctttgacc 1980

cattatttca gacttaaaaa caagcatgtt ttcaaatggc actatgagct gccaatgatg 2040

tatcaccacc atatctcatt attctccagt aaatgtgata ataatgtcat ctgttaacat 2100

aaaaaaagtt tgacttcaca aaagcagctg gaaatggaca accacaatat gcataaatct 2160

aactcctacc atcagctaca cactgcttga catatattgt tagaagcacc tcgcatttgt 2220

gggttctctt aagcaaaata cttgcattag gtctcagctg gggctgtgca tcaggcggtt 2280

tgagaaatat tcaattctca gcagaagcca gaatttgaat tccctcatct tttaggaatc 2340

atttaccagg tttggagagg attcagacag ctcaggtgct ttcactaatg tctctgaact 2400

tctgtccctc tttgtgttca tggatagtcc aataaataat gttatctttg aactgatgct 2460

cataggagag aatataagaa ctctgagtga tatcaacatt agggattcaa agaaatatta 2520

gatttaagct cacactggtc aaaaggaacc aagatacaaa gaactctgag ctgtcatcgt 2580

ccccatctct gtgagccaca accaacagca ggacccaacg catgtctgag atccttaaat 2640

caaggaaacc agtgtcatga gttgaattct cctattatgg atgctagctt ctggccatct 2700

ctggctctcc tcttgacaca tattagcttc tagcctttgc ttccacgact tttatctttt 2760

ctccaacaca tcgcttacca atcctctctc tgctctgttg ctttggactt ccccacaaga 2820

atttcaacga ctctcaagtc ttttcttcca tccccaccac taacctgaat gcctagaccc 2880

ttatttttat taatttccaa tagatgctgc ctatgggcta tattgcttta gatgaacatt 2940

agatatttaa agctcaagag gttcaaaatc caactcatta tcttctcttt ctttcacctc 3000

cctgctcctc tccctatatt actgattgca ctgaacagca tggtccccaa tgtagccatg 3060

caaatgagaa acccagtggc tccttgtggt acatgcatgc aagactgctg aagccagaag 3120

gatgactgat tacgcctcat gggtggaggg gaccactcct gggccttcgt gattgtcagg 3180

agcaagacct gagatgctcc ctgccttcag tgtcctctgc atctcccctt tctaatgaag 3240

atccatagaa tttgctacat ttgagaattc caattaggaa ctcacatgtt ttatctgccc 3300

tatcaatttt ttaaacttgc tgaaaattaa gttttttcaa aatctgtcct tgtaaattac 3360

tttttcttac agtgtcttgg catactatat caactttgat tctttgttac aacttttctt 3420

actcttttat caccaaagtg gcttttattc tctttattat tattattttc ttttactact 3480

atattacgtt gttattattt tgttctctat agtatcaatt tatttgattt agtttcaatt 3540

tatttttatt gctgactttt aaaataagtg attcgggggg tgggagaaca ggggagggag 3600

agcattagga caaataccta atgcatgtgg gacttaaaac ctagatgatg ggttgatagg 3660

tgcagcaaac cactatggca cacgtatacc tgtgtaacaa acctacacat tctgcacatg 3720

tatcccagaa cgtaaagtaa aatttaaaaa aaagtgaaaa aaaaaaaaaa 3770

<210> 4

<211> 1483

<212> DNA

<213> human (homosapiens)

<400> 4

agccccaagc ttaccacctg cacccggaga gctgtgtcac catgtgggtc ccggttgtct 60

tcctcaccct gtccgtgacg tggattggtg ctgcacccct catcctgtct cggattgtgg 120

gaggctggga gtgcgagaag cattcccaac cctggcaggt gcttgtggcc tctcgtggca 180

gggcagtctg cggcggtgtt ctggtgcacc cccagtgggt cctcacagct gcccactgca 240

tcaggaacaa aagcgtgatc ttgctgggtc ggcacagcct gtttcatcct gaagacacag 300

gccaggtatt tcaggtcagc cacagcttcc cacacccgct ctacgatatg agcctcctga 360

agaatcgatt cctcaggcca ggtgatgact ccagccacga cctcatgctg ctccgcctgt 420

cagagcctgc cgagctcacg gatgctgtga aggtcatgga cctgcccacc caggagccag 480

cactggggac cacctgctac gcctcaggct ggggcagcat tgaaccagag gagttcttga 540

ccccaaagaa acttcagtgt gtggacctcc atgttatttc caatgacgtg tgtgcgcaag 600

ttcaccctca gaaggtgacc aagttcatgc tgtgtgctgg acgctggaca gggggcaaaa 660

gcacctgctc gggtgattct gggggcccac ttgtctgtaa tggtgtgctt caaggtatca 720

cgtcatgggg cagtgaacca tgtgccctgc ccgaaaggcc ttccctgtac accaaggtgg 780

tgcattaccg gaagtggatc aaggacacca tcgtggccaa cccctgagca cccctatcaa 840

ccccctattg tagtaaactt ggaaccttgg aaatgaccag gccaagactc aagcctcccc 900

agttctactg acctttgtcc ttaggtgtga ggtccagggt tgctaggaaa agaaatcagc 960

agacacaggt gtagaccaga gtgtttctta aatggtgtaa ttttgtcctc tctgtgtcct 1020

ggggaatact ggccatgcct ggagacatat cactcaattt ctctgaggac acagatagga 1080

tggggtgtct gtgttatttg tggggtacag agatgaaaga ggggtgggat ccacactgag 1140

agagtggaga gtgacatgtg ctggacactg tccatgaagc actgagcaga agctggaggc 1200

acaacgcacc agacactcac agcaaggatg gagctgaaaa cataacccac tctgtcctgg 1260

aggcactggg aagcctagag aaggctgtga gccaaggagg gagggtcttc ctttggcatg 1320

ggatggggat gaagtaagga gagggactgg accccctgga agctgattca ctatgggggg 1380

aggtgtattg aagtcctcca gacaaccctc agatttgatg atttcctagt agaactcaca 1440

gaaataaaga gctgttatac tgtgaaaaaa aaaaaaaaaa aaa 1483

<210> 5

<211> 173

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gagggaagaa ggaacagtgg aggcgggacg ccctccatct cctcggagcc ccgcgaggtc 60

cggcccagca acttcccggc atccgcgctc tagcctgaac cctggcctgg gccgagcagt 120

ggcagcagag agtggcctcg gagggaagcc actgccacct gagacagccc aag 173

<210> 6

<211> 222

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gttccagaac cgccggatga agtggaaaaa agaatctaat ctcacatcca ctctctcggg 60

gggcggcgga ggggccaccg ccgacagcct gggcggaaaa gaggaaaagc gggaagagac 120

agaagaggag aagcagaaag agtgaccagg actgtccctg ccacccctct ctccctttct 180

ccctcgctcc ccaccaactc tcccctaatc acacactctg ta 222

<210> 7

<211> 245

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

acaaattact ctggttgctg tgttgatagg acagcagaag ggtaggagca gggacaccag 60

ttaagttatt gcaatggtta aggtgagagg tggtggctgg gcctaggcct ttttggagtg 120

aacaagctat ttgcttggct tccatgatcc ttctccctcc actggaatgg aaggtacttg 180

atattagaga tttttgtctg tttcatcact actgtaaccc caatgtctac aacagactct 240

ggcac 245

<210> 8

<211> 136

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tctgcggcgg tgttctggtg cacccccagt gggtcctcac agctgcccac tgcatcagga 60

acaaaagcgt gatcttgctg ggtcggcaca gcctgtttca tcctgaagac acaggccagg 120

tatttcaggt cagcca 136

<210> 9

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gagggaagaa ggaaca 16

<210> 10

<211> 15

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cttgggctgt ctcag 15

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cggcatccgc gctctagcct 20

<210> 12

<211> 14

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gttccagaac cgcc 14

<210> 13

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tacagagtgt gtgattagg 19

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gggccaccgc cgacagcc 18

<210> 15

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

acaaattact ctggttg 17

<210> 16

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gtgccagagt ctgttgt 17

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ccatgatcct tctccctcca c 21

<210> 18

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

tctgcggcgg tgttct 16

<210> 19

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tggctgacct gaaata 16

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gcccactgca tcaggaaca 19

Claims (5)

1. The application of DLX1, HOXC6 and PCA3 quantitative detection reagents in preparing a combined diagnosis kit for prostate cancer is disclosed, wherein the nucleotide sequence of DLX1 is shown as SEQ ID NO.1, the nucleotide sequence of HOXC6 is shown as SEQ ID NO.2, and the nucleotide sequence of PCA3 is shown as SEQ ID NO. 3.
2. 2. Use of a quantitative detection reagent for DLX1, HOXC6 and PCA3 according to claim 1 for the preparation of a kit for the combined diagnosis of prostate cancer, characterized in that said kit comprises quantitative detection reagents for DLX1mRNA, HOXC6mRNA and PCA3 mRNA; the DLX1mRNA quantitative detection reagent comprises detection primers shown in SEQ ID NO.9 and SEQ ID NO.10 and a TaqMAN probe with a nucleotide sequence shown in SEQ ID NO. 11; the HOXC6mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.12 and SEQ ID NO.13 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 14; the PCA3 mRNA quantitative detection reagent comprises detection primers shown as SEQ ID NO.15 and SEQ ID NO.16 and a TaqMAN probe with a nucleotide sequence shown as SEQ ID NO. 17.
3. 3. The use of the quantitative detection reagent of DLX1, HOXC6 and PCA3 according to claim 2 for preparing a kit for combined diagnosis of prostate cancer, wherein the kit further comprises a PSA mRNA quantitative detection reagent comprising a detection primer as shown in SEQ ID No.18 and SEQ ID No.19 and a TaqMAN probe having a nucleotide sequence as shown in SEQ ID No. 20;

   the judging formula of the combined application of DLX1, HOXC6 and PCA3 is p=1/[ 1+exp (-z) ], and z=1.741 x A+0.03 x B+0.489 x C-4.652, wherein A is a DLX1 score, B is a HOXC6 score, C is a PCA3 score, and DLX1 score=DLX 1 copy number/PSA copy number; HOXC6 score = HOXC6 copy number/PSA copy number; PCA3 score = PCA3 copy number/PSA copy number.
4. 4. Use of a quantitative detection reagent for DLX1, HOXC6 and PCA3 according to claim 1 for the preparation of a kit for the combined diagnosis of prostate cancer, characterized in that: the kit also comprises a one-step RT-PCR reaction solution, a SuperScript IV one-step RT-PCR system and Mg ²⁺ And water.
5. 5. Use of a quantitative detection reagent for DLX1, HOXC6 and PCA3 according to claim 1 for the preparation of a kit for the combined diagnosis of prostate cancer, characterized in that: the kit also comprises a standard substance, a negative quality control substance and a positive quality control substance; the standard substance comprises a DLX1 gene recombinant plasmid shown as SEQ ID NO.5, a HOXC6 gene recombinant plasmid shown as SEQ ID NO.6, a PCA3 gene recombinant plasmid shown as SEQ ID NO.7 and a PSA gene recombinant plasmid shown as SEQ ID NO. 8; the positive quality control product is cDNA after reverse transcription of total LNcap RNA of a prostate cancer cell strain, and the negative quality control product is cDNA after reverse transcription of total RWPE-1 RNA of a normal prostate epithelial cell strain.

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