CN113528660A - Risk assessment device for prostate cancer - Google Patents
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Abstract
A risk assessment device for prostate cancer, comprising a collection device; a nucleic acid extraction device; the data acquisition and analysis device is used for quantitatively detecting the expression quantities of a PCA3 gene, an ERG gene and an SPDEF gene in a sample RNA solution and calculating the expression quantities of the PCA3 gene and the ERG gene relative to the SPDEF gene, wherein the delta CT of the PCA3 gene and the ERG gene is calculated by recording the Ct values of the PCA3, the ERG and the SPDEF, the sum of the relative expression quantities of the PCA3 gene and the ERG gene is calculated by using a calculation formula of the relative expression quantities, namely delta CT (PCA3) +2 delta Ct (ERG), and then a score is calculated by using a formula (16.92-delta Ct) × 1.83, wherein the score can reflect the risk level of the occurrence of the prostate cancer.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a risk assessment device for prostate cancer.
Background
Globally, prostate cancer is the second most prevalent malignancy among men than lung cancer. In recent years, with the aging population, the change of diet and life style, the improvement of diagnosis and treatment technology and the enhancement of health concept, the incidence rate and the diagnosis rate of prostate cancer have increased remarkably. Early diagnosis is an extremely important ring for prostate cancer, but relevant diagnostic tools for distinguishing high-grade prostate cancer from low-grade prostate cancer have been lacking. Prostate Specific Antigen (PSA) screening is currently the most common test for determining whether a prostate biopsy should be performed in men, but it is highly false positive and false negative, and therefore does not accurately predict the presence of an aggressive disease. However, most men with elevated PSA levels will undergo an immediate tissue biopsy, even though many of them have only low grade prostate cancer or none at all. Overtreatment often leads to complications such as infection, sepsis and even death, and unnecessary invasive treatment also has side effects such as impotence and urinary incontinence, and the psychological burden on patients is greatly increased. A negative result on prostate puncture does not completely exclude prostate cancer, and only 25% of men with prostate biopsies have high grade prostate cancer.
Therefore, there is a need for a non-invasive means based liquid biopsy test that accurately predicts the aggressiveness of prostate cancer and avoids unnecessary biopsies for low risk or benign patients.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a risk assessment device for prostate cancer.
The invention provides a risk assessment device for prostate cancer, which comprises a collecting device, a nucleic acid acquiring device, an amplifying device and a data acquiring and analyzing device.
The collecting device is used for collecting the exosome solution of the testee.
The nucleic acid acquisition device is used for extracting RNA of the exosome solution to obtain a sample RNA solution.
The amplification device is used for quantitatively detecting the target region in the sample RNA solution, wherein the target region comprises PCA3 gene, ERG gene and SPDEF gene.
The data acquisition and analysis device is used for acquiring the expression levels of the PCA3 gene, the ERG gene and the SPDEF gene and calculating the expression levels of the PCA3 gene and the ERG gene relative to the SPDEF gene.
The data acquisition and analysis device normalizes the expression levels of the PCA3 gene and the ERG gene to the SPDEF gene by recording the Ct values of the PCA3 gene, the ERG gene and the SPDEF gene, respectively calculates the delta CT values of the PCA3 gene and the ERG gene, calculates the sum of the relative expression amounts of the PCA3 gene and the ERG gene by using a calculation formula of the relative expression amounts, namely delta CT (2 ^ delta Ct (PCA3) +2^ delta Ct (ERG)), and then uses the formula: (16.92- Δ Δ Ct) × 1.83 and a score was calculated which reflects the risk level for prostate cancer.
In an embodiment of the present application, the amplification device comprises a qRT-PCR amplification system.
In an embodiment of the present application, the method for extracting the sample RNA solution in the exosome solution by the nucleic acid extraction device comprises:
adding 600-800 mu l of Trizol solution into the exosome solution, incubating and homogenizing at 18-25 ℃ for 3-5 min, adding 100-220 mu l of chloroform, shaking and uniformly mixing for 10-20 s, centrifuging at 2-6 ℃ and 10000-12000 rpm for 10-15 min, taking the upper clear solution, and carrying out RNA purification on the upper clear solution to obtain a sample RNA solution.
In an embodiment of the present application, the method for amplifying the sample RNA solution by the amplification device comprises:
adding the sample RNA solution into a qRT-PCR amplification system, firstly amplifying for 10-30 min at the temperature of 30-50 ℃, and then amplifying for 2-10 min at the temperature of 90-95 ℃.
Further amplifying for 10-30 s at 90-95 ℃, and finally amplifying for 30-60 s at 55-65 ℃, wherein the step is circulated for 30-50 times to obtain a PCR product.
Wherein, the fluorescence signal is collected in the step of amplifying at 55-65 ℃.
In the embodiment of the application, the qRT-PCR amplification system comprises a primer mixed solution, a PCR buffer solution, a PCR reaction solution and an enzyme mixed solution.
In an embodiment of the present application, the primer mixture includes, by concentration: 100nM to 500nM ERG F, 100nM to 500nM ERG R, 100nM to 500nM ERG Pb, 100nM to 500nM SPDEF R, 100nM to 500nM SPDEF Pb, 100nM to 500nM PCA 3F, 100nM to 500nM PCA 3R, 100nM to 500nM PCA3 Pb, 0.1 to 10ROX and the balance water.
In an embodiment of the present application, the PCR buffer comprises a Tris-HCl solution having a pH of 7.5 to 9.0 at a concentration of 50mM to 800mM, KCl at a concentration of 50mM to 800mM, ammonium sulfate at a concentration of 50mM to 500mM, and the balance water.
In the embodiment of the present application, the PCR reaction solution comprises 1-10 mM PCR buffer solution and 1 mM-6 mM MgCl20.5 to 5 weight percent of glycerin, 0.1 to 1PC300 and the balance of water.
In the embodiment of the application, the enzyme mixture comprises Taq-HS with the concentration of 1U-10U, RTase with the concentration of 1U-50U, Taq Buffer, 0.1 mM-1 mM dNTPs, 0.1-10 PC300 and the balance of water.
In the embodiment of the application, the collecting device is further used for carrying out pretreatment on the exosome solution, and the pretreatment comprises purification of the exosome solution.
Compared with the prior art, the invention has the beneficial effects that: by directly extracting exosomes from human urine, the urine does not need DRE pre-collection or special treatment; by measuring the contents of three preferred targets PCA3 gene, ERG gene and SPDEF gene in exosome, the relative expression quantity of PCA3 gene and ERG gene is calculated, invasive means is not needed, preliminary biopsy and non-invasive liquid biopsy are avoided for men suffering from negative and/or inert prostate cancer, only urine samples are collected, and high compliance is achieved; the detection can be finished by using a common fluorescent quantitative PCR instrument without purchasing additional equipment; the detection is carried out by multiple one-step methods, the method is simple and quick, and the closed-tube operation is good to avoid pollution.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The names of technical means used in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.
The embodiments described below and the features of the embodiments can be combined with each other without conflict.
The invention provides a risk assessment device for prostate cancer, which comprises a collecting device, a nucleic acid acquiring device, an amplifying device and a data acquiring and analyzing device.
The collecting device is used for collecting exosome solution of a tester.
In the embodiment, 5-50 mL of urine of a tester is collected, an exosome solution in the urine is extracted by an ultrafiltration method, and the aperture of a filter membrane is 20 nm. In addition to ultrafiltration, the exosome solution may be purified and concentrated by ultracentrifugation, membrane affinity, polymer sedimentation, exclusion chromatography, immunocapture, and the like. Finally obtaining 100-500 mul of purified exosome solution.
The nucleic acid acquisition device is used for extracting RNA of the exosome solution to obtain a sample RNA solution.
In this embodiment, all exosome RNAs from which ribosomal RNAs were removed were extracted from purified exosomes using miRNeasy mini kit (Qiagen).
The method comprises the following specific steps: adding 200-1000 mul of Trizol to a sample exosome solution to crack the exosome solution, incubating and homogenizing at room temperature (18-25 ℃) for 2-15 min, reversing up and down, shaking and uniformly mixing for 15-30 s, centrifuging for 5-20 min at 2-8 ℃ and 8000-14000 rpm, taking an upper clear solution, and placing the upper clear solution on an RNeasy micro column for RNA purification to obtain a sample RNA solution.
The amplification device is used for quantitatively detecting the target region in the sample RNA solution, wherein the target region comprises PCA3 gene, ERG gene and SPDEF gene.
In the embodiment, the expression amounts of the PCA3 gene, the ERG gene, the SPDEF gene, the KLK3 gene, the AMACR gene, the BIRC5 gene, the HOXC6 gene and the SPARCL1 gene in the sample RNA solution are quantitatively detected by a real-time PCR technology, and the PCA3 gene, the ERG gene and the SPDEF gene are preferably used as targets to calculate the expression amounts of the PCA3 gene and the ERG gene relative to the SPDEF gene. The reduction or loss of E-cadherin expression in prostate cancer is a common phenomenon of tumor cell conversion to invasive cells. SAM is directed to ETS transcription factor (SPDEF) of domain as a molecular switch of E-cadherin expression, thus having tumor invasiveness. In most prostate cancer samples, the level of SPDEF expression is higher than normal prostate epithelial tissue, indicating that SPDEF is up-regulated in cancer. In contrast to benign prostate tissue, SPDEF expression increases in prostate cancer and increases with prostate cancer grade, in contrast, loss of SPDEF correlates with increased aggressiveness and is characterized by a tumor metastasis suppressor function, and SPDEF expression is negatively correlated with tumor aggressiveness and patient prognosis, indicating that SPDEF can be a prognostic marker for aggressive prostate cancer, and thus SPDEF expression can be a diagnostic or prognostic marker for the more lethal aggressive phenotype that distinguishes indolent disease from prostate cancer. The PCA3(Prostate cancer gene 3) gene is a Prostate-specific, non-coding mRNA that is highly expressed (> 95%) in Prostate cancer tissue, and many studies have shown that PCA3 can improve the accuracy of cancer prediction at the time of primary biopsy and is associated with more aggressive cancers in prostatectomy. ERG is currently considered to be a key oncogene of prostate cancer, and TMPRSS2_ ERG fusion gene is the most common fusion gene among them, which is formed by fusion of transmembrane serine protease 2(TMPRSS2) gene with ETS transcription factor ERG, and occurs in about 50% of prostate cancer patients. TMPRSS2_ ERG fusion resulted in prostate cancer with a more aggressive cancer phenotype, associated with higher tumor stage and prostate cancer specific death. The PCA3 gene, the ERG gene and the SPDEF gene show unique features and good application prospects in the aspects of early diagnosis, prognosis judgment and the like of the prostatic cancer. However, if a single index is adopted for detection, the accuracy of the method is possibly not enough, and the method combines the clinical significance of the three indexes to carry out combined detection, so that the detection accuracy is higher.
The data acquisition and analysis device is used for acquiring the expression levels of the PCA3 gene, the ERG gene and the SPDEF gene and calculating the expression levels of the PCA3 gene and the ERG gene relative to the SPDEF gene.
The data acquisition and analysis device normalizes the mRNA expression levels of the PCA3 gene and the ERG gene to the SPDEF gene by recording the Ct values of the PCA3 gene, the ERG gene and the SPDEF gene, respectively calculates the delta CT values of the PCA3 gene and the ERG gene, calculates the relative expression of the PCA3 gene and the ERG gene by using a calculation formula delta Ct of the relative expression 2^ delta CT, and then uses the formula: (16.92- Δ Δ Ct) × 1.83 and a score corresponding to the PCA3 gene and the ERG gene was calculated and reflected the risk level for prostate cancer development. In particular, patients who have a score of less than 10 calculated according to the algorithm described above may be identified as having a low risk of prostate cancer, while patients who have a score of 10 or higher may be identified as having a higher risk of prostate cancer.
In this embodiment, adopt this application the device, the urine does not need DRE to collect in advance or special treatment, adopts more ripe RT-PCR method, and multiple one-step process detects, and is simple quick, does not have the liquid biopsy of wound, only needs to collect the urine sample, has very high compliance.
In this embodiment, the qRT-PCR amplification system used in the real-time PCR technique includes a primer mixture, a PCR buffer, a PCR reaction solution, and an enzyme mixture.
The primer mixture contains the components shown in table 1:
TABLE 1
Components | Concentration (nM) |
ERG F | 100~500 |
ERG R | 100~500 |
ERG Pb | 100~500 |
SPDEF F | 100~500 |
SPDEF R | 100~500 |
SPDEF Pb | 100~500 |
PCA3 F | 100~500 |
PCA3 R | 100~500 |
PCA3 Pb | 100~500 |
ROX | 0.1~10 |
H2O | / |
The primer/probe sequence set in the kit for testing three preferred targets, the urine exosome PCA3 gene, ERG gene and SPDEF gene, is shown in table 2, which comprises: a primer of a nucleotide sequence shown as SEQ1-8 and a probe of a nucleotide sequence shown as SEQ 9-12. The abbreviations for the individual nucleotide sequences in Table 2 are from Life Technologies, with the primer probes designated as "LT", 5 '-FAM/JOE/CY 5 in the sequences referring to the 5' reporter group, and 3 '-BHQ 1/BHQ2 referring to the 3' -quencher group.
TABLE 2
The PCR buffer contained the components shown in table 3:
TABLE 3
Components | Concentration of | PH |
Tris-HCl | 50mM~800mM | 7.5~9.0 |
KCl | 50mM~800mM | / |
Ammonium sulfate | 50mM~500mM | / |
H2O | / | / |
The PCR reaction solution contained the components shown in table 4:
TABLE 4
Components | Concentration of |
PCR buffer solution | 1~10 |
MgCl2 | 1mM~6mM |
Glycerol | 0.5wt%~5wt% |
PC300 | 0.1~1 |
H2O | / |
The enzyme mixture contained the components shown in table 5:
TABLE 5
Components | |
Taq-HS | 1U~10U |
RTase | 1U~50U |
Taq Buffer | / |
dNTPs | 0.1mM~1mM |
PC300 | 0.1~10 |
H2O | / |
The qRT-PCR amplification reaction comprises the following steps:
the first step is as follows: in a PCR instrument, firstly amplifying at 30-50 ℃ for 10-50 min, and then amplifying at 90-95 ℃ for 2-10 min;
the second step is that: and amplifying for 10-30 s at 90-95 ℃, and amplifying for 30-60 s at 55-65 ℃, wherein the step is circulated for 35-50 times to obtain a PCR product.
Wherein, fluorescent signals are collected in the step of amplifying at 55-65 ℃, and Ct values of PCA3 gene, ERG gene and SPDEF gene are recorded, wherein Ct is-1/lg (1+ Ex) × lgX + lgN/lg (1+ Ex), wherein N is the cycle number of the amplification reaction, X is the initial template amount, Ex is the amplification efficiency, and N is the amount of the amplification product when the fluorescent amplification signals reach the threshold intensity. The experimental results were analyzed by a relative quantitative method, and the PCA3 gene and the ERG gene were used as the target genes and the SPDEF gene was used as the reference gene in the present invention. Subtracting the Ct value of the SPDEF gene of each sample from the Ct value of the PCA3 gene and the ERG gene of each sample to obtain delta Ct, wherein the delta Ct is Ct (target gene) -Ct (internal reference gene) (namely, the delta Ct is Ct (PCA3) or Ct (ERG) -Ct (SPDEF)), and then calculating the delta CT through the delta Ct, wherein the delta Ct is 2^ delta Ct (PCA3) +2^ delta Ct (ERG), and the delta Ct is the sum of the relative expression quantity of the PCA3 gene and the ERG gene of each sample by using the formula: (16.92- Δ Δ Ct) × 1.83 and a score was calculated which reflects the risk level for prostate cancer. Specifically, patients having a score of less than 10 calculated according to the above algorithm may be identified as having a low risk of prostate cancer, while patients having a score of 10 or higher may be identified as having a higher risk of prostate cancer.
Specifically, in this embodiment, the gene expression results from the RT-PCR experiment are analyzed in multiplex, and the normalization gene used is SPDEF. Calculating the expression amount of each normalized SPDEF according to the Ct value of each target obtained by the experiment, specifically, subtracting the Ct value of the SPDEF gene of each sample from the Ct value of the PCA3 gene and the ERG gene of each sample to obtain delta Ct, wherein the delta Ct is Ct (target gene) -Ct (internal reference gene) (namely, the delta Ct is Ct (PCA3) or Ct (ERG) -Ct (SPDEF)), and the relative expression amounts of the PCA3 is 2 delta Ct (PCA3) and the ERG is 2 delta Ct (ERG)). The test (LDT) score (referred to herein as the PCa score) is recalculated using the following formula: the PCa fraction was (16.92- Δ Δ Ct) × 1.83, where Δ Δ Ct is the sum of the relative expression of PCa3 and ERG. Patient samples were scored according to PCa score, with a cutoff value of 10, PCa scores less than 10 being scores associated with low prostate cancer risk, and PCa scores greater than or equal to 10 being scores associated with higher prostate cancer risk.
The relative expression and assessment scores of the genes in the sample, combined with other clinically standard treatment factors including age, race and family history, can be used to guide prostate biopsy decisions, which may avoid preliminary biopsies in men with negative and/or indolent prostate cancer when stratifying the risk of high grade prostate cancer.
The invention also provides a method for detecting the relative expression quantity of a target area by using the prostate cancer risk assessment device, which comprises the following steps:
s1, collecting urine of the testee, and purifying the exosome solution from the urine.
In the embodiment, 5-50 mL of urine of a tester is collected, an exosome solution in the urine is extracted by an ultrafiltration method, and the aperture of a filter membrane is 20 nm. In addition to ultrafiltration, the exosome solution may be purified and concentrated by ultracentrifugation, membrane affinity, polymer sedimentation, exclusion chromatography, immunocapture, and the like. Finally obtaining 100-500 mul of purified exosome solution.
S2, extracting RNA of the exosome solution obtained in the S1 to obtain a sample RNA solution.
In this embodiment, all exosome RNAs from which ribosomal RNAs were removed were extracted from purified exosomes using miRNeasy mini kit (Qiagen).
The method comprises the following specific steps: adding 200-1000 mul of Trizol to a sample exosome solution to crack the exosome solution, incubating and homogenizing at room temperature (18-25 ℃) for 2-15 min, reversing up and down, shaking and uniformly mixing for 10-60 s, centrifuging for 5-20 min at 2-8 ℃ and 8000-14000 rpm, taking an upper clear solution, and placing the upper clear solution on an RNeasy micro column for RNA purification to obtain a sample RNA solution.
S3, taking the sample RNA solution obtained in S2, and quantitatively detecting the expression levels of the PCA3 gene, the ERG gene, the SPDEF gene, the KLK3 gene, the AMACR gene, the BIRC5 gene, the HOXC6 gene and the SPARCL1 gene in the sample RNA solution by a real-time PCR technology, preferably taking the PCA3 gene, the ERG gene and the SPDEF gene as targets, and calculating the expression levels of the PCA3 gene and the ERG gene relative to the SPDEF gene.
The method comprises the steps of recording Ct values of PCA3, ERG and SPDEF, wherein delta Ct is Ct (target gene) -Ct (reference gene) (namely delta Ct is Ct (PCA3) or Ct (ERG) -Ct (SPDEF)), calculating delta CT through the delta Ct, and the delta Ct is the sum of relative expression amounts of PCA3 gene and ERG gene of each sample by using a formula: (16.92- Δ Δ Ct) × 1.83 and a score was calculated which reflects the risk level for prostate cancer. Specifically, patients having a score of less than 10 calculated according to the above algorithm may be identified as having a low risk of prostate cancer, while patients having a score of 10 or higher may be identified as having a higher risk of prostate cancer.
The method for detecting the relative expression level of the target region by the prostate cancer risk assessment device of the present application is specifically described below by specific examples.
Example 1
S1, collecting urine of the testers, and purifying the exosome solution from the urine.
Collecting 5-50 mL of urine of a tester, extracting an exosome solution from the urine by using an ultrafiltration method, wherein the aperture of a filter membrane is 10-50 nm, and finally obtaining 100-500 mu l of purified exosome solution.
S2, extracting RNA of the exosome solution obtained in the S1 to obtain a sample RNA solution.
Adding 200-1000 mul of Trizol to a sample exosome solution to crack the exosome solution, incubating and homogenizing at room temperature (18-25 ℃) for 5-15 min, reversing up and down, shaking and uniformly mixing for 15-30 s, centrifuging at 2-8 ℃ and 8000-14000 rpm for 2-15 min, taking an upper clear solution, and placing the upper clear solution on an RNeasy micro column for RNA purification to obtain a sample RNA solution.
S3, adding the sample RNA solution obtained in S2 into a qRT-PCR amplification system, amplifying at 30-50 ℃ for 5-50 min, and then amplifying at 90-95 ℃ for 2-10 min;
and amplifying for 10-30 s at 90-95 ℃, and amplifying for 30-60 s at 55-65 ℃, wherein the steps are circulated for 30-50 times to obtain a PCR product.
Wherein, fluorescence signals are collected in the step of amplifying at 55-65 ℃, Ct values of PCA3 gene, ERG gene and SPDEF gene are recorded, then the Ct values are calculated to obtain the delta Ct, the delta Ct is Ct (target gene) -Ct (internal reference gene) (namely, the delta Ct is Ct (PCA3) or Ct (ERG) -Ct (SPDEF)), then the delta CT is calculated through the delta Ct, the delta CT is 2^ delta Ct (PCA3) +2^ delta Ct (ERG), the delta Ct represents the sum of relative expression of the PCA3 gene and the ERG gene of each sample, and the formula is utilized: (16.92- Δ Δ Ct) × 1.83, a score was calculated and judged.
In this embodiment, the qRT-PCR amplification system used in the real-time PCR technique includes a primer mixture, a PCR buffer, a PCR reaction solution, and an enzyme mixture, and the specific components of the primer mixture, the PCR buffer, the PCR reaction solution, and the enzyme mixture are as described above.
In conclusion, the beneficial effects of the invention are as follows: by directly extracting exosomes from human urine, the urine does not need DRE pre-collection or special treatment; by measuring the contents of three preferred targets PCA3 gene, ERG gene and SPDEF gene in exosome, the relative expression quantity of PCA3 gene and ERG gene is calculated, invasive means is not needed, preliminary biopsy and non-invasive liquid biopsy are avoided for men suffering from negative and/or inert prostate cancer, only urine samples are collected, and high compliance is achieved; the detection can be finished by using a common PCR instrument without purchasing additional equipment; the detection is carried out by multiple one-step methods, the method is simple and quick, and the closed-tube operation is good to avoid pollution.
The above description of the examples and comparative examples is only intended to aid in the understanding of the process of the invention and its core ideas; in addition, it is obvious to those skilled in the art that other various corresponding changes and modifications can be made according to the technical idea of the present invention, and all such changes and modifications should fall within the scope of the claims of the present invention.
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Claims (10)
1. A risk assessment device for prostate cancer, comprising:
the collecting device is used for collecting the exosome solution of the testee;
the nucleic acid acquisition device is used for extracting RNA of the exosome solution to obtain a sample RNA solution;
amplification means for quantitatively detecting the target region in the sample RNA solution, the target region comprising PCA3 gene, ERG gene, and SPDEF gene;
a data acquisition and analysis device for acquiring the expression level of PCA3 gene, ERG gene and SPDEF gene, and calculating the expression level of PCA3 gene and ERG gene relative to SPDEF gene,
the data acquisition and analysis device normalizes the expression levels of the PCA3 gene and the ERG gene to the SPDEF gene by recording the Ct values of the PCA3 gene, the ERG gene and the SPDEF gene, respectively calculates the delta CT values of the PCA3 gene and the ERG gene, calculates the sum of the relative expression amounts of the PCA3 gene and the ERG gene by using a calculation formula of the relative expression amounts, namely delta CT (2 ^ delta Ct (PCA3) +2^ delta Ct (ERG)), and then uses the formula: (16.92- Δ Δ Ct) × 1.83 and a score was calculated which reflects the risk level for prostate cancer.
2. The prostate cancer risk assessment device according to claim 1, wherein said amplification device comprises a qRT-PCR amplification system.
3. The prostate cancer risk assessment device according to claim 2, wherein said nucleic acid extraction means extracting said sample RNA solution in said exosome solution comprises:
adding 600-800 mu l of Trizol solution into the exosome solution, incubating and homogenizing at 18-25 ℃ for 3-5 min, adding 100-220 mu l of chloroform, shaking and uniformly mixing for 10-20 s, centrifuging at 2-6 ℃ and 10000-12000 rpm for 10-15 min, taking the upper clear solution, and carrying out RNA purification on the upper clear solution to obtain a sample RNA solution.
4. The prostate cancer risk assessment device according to claim 2, wherein the amplification method of the sample RNA solution by the amplification device comprises:
adding the sample RNA solution into a qRT-PCR amplification system, firstly amplifying for 10-30 min at the temperature of 30-50 ℃, and then amplifying for 2-10 min at the temperature of 90-95 ℃;
further amplifying for 10-30 s at 90-95 ℃, and finally amplifying for 30-60 s at 55-65 ℃, wherein the step is circulated for 30-50 times to obtain a PCR product;
wherein, the fluorescence signal is collected in the step of amplifying at 55-65 ℃.
5. The prostate cancer risk assessment device according to claim 2, wherein the qRT-PCR amplification system comprises a primer mixture, a PCR buffer, a PCR reaction solution, and an enzyme mixture.
6. The prostate cancer risk assessment device according to claim 5, wherein the primer mixture includes, by concentration: 100nM to 500nM ERG F, 100nM to 500nM ERG R, 100nM to 500nM ERG Pb, 100nM to 500nM SPDEF R, 100nM to 500nM SPDEF Pb, 100nM to 500nM PCA 3F, 100nM to 500nM PCA 3R, 100nM to 500nM PCA3 Pb, 0.1 to 10ROX and the balance water.
7. The prostate cancer risk assessment device according to claim 5, wherein said PCR buffer solution comprises Tris-HCl solution with a pH value of 7.5-9.0 at a concentration of 50 mM-800 mM, KCl at a concentration of 50 mM-800 mM, ammonium sulfate at a concentration of 50 mM-500 mM, and the balance water.
8. The device for risk assessment of prostate cancer according to claim 5, wherein said PCR reaction solution comprises 1-10 PCR buffer solution, 1 mM-6 mM MgCl20.5 to 5 weight percent of glycerin, 0.1 to 1PC300 and the balance of water.
9. The device for risk assessment of prostate cancer according to claim 5, wherein the enzyme mixture comprises Taq-HS at a concentration of 1U to 10U, RTase at a concentration of 1U to 50U, Taq Buffer, 0.1mM to 1mM dNTPs, 0.1 to 10PC300, and the balance water.
10. The prostate cancer risk assessment device according to claim 1, wherein said collection device is further configured to perform a pre-treatment of said exosome solution, said pre-treatment comprising purification of said exosome solution.
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