CN115851926B - Real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer and special primer and probe thereof - Google Patents

Real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer and special primer and probe thereof Download PDF

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CN115851926B
CN115851926B CN202210931213.0A CN202210931213A CN115851926B CN 115851926 B CN115851926 B CN 115851926B CN 202210931213 A CN202210931213 A CN 202210931213A CN 115851926 B CN115851926 B CN 115851926B
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primer
spdef
hoxc6
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CN115851926A (en
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居金良
崔振玲
张适麒
冉鹏展
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Shanghai Rendu Biotechnology Co ltd
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Abstract

The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer and a special primer and a special probe thereof, belonging to the technical field of tumor diagnosis. The provided kit comprises reagents for respectively and specifically detecting the following genes based on a real-time fluorescent nucleic acid isothermal amplification method: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and optionally SPDEF and/or KLK3, which can detect prostate cancer (including blood PSA gray zone) with high sensitivity using random urine of patients as a sample to be tested and has extremely high negative predictive value, thus effectively avoiding unnecessary prostate puncture biopsy of negative subjects.

Description

Real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer and special primer and probe thereof
Technical Field
The invention belongs to the technical field of tumor diagnosis, and particularly relates to a prostate cancer detection kit suitable for a real-time fluorescent nucleic acid isothermal amplification detection system, and a special primer and a special probe thereof.
Background
The incidence of prostate cancer (PCa) is the top of urinary system tumor incidence. The current clinical diagnosis method of the prostate cancer mainly comprises the following steps: digital rectal examination, ultrasonic B-mode and multiparameter nuclear magnetic resonance imaging examination of the prostate, and blood PSA (Prostate Specific Antigen) detection. The sensitivity and specificity of the rectal examination to the diagnosis of the prostate cancer are low, and the rectal examination is greatly influenced by the artificial factors; imaging examination represented by multi-parameter nuclear magnetic resonance has good diagnosis effect only on clinically significant prostate cancer, but has certain limitation on diagnosis of some early-stage prostate cancers; detection of blood PSA by prostate needle biopsy is the gold standard for current diagnosis of prostate cancer, but only about 25% of patients in the blood PSA gray area (4-10 ng/mL) population have a positive detection rate, resulting in about 75% of patients experiencing unnecessary needle biopsies. In addition, prostate biopsy can cause great pain to the patient and risk infection. Thus, there is an urgent need for a method of prostate cancer detection that is non-invasive and suitable for patients in the blood PSA gray area population.
Patent document CN111518908A (hereinafter referred to as document 1) discloses a urine prostate cancer marker combination and its use in preparing a precise diagnostic reagent, and this document 1 uses TMPRSS2-ERG (T2 ERG), PCA3, SChLAP1, malt 1, TTTY15-USP9Y, HOXC6 and DLX1 as marker combinations for detecting prostate cancer, can use urine as a detection sample, and can screen out 87.8% of positive patients (including blood PSA gray area patients) with high specificity (78.0%) and high sensitivity (87.8%, meaning that 87.8% of positive patients can be screened out), and the AUC is 0.87 (95% ci:0.78-0.93 at maximum). However, on one hand, the document 1 discloses a combination of markers for detecting prostate cancer, which is suitable for a PCR reaction system, and requires temperature rise and fall and circulation during the PCR reaction, so that the required detection time is longer, the efficiency is lower, and meanwhile, a fluorescent quantitative PCR instrument is required, thereby increasing the detection cost; in addition, the reaction product of PCR is DNA, which is not easy to degrade and easily causes sample cross contamination and experimental environment pollution; on the other hand, although this document 1 can achieve the object of detecting prostate cancer with high accuracy, the sensitivity of detection is still low and the negative predictive value is also low (81.3% indicating that only 81.3% of the detected objects among the actual negative objects are judged to be true negative), so that more positive patients are not screened and more negative objects need to undergo unnecessary puncture biopsy.
Disclosure of Invention
In view of one or more problems existing in the prior art, one aspect of the present invention provides a kit for real-time fluorescent nucleic acid isothermal amplification detection of prostate cancer, comprising reagents for respectively and specifically detecting the following genes based on a real-time fluorescent nucleic acid isothermal amplification method: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, and MALAT1; optionally, the kit further comprises a reagent for specifically detecting SPDEF genes and/or KLK3 genes based on a real-time fluorescent nucleic acid isothermal amplification method, wherein the SPDEF genes and/or KLK3 genes serve as detection reference genes.
In some embodiments, the specific detection reagent for each gene comprises a nucleic acid corresponding to that gene:
(1) Nucleic acid extract: comprising a solid support comprising a specific capture probe for capturing a gene sequence and a first primer for specifically binding to a target sequence in the gene sequence;
(2) Detection liquid a: comprising a second primer that cooperates with the first primer for amplifying a target sequence;
(3) Detection liquid b: comprising a first primer and a target detection probe, wherein the target detection probe specifically binds to an amplified product RNA copy of a target;
Optionally, the kit further comprises:
(4) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase.
In some embodiments of the present invention, in some embodiments,
the nucleotide sequences of specific capture probes for specifically detecting the following genes are shown in SEQ ID NOs 1-10 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of the first primers for specifically detecting the following genes are shown in SEQ ID NOs 11-20 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of the second primers for specifically detecting the following genes are shown in SEQ ID NOs 21-30 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of target detection probes for specifically detecting the following genes are respectively shown in SEQ ID NO. 31-40: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3, and a fluorescent reporter group and a quenching group are respectively carried at two ends of the nucleotide sequence of the target detection probe.
In some embodiments, the kit further comprises an exogenous internal standard having a nucleotide sequence set forth in SEQ ID NO. 61;
Optionally, the kit further comprises an internal standard capture probe, a first internal standard primer, a second internal standard primer and an internal standard detection probe for specifically detecting the exogenous internal standard, wherein the nucleotide sequences of the internal standard capture probe, the first internal standard primer, the second internal standard primer and the internal standard detection probe are respectively shown as SEQ ID NO. 62-65, and fluorescent reporter groups and quenching groups are respectively carried at two ends of the nucleotide sequence of the internal standard detection probe.
In some embodiments, the kit further comprises:
(5) Washing liquid: it contains NaCl and SDS, optionally 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(6) Mineral oil; and/or
(7) Positive control: a system for in vitro transcription of RNA comprising the following gene nucleic acids, respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3; and/or
(8) Negative control: a system that does not contain the following genetic nucleic acids: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1; and/or
(9) Positive standard: the concentration gradients were 10 respectively 2 -10 7 Copy/. Mu.L of in vitro transcribed RNA system comprising the following gene nucleic acids: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3; and/or
(10) Standard curve: standard curves with the ordinate of the following genes as target dt value/internal standard dt value and the abscissa as concentration log value: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3.
In some embodiments of the present invention, in some embodiments,
the nucleic acid extraction solution comprises the following components: 250-800mM HEPES, 4-10% lithium dodecyl sulfate, 1-50 μm of the specific capture probe, 50-500mg/L magnetic beads, 25-150pmol/mL of the first primer; optionally, the components of the nucleic acid extract further comprise the exogenous internal standard, 1-50 mu m of the internal standard capture probe and 25-150pmol/mL of the first internal standard primer;
the components of the detection liquid a comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of said second primer; optionally, the component of the detection solution a further comprises 250-750pmol/mL of the second internal standard primer;
the components of the detection liquid b comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the first primer, 143-857pmol/mL of the target detection probe; optionally, the components of the detection solution b further comprise 143-857pmol/mL of the first internal standard primer and 143-857pmol/mL of the internal standard detection probe;
The SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
In another aspect, the present invention provides a sequence combination for real-time fluorescent nucleic acid isothermal amplification detection of prostate cancer according to the above kit, comprising:
a specific capture probe with nucleotide sequences shown as SEQ ID NO. 1-10, a first primer with nucleotide sequences shown as SEQ ID NO. 11-20, a second primer with nucleotide sequences shown as SEQ ID NO. 21-30, and a target detection probe with nucleotide sequences shown as SEQ ID NO. 31-40;
optionally, the sequence combination further comprises an exogenous internal standard with a nucleotide sequence shown as SEQ ID NO. 61, an internal standard capture probe shown as SEQ ID NO. 62, a first internal standard with a nucleotide sequence shown as SEQ ID NO. 63, a second internal standard with a nucleotide sequence shown as SEQ ID NO. 64 and an internal standard detection probe shown as SEQ ID NO. 65.
The invention also provides a biomarker for detecting prostate cancer based on a real-time fluorescent nucleic acid isothermal amplification detection principle, which is a combination of the following genes: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and optionally SPDEF and/or KLK3.
The use of the above-mentioned biomarkers for the preparation of a reagent for the detection of prostate cancer for the specific detection of PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and optionally SPDEF and/or KLK3, respectively, in a sample is also within the scope of the present invention.
In some embodiments, the sample comprises urine.
The real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer provided by the technical scheme can detect and screen mRNA of PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1 and optional mRNA of SPDEF and/or KLK3 serving as detection reference genes respectively, comprehensively analyze detection results of the target genes, can realize high sensitivity (92.86%, which is obviously higher than 87.8% of the method of the document 1) and high accuracy (AUC is 0.880 (95% CI: 0.759-0.954) when SPDEF is used for detecting the reference genes, AUC is 0.861 (0.735-0.942)) when KLK3 is used for detecting the reference genes, can realize high sensitivity (96% or more when SPDEF is used for detecting the reference genes, 96.4% when the SPDEF is used for detecting the reference genes, and can realize high accuracy (96.97% when the SPDEF is used for detecting the reference genes), can realize high accuracy (0.861.861% when the KLK3 is used for detecting the reference genes), and can realize high detection accuracy (96% when the contrast is not higher than the positive detection result, and can realize high detection accuracy of the detection result when the detection kit is actually used for detecting contrast of the contrast 1.96%. In addition, the detection sample of the kit can be urine (such as random urine of a detection object), so that a noninvasive sampling mode can be adopted when the kit is used for screening a prostate cancer patient, and prostate puncture biopsy is not needed, so that puncture pain or infection risk to the patient can be avoided. On the other hand, compared with the PCR detection method disclosed in the above document 1, the real-time fluorescent nucleic acid isothermal amplification detection kit for prostate cancer provided by the invention has the following advantages: (1) The invention determines that the real-time fluorescent nucleic acid isothermal amplification detects specific gene combinations and internal reference genes, so that the amplification and detection of nucleic acid can be synchronously carried out in the same closed system by using specific probes and primers, the whole process has no heating and cooling processes, the amplification and detection time is greatly shortened (the detection can be completed within 40 minutes, and even within 30 minutes), and the detection efficiency is improved; meanwhile, the design and production cost of the PCR instrument are reduced; (2) The amplified product of the invention is RNA which is easy to degrade in nature, and has easy pollution and small cross influence compared with DNA amplified by PCR.
Drawings
FIGS. 1-10 are SAT amplification curves for the following 10 genes, group 1 and group 2 primers and probes, respectively: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1; wherein in FIGS. 1-10, panel A shows SAT amplification curves of the primers and probes of group 1, respectively, and panel B shows SAT amplification curves of the primers and probes of group 2, respectively;
FIG. 11 is a standard curve of 10 genes, wherein A-J panels are directed to the following genes, respectively: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1;
FIG. 12 is a SAT amplification curve of an exogenous internal standard in 6 concentration-positive standards of 10 genes, wherein A-J panels are directed to the following genes, respectively: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1;
FIG. 13 is a scatter plot of relative expression levels of PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 in the positive and negative groups of prostate puncture biopsies;
FIG. 14 is a ROC curve of a predictive model of 8 gene combinations including PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 when SPDEF is used as a reference gene;
FIG. 15 is a ROC curve of a predictive model of 8 gene combinations including PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 when KLK3 is used as a reference gene.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings.
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The various biomaterials described in the examples were obtained by merely providing a means of experimental acquisition for the purposes of specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
All primers, probes and in vitro transcribed RNA products mentioned in the present invention are synthesized using the prior art.
Example 1: determination of biomarkers for detection of prostate cancer based on real-time fluorescent nucleic acid isothermal amplification detection principle
In this example, the inventors collected a large number of clinical cases and selected a large number of biomarkers (e.g., DLX1, SChLAP1, TDRD1, TTTY15-USP9Y, SPON2, PCAT14, PCA3, OR51E2, ERG, RPL7P16, PIP5K1A, CCND1, GSTP1, CST3, CST4, HOXC6, HOXC4, CCNA1, LMTK2, MYO6, HPN, CDK1, PSCA, PTEN, GOLM1, PMP22, EZH2, FGFR1, FN1, VEGFA, TMPRSS2, ANXA3, CRISP3, BIRC5, AMACR, HIF1A, KLK3, KLK2, MSMB, FLT1, MMP9, AR, TERT, PGC, SPINK, STAT3, STAT5, TFF3, RELA, NDB 4, EHD3, PFKL, RAN, ACSM, EHNA 1, APL 1, SSID 1, SSF 9, etc.) as a fluorescent marker for detection of prostate cancer and the like, and amplified in real-time samples, and further, a group of biomarkers which can detect the prostate cancer with high accuracy and high sensitivity and have extremely high negative predictive value are determined, and finally 8 genes of PCA3, ERG, HOXC6, HOXC4, DLX1, TDRD1, AMACR and MALAT1 are used as detection genes (hereinafter also referred to as targets), and SPDEF and/OR KLK3 are used as detection reference genes. Urine is taken as a sample to be detected, and the biomarker genes are detected, so that the fact that the relative expression amounts of the biomarkers in the detection of the prostate puncture positive group and the prostate puncture negative group are obviously different is also proved, so that the detection of the 8 genes has important clinical significance for diagnosing the prostate cancer, and the genes can be used for carrying out joint detection on the prostate cancer.
In this embodiment, the steps for detecting the urine sample to be tested using one or both of the above 8 genes and 2 detection internal genes are as follows:
1.1 collecting samples to be tested (random pre-urinating urine of test subjects)
125 cases of detection subjects (including 58 cases of positive prostate cancer and 67 cases of negative prostate cancer) of the blood PSA detection are taken, in these detection subjects, 51 cases of blood PSA gray area patients (including 14 cases of positive and 40 cases of negative prostate cancer) and 53 cases of high-fraction prostate cancer patients (all positive cases, wherein 12 cases of blood PSA gray area high-fraction prostate cancer patients) are taken, 10mL-20mL of random urine front-section urine is added into a sample preservation solution (which contains a high-concentration detergent and is commercially available from Shanghai Reed Biotech Co., ltd.) in a ratio of 1:1, and the mixture is uniformly mixed to serve as a sample to be detected, and the sample is frozen and preserved at-70 ℃.
1.2 preparation of test samples
400. Mu.L of PCA3, ERG, SPDEF, KLK, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 gene positive control (see detailed below), 400. Mu.L of six concentration positive standards for each gene (see detailed below), 400. Mu.L of negative control (sample preservation solution), and 400. Mu.L of test sample (10 tubes for each test object) were placed in sample treatment tubes, respectively, and 1330 tubes (positive control 10 tubes, positive standard 60 tubes, negative control 10 tubes, test sample 1250 tubes) were used for test samples.
1.3 nucleic acid extraction
For each sample processing tube for 1.2 standby, the following operations were performed:
(1) 100. Mu.L of nucleic acid extract was added to the sample processing tube: HEPES 500mM, LLS 8%, specific capture probes corresponding to the detected genes (see example 2) 25. Mu.M, 10 5 Copy/. Mu.L of exogenous internal standard (see detailed below), internal standard capture probe (see detailed below) 15. Mu.M, magnetic beads 150mg/L, 100pmol/mL of first primer corresponding to the detected gene (see example 2), 100pmol/mL of first internal standard primer (see detailed below), and mixing. Preserving heat for 10 minutes at 60 ℃ and standing for 5-10 minutes at room temperature;
(2) The sample treatment tube is placed on a magnetic bead separation device and kept stand for 2-5 minutes. After the magnetic beads are adsorbed on the tube wall, the sample treatment tube is kept on the magnetic bead separation device, the liquid is sucked and discarded, and the magnetic beads are reserved. Adding 1mL of washing solution (HEPES 25mM, naCl 150mM, 1% SDS, EDTA 2.5 mM), shaking uniformly, standing for 2-5 min, discarding the liquid, retaining the magnetic beads, then adding 800 μL of washing solution and 150 μL of mineral oil, standing for 2-5 min after shaking uniformly, discarding the liquid, and retaining the magnetic beads;
(3) The sample processing tube is removed from the magnetic bead separation device, and the magnetic bead-nucleic acid complex is contained in the tube for standby.
1.4 SAT amplification assay
For each sample processing tube for 1.3 spares, the following operations were performed:
(1) To each of the sample processing tubes containing the magnetic bead-nucleic acid complex, 40. Mu.L of the detection solution a: tris 15mM, mgCl 2 15mM, dNTP 2.5mM, NTP 3mM, PVP 40% and KCl 10mM, a second primer (see example 2) corresponding to the detected gene 500pmol/mL, a second internal primer (see detailed below) 500pmol/mL, and the magnetic beads were resuspended by shaking;
(2) Adding 40 mu L of the reaction detection solution a which is uniformly mixed by vibration into a clean micro-reaction tube, and adding 50 mu L of mineral oil into each reaction tube at 42 ℃ for 5-10min. To the microreactor tube, 25. Mu.L of SAT enzyme solution (preheated at 42℃in advance, containing M-MLV reverse transcriptase 60000U/mL, T7 RNA polymerase 40000U/mL, 10mM HEPES pH7.5, 15mM N-acetyl-L-cysteine, 0.15mM zinc acetate, 20mM trehalose, 100mM Tris-HCl pH 8.0, 80mM KCl, 0.25mM EDTA, 0.5% (v/v) Triton X-100 and 30% (v/v) glycerol) was added at 42℃for 5 to 10min;
(3) To the microreactor tube was added 35. Mu.L of detection solution b: tris 15mM, mgCl 2 15mM, dNTP 2.5mM, NTP 3mM, PVP 40% and KCl 10mM, first primer 429pmol/mL corresponding to the detection gene, first inner marker 429pmol/mL, target detection probe (see example 2) 429pmol/mL corresponding to the detection gene, and inner marker detection probe (see detailed below) 429pmol/mL, the reaction tube is rapidly transferred to a constant temperature fluorescence detection instrument, and the reaction is carried out at 42 ℃ for 40 minutes, and fluorescence is detected every 1 minute for 40 times; the fluorescein channel was selected from FAM and HEX channels.
The specific capture probes, first primers, second primers and target detection probes for each gene referred to in steps 1.3 and 1.4 above are determined in example 2 below, see the detailed description of example 2.
The positive standard and positive control of the gradient concentration in this example were prepared by the following steps:
(a) PCA3 (GenBank: NR_ 132313.1), ERG (GenBank: NM_ 001243428.1), SPDEF (GenBank: NM_ 012391.3), KLK3 (GenBank: NM_ 001030047.1), DLX1 (GenBank: NM_ 178120.5), HOXC6 (GenBank: NM_ 153693.5), HOXC4 (GenBank: NM_ 014620.6), TDRD1 (GenBank: NM_ 001385372.1), AMACR (GenBank: NM_ 014324.6), MALAT1 (NR_ 002819.4) gene fragments were synthesized by chemical synthesis methods, respectively;
(b) Cloning each gene fragment synthesized in the step (2.1) into each of the gene fragmentsConstructing respective positive plasmids in the vector;
(c) Each positive plasmid was transformed into E.coli DH 5. Alpha. And designatedStrains, stored at-70 ℃;
(d) From the genes corresponding to eachExtraction of the Strain->And (3) carrying out transcription RNA purification on the extracted plasmid to remove DNA, and quantifying and identifying in vitro transcription RNA. RNA concentration was analyzed by a NanoDrop 2000 spectrophotometer, OD thereof 260 Values between 1.8 and 2.0 indicate better RNA purity;
(e) Quantifying high concentration standard substances of transcribed RNA of each gene of PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1 by ultraviolet spectrophotometry, and respectively diluting to 10 2 -10 7 The concentration of copy/. Mu.L was 10 in step 1.3 as a positive standard 5 Copy/. Mu.L of positive standard was used as positive control.
Exogenous internal standard, and primers and probes thereof:
preparing an exogenous internal standard according to the steps (a) - (e), wherein the nucleotide sequence of the exogenous internal standard is shown as SEQ ID NO: shown at 61. Based on the sequence of the exogenous internal standard, a primer and a probe which are suitable for a real-time fluorescent nucleic acid isothermal amplification detection system and aim at the exogenous internal standard are designed, wherein the primer and the probe comprise an internal standard capture probe, a first internal standard primer, a second internal standard primer and an internal standard detection probe, and the nucleotide sequences of the primer and the probe are respectively shown as SEQ ID NO: 62-65. The exogenous internal standard is in vitro transcribed RNA, has no biological activity, and can be used for controlling the variation difference of the detection sample during the extraction, amplification and detection (the better the exogenous internal standard amplification consistency is, the higher the consistency of the detection sample during the extraction and amplification is shown).
1.5, result determination
1.5.1 drawing of a Standard Curve
As shown in panels A of FIGS. 1 to 10, the SAT (real-time fluorescent nucleic acid isothermal) amplification curves of six concentration-positive standards of PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 are shown, respectively, and the abscissa indicates time and the ordinate indicates relative fluorescence intensity, so that the amplification effect of each concentration-positive standard for each gene is good, and the dt value is automatically read by the instrument based on these amplification curves. The results of plotting the standard curves for each gene (PCA 3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT 1) with the ordinate as the target dt value/internal standard dt value, and the abscissa as the log value of the concentration are shown in FIG. 11, wherein the A-J panels represent the standard curves for PCA3, ERG, SPDEF, KLK, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, respectively. In fig. 12, panels a-J respectively show SAT amplification curves for the exogenous internal standard, and it can be seen that the exogenous internal standard has good consistency in amplification in the standard of each concentration, and good repeatability, indicating that the positive standard of each concentration has good consistency in the extraction and amplification processes.
1.5.2 calculation of relative expression levels of PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1 in the sample to be tested
According to the standard curve drawn in the above step 1.5.1, taking SPDEF as a reference gene (the SPDEF quantitative value needs to be removed to be less than 1000 copies/reaction (equivalent to 2500 copies/mL) of a sample to be tested, so as to ensure that enough prostate cells are present in the sample to be tested, and 124 cases are used as effective samples in the embodiment), the relative expression amounts of PCA3, ERG, AMACR, MALAT1, TDRD1, DLX1, HOXC6 and HOXC4 in a positive sample (58 cases) and a negative sample (66 cases) are calculated respectively, and the results are shown in A-J panels in FIG. 13 respectively, so that the relative expression amounts of 8 genes above have significance differences in the positive sample and the negative sample, and the 8 genes are proved to have clinical significance in prostate cancer detection and diagnosis, so that the method can be used for detecting and diagnosing prostate cancer.
Example 2: design of special primer and probe for real-time fluorescent nucleic acid isothermal amplification detection of prostate cancer
Based on the biomarkers for detection of prostate cancer (including 8 genes of PCA3, ERG, HOXC6, HOXC4, DLX1, TDRD1, AMACR, MALAT1 and SPDEF and/or KLK3 as reference genes) determined in example 1 above, this example designed and determined a constant temperature for fluorescent nucleic acids suitable for use in real time for these biomarker genes Primers and probes for an amplification detection system. Wherein 15 sets of primer and probe combinations are designed for each gene, and positive standards of PCA3, AMACR, ERG, MALAT1, TDRD1, DLX1, HOXC4, HOXC6, SPDEF and KLK3 mRNA are respectively used (in vitro transcription RNA standard of nucleic acid of each gene, the concentration is 10 respectively) 2 -10 7 Copy/. Mu.L, six concentration gradients in total, prepared as described in example 1 above) were used as templates to verify the detection sensitivity and specificity effects of the 15 sets of primer probes, from which the corresponding optimal primer and probe combinations for detecting the above 10 genes, respectively, were determined. As an example, 2 of which are listed for each gene, group 1 listed below is the best combination of genes, group 2 shows the control combination of genes as an example of the other 13 groups (where 15 groups all use the same gene-specific capture probe and target detection probe), and the detection sensitivity and specific effect of these 2 groups of primer probes were verified using positive standards of PCA3, AMACR, ERG, MALAT1, TDRD1, DLX1, HOXC4, HOXC6, SPDEF, KLK3 mRNA as templates, respectively.
Table 1: primers and probes for different biomarkers for detecting prostate cancer in group 1
Table 2: primers and probes for different biomarkers for detecting prostate cancer in group 2
Gene Nucleotide sequence of first primer Sequence numbering
PCA3 TTTCCAGCCCCTTTAAATATC SEQ ID NO:41
ERG GACCAGCGTCCTCAGTTAGATCC SEQ ID NO:42
DLX1 CGGAGCTCGCGGCCTCTTTGG SEQ ID NO:43
HOXC6 GGGTCGGCTACGGAGCGGACCGG SEQ ID NO:44
HOXC4 CTCCAGCGCCGCCAGCAAGCAACCC SEQ ID NO:45
TDRD1 TCTTGGAAGAGGAAGTGGTT SEQ ID NO:46
AMACR GCACTGGGCATTATAATGGCTCTTT SEQ ID NO:47
MALAT1 CTGCTAAAATTTACATGT SEQ ID NO:48
SPDEF AGATCCCATGGACTGGAGCCC SEQ ID NO:49
KLK3 GTGGGTCCTCACAGCTGCC SEQ ID NO:50
Nucleotide sequence of the second primer Sequence numbering
PCA3 AATTTAATACGACTCACTATAGGGAGAGCTCATCGATGACCCAAGATGG SEQ ID NO:51
ERG AATTTAATACGACTCACTATAGGGAGAGCCTGGATTTGCAAGGCGGCTACTT SEQ ID NO:52
DLX1 AATTTAATACGACTCACTATAGGGAGAAACGCACTACCCTCCAGAGCCGCCC SEQ ID NO:53
HOXC6 AATTTAATACGACTCACTATAGGGAGAGCGATCTCGATGCGCCGGCGC SEQ ID NO:54
HOXC4 AATTTAATACGACTCACTATAGGGAGATTGGGGTTCACCGTGCTAA SEQ ID NO:55
TDRD1 AATTTAATACGACTCACTATAGGGAGAATTTCTATAAGCACATGGTCTAAAA SEQ ID NO:56
AMACR AATTTAATACGACTCACTATAGGGAGACACAGAAAAGAACTTAAATATG SEQ ID NO:57
MALAT1 AATTTAATACGACTCACTATAGGGAGACCCCCCAAGATTGCCCCAA SEQ ID NO:58
SPDEF AATTTAATACGACTCACTATAGGGAGAGGTATTGGTGCTCTGTCCACAGG SEQ ID NO:59
KLK3 AATTTAATACGACTCACTATAGGGAGATGTGTCTTCAGGATGAAACAG SEQ ID NO:60
Positive standards of the respective genes prepared in example 1 above were prepared by using the two sets of primers and probes (set 1 and set 2) of tables 1 and 2 above (concentrations were 10, respectively 7 Copy/. Mu.L, 10 6 Copy/. Mu.L, 10 5 Copy/. Mu.L, 10 4 Copy/. Mu.L, 10 3 Copy/. Mu.L, 10 2 Copy/. Mu.L) was subjected to real-time fluorescent nucleic acid isothermal amplification detection (specific detection methods are described in example 1 above).
The results are shown in FIGS. 1-10, wherein the A-panels as in FIGS. 1-10 represent the primers and probes for PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, group 1, respectivelyAmplification curves ("S" curves represent concentration 10 from left to right, respectively) 7 Copy/. Mu.L, 10 6 Copy/. Mu.L, 10 5 Copy/. Mu.L, 10 4 Copy/. Mu.L, 10 3 Copy/. Mu.L, 10 2 Copy/. Mu.L), panels B in FIGS. 1-10 represent the amplification curves for the primers and probes for set 2 of PCA3, ERG, SPDEF, KLK, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, respectively. It can be seen that the detection sensitivity of the primers and probes of group 1 was significantly better than that of the primers and probes of group 2 for any one of the genes PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, and then the specificity of the primers and probes of group 1 was also detected, which showed good specificity, so that the primers and probes of group 1 were determined for each of the above 10 genes, respectively, for real-time fluorescent nucleic acid isothermal amplification detection of each gene. The primers and probes of group 1 were used in the above example 1, and the primers and probes of group 1 were also used in the following examples.
Example 3: establishment of a model for diagnosing prostate cancer in combination with PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF genes
This example uses the biomarker genes for detecting prostate cancer determined in example 1 above and the primers and probes for each biomarker gene (primer and probe of set 1) determined in example 2 above to perform SAT quantitative detection on prostate cancer clinical urine samples (51 samples from blood PSA gray area patients, including 14 positive samples and 37 negative samples) to obtain the relative expression amount of each biomarker gene in the clinical urine samples. Since the SChLAP1 mRNA in the above document 1 can also be used as a biomarker gene for detecting prostate cancer, the relative expression amount thereof in a clinical sample is also detected using a primer and a probe for this gene in this example, and the nucleotide sequences of a specific capture probe, a first primer, a second primer and a target detection probe for SChLAP1 (GenBank: nr_ 104320.1) are respectively set as SEQ ID NO: 66-69. Specific assay methods as described in example 1 above, a urine sample was divided into 10 portions and assayed separately, and the score value of each biomarker gene (exemplified by SPDEF as the reference gene to be assayed) was calculated as follows:
PCA3 score = PCA3 copy number/SPDEF copy number 1000; AMACR score = AMACR copy number/SPDEF copy number 1000; ERG score = ERG copy number/SPDEF copy number 1000; MALAT1 score = MALAT1 copy number/SPDEF copy number 1000; TDRD1 score = DRD1 copy number/SPDEF copy number 1000; HOXC6 score = HOXC6 copy number/SPDEF copy number 1000; HOXC4 score = HOXC4 copy number/SPDEF copy number 1000; DLX1 score = DLX1 copy number/SPDEF copy number x 1000; SChLAP1 score = SChLAP1 copy number/SPDEF copy number 1000.
Establishing a scoring value regression model of the 9 biomarker genes by using a Logistic regression method, and calculating a comprehensive scoring value by using a comprehensive scoring formula, wherein:
malt 1 and HOXC6 combined composite score z=0.027×a+0.087×b-3.118;
MALAT1, HOXC6 and ERG combined composite score z=0.024 x a+0.105 x b+0.08 x c-3.686;
malt 1, HOXC6, ERG, and PCA3 combined composite score z=0.025 x a+0.144 x b+0.095 x c+0.002 x d-4.564;
malt 1, HOXC6, ERG, PCA3 and HOXC4 combined composite score z=0.026 a+0.116 b+0.087 c+0.002 d+0.017 e-5.01;
malt 1, HOXC6, ERG, PCA3, HOXC4 and DLX1 combined composite score z=0.024×a+0.063×b+0.086×c+0.002×d+0.012×e+0.078×f-5.1;
Malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1 and TDRD1 combined composite score z=0.025×a+0.089×b+0.085×c+0.002×d+0.016×e+0.098×f-0.053×g-5.108;
malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1 and AMACR combined composite score z=a 0.028+b 0.08+c 0.098+d 0.002+e 0.019+f 0.0163-G0.033+h 0.063-8.943;
malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1, AMACR, and SChLAP1 combined composite score z=a 0.025+b 0.117+c 0.09+d 0.002+e 0.015+f 0.168-G0.06+h 0.079+i 0.107-10.839;
wherein A is MALAT1, B is HOXC6, C is ERG, D is PCA3, E is HOXC4, F is DLX1, G is TDRD1, H is AMACR, and I is SChLAP 1.
Statistical analysis was performed using SPSS (version 21.0). The clinical gold standard prostate puncture biopsy results and the composite score value of the above 9 biomarker genes are used to evaluate the sensitivity, specificity, negative predictive value and positive predictive value of the composite score of the 9 biomarker genes by a subject operating characteristic (ROC) curve to determine the final cutoff value. The cutoff value can be used as a diagnostic value for a prostate cancer patient. Wherein:
sensitivity (SE) =number of samples above cutoff in tumor patients/number of patient samples;
Specificity (SP) =number of samples below cut-off value/number of control samples in tumor patients;
positive Predictive Value (PPV) =number of samples above the cutoff/number of all samples above the cutoff in tumor patients;
negative Predictive Value (NPV) =number of samples below the cutoff value/number of all samples below the cutoff value in tumor patients;
accuracy= (number of samples above the cut-off in tumor patients + number of samples below the cut-off in tumor patients)/number of all samples.
The results are shown in table 3 below, and as a result of correlation analysis of prostate cancer diagnosis using the above 9 biomarker genes, it was shown that the above 9 biomarker genes have significant diagnostic value for prostate cancer, wherein the area under the ROC curve (AUC) of the malt 1 score is most ranked first, and the rest (except for SChLAP 1) is ranked from the AUC value from the large to the small. And as is apparent from the results shown in table 3 below, the correlation results (including the order of comparing the degree of influence of the respective genes) obtained based on the real-time fluorescence isothermal amplification detection method of the present invention for diagnosis of prostate cancer are different from those of the above-mentioned document 1 based on the PCR method, for the same biomarker genes, which reflects that the combination of biomarkers for detection of prostate cancer, which are disclosed in the above-mentioned document 1 as suitable for the PCR detection system, may not be suitable for the real-time fluorescence isothermal amplification detection system based on the present invention.
Table 3: correlation analysis results of 9 biomarker genes on prostate cancer diagnosis
SPDEF internal reference SE SP PPV NPV AUC(95％CI)
MALAT1 78.57 62.61 44 88.5 0.726(0.583-0.841)
HOXC6 85.71 59.46 44.4 91.7 0.701(0.556-0.821)
ERG 78.57 70.27 50 89.7 0.701(0.556-0.821)
PCA3 85.71 51.35 40 90.5 0.697(0.552-0.818)
HOXC4 78.57 62.16 44 88.5 0.691(0.546-0.813)
DLX1 78.57 56.76 40.7 87.5 0.663(0.517-0.789)
TDRD1 71.43 48.65 34.5 81.8 0.660(0.520-0.792)
AMACR 78.57 70.27 50 89.7 0.656(0.510-0.784)
SChLAP1 78.57 48.65 36.7 85.7 0.664(0.518-0.790)
The diagnostic performance of different gene combination models was further assessed using multivariate Logistic regression. SPDEF is used as a detection reference gene, and other detection genes determined by the invention are added into a regression model one by one according to AUC sequencing on the basis of MALAT1 single gene. As shown in table 4, it can be seen that in the multiplex logistic regression analysis, the predictive model of 8 gene combinations including MALAT1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1, and AMACR is optimal, as shown in fig. 14, the ROC curve of the predictive model, whose AUC is 0.880 (95% ci: 0.759-0.954), sensitivity (SE), specificity (SP), positive predictive value (positive predictive value, PPV), negative predictive value (negetive predictive value, NPV) are 92.86%, 72.97%, 56.5%, and 96.4%, respectively, is shown, and it can be seen that the model has extremely high Sensitivity and negative predictive value, can detect 92.86% of positive patients, and can determine 96.4% of the detected objects among all the actual negative objects as true negative results, thereby avoiding that most negative objects undergo unnecessary puncture biopsy. However, when the SChLAP1 gene was added continuously to the model, the 9 gene models were constructed, which resulted in a decrease in AUC area (0.857 (95% CI: 0.731-0.939)), and a decrease in specificity (40.54%), positive predictive value (37.1%) and negative predictive value (93.7%), and therefore the overall diagnostic effect was decreased. Thus, the present invention determines that a combined model of the above 8 genes (including malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1, and AMACR) is used for detection and screening of prostate cancer, which has extremely high sensitivity and negative predictive value, can detect 92.86% of positive patients, and can avoid that 96.4% of detected objects among actual negative objects undergo unnecessary puncture biopsies, and determines its optimal cutoff value (cutoff) as 0.21605 according to the model, namely: positive samples when the number of the samples is greater than 0.21605; and 0.21605 or less is a negative sample.
Table 4: correlation analysis results of different gene combinations on prostate cancer diagnosis
Example 4: establishment of a model for diagnosis of prostate cancer in combination with PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, KLK3 genes
This example uses the same method as that of example 3 above, and uses the biomarker genes (including PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, KLK3 gene as an internal reference detection gene) determined in example 1 above and the primers and probes (primer and probes of group 1) for each biomarker gene determined in example 2 to perform SAT quantitative detection on prostate cancer clinical urine samples (including 53 high-score prostate cancer patient samples and 67 negative samples) to obtain the relative expression level of each biomarker gene in the clinical urine samples, and additionally supplements the use of SCHLAP1 mRNA as a biomarker gene for prostate cancer detection.
As a result, as shown in table 5, it was found that in the multiple logistic regression analysis, the prediction model of the 8 gene combinations including MALAT1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1, and AMACR was also good when KLK3 gene was used as the reference detection gene (the overall score z=a=0.058+b+c+0.091+d+e+0.012+e+0.029+f 0.0563-G0.053+h+0.085-6.543, where a is the MALAT1 score, B is the HOXC6 score, C is ERG score, D is PCA3 score, E is HOXC4 score, F is DLX1 score, G is TDRD1 score, H is AMACR score), as shown in FIG. 15, the ROC curve of the prediction model is shown, the AUC is 0.861 (95% CI: 0.735-0.942), the sensitivity, specificity, positive predictive value, negative predictive value of diagnosing prostate cancer are 92.86%, 51.35%, 41.9% and 97.8%, respectively, and the model is also seen to have extremely high sensitivity and negative predictive value. 92.86% of positive patients can be detected and 97.8% of the detected subjects can be judged as true negative results among all actual negative subjects, thereby avoiding that most negative subjects undergo unnecessary needle biopsies. However, when the SChLAP1 gene was added continuously to the model, the 9 gene models were constructed, which resulted in a decrease in AUC area (0.855 (95% CI: 0.728-0.938)), and a decrease in specificity (43.24%), positive predictive value (38.2%) and negative predictive value (94.1%), and therefore the overall diagnostic effect was decreased. Thus, the present invention determines that a combined model of the above 8 genes (including malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1, and AMACR) is used for detection and screening of prostate cancer, which has extremely high sensitivity and negative predictive value, can detect 92.86% of positive patients, and can avoid that 97.8% of detected subjects among actual negative subjects undergo unnecessary puncture biopsies, and determines its optimal cutoff value (cutoff) as 0.56704 according to the model, namely: positive samples when the number of the samples is greater than 0.56704; and 0.56704 or less is a negative sample.
Table 5: correlation analysis results of different gene combinations on prostate cancer diagnosis
Example 5: kit for detecting prostate cancer by real-time fluorescent nucleic acid isothermal amplification
From the results of examples 3 and 4 above, it is understood that the prediction effect of the prediction model comprising 8 gene combinations of malt 1, HOXC6, ERG, PCA3, HOXC4, DLX1, TDRD1 and AMACR is better, and the accuracy of detecting prostate cancer is significantly higher than that of the case of using a single biomarker, and also significantly higher than that of the case of using other biomarkers in combination, regardless of whether SPDEF is used as the detection reference gene or KLK3 is used as the detection reference gene. And when the biomarkers are used for detecting the prostate cancer, urine (such as random urine anterior segment urine) can be used as a detection sample, and prostate puncture biopsy is not needed to be carried out on patients, so that the large-scale physical examination of people is facilitated. Based on this, this embodiment provides a prostate cancer detection kit based on the principle of real-time fluorescent nucleic acid isothermal amplification detection, which may include:
(5.1) reagents for the specific detection of the following genes, respectively, based on the real-time fluorescent nucleic acid isothermal amplification method: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, and MALAT1; optionally, the kit further comprises a reagent for specifically detecting the human SPDEF gene and/or the human KLK3 gene based on a real-time fluorescent nucleic acid isothermal amplification method, wherein the human SPDEF gene and/or the human KLK3 gene is used as a detection reference gene. Specifically, the specific detection reagent for each of the above genes includes a reagent corresponding to the gene:
(1) Nucleic acid extract: comprising a solid support comprising a specific capture probe for capturing a gene sequence and a first primer for specifically binding to a target sequence in the gene sequence;
(2) Detection liquid a: comprising a second primer that cooperates with the first primer for amplifying a target sequence;
(3) Detection liquid b: comprising a first primer and a target detection probe, wherein the target detection probe specifically binds to an amplified product RNA copy of a target;
may further comprise:
(4) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase.
More specifically, the nucleotide sequences of specific capture probes for specifically detecting genes PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3 are respectively shown in SEQ ID NO. 1-10, the nucleotide sequences of a first primer are respectively shown in SEQ ID NO. 11-20, the nucleotide sequences of a second primer are respectively shown in SEQ ID NO. 21-30, the nucleotide sequences of target detection probes are respectively shown in SEQ ID NO. 31-40, and fluorescent reporter groups and quenching groups are respectively carried at both ends of the nucleotide sequences of the target detection probes;
Still more particularly, the embodiment provides a specific detection reagent for each of the above genes comprising:
(1) A nucleic acid extract comprising the components: 250-800mM HEPES, 4-10% lithium dodecyl sulfate, 1-50 μm of the specific capture probe, 50-500mg/L magnetic beads, 25-150pmol/mL of the first primer; optionally, the components of the nucleic acid extract further comprise the exogenous internal standard, 1-50 mu m of the internal standard capture probe and 25-150pmol/mL of the first internal standard primer;
(2) The detection liquid a comprises the following components: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of said second primer; optionally, the component of the detection solution a further comprises 250-750pmol/mL of the second internal standard primer;
(3) The detection liquid b comprises the following components: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the first primer, 143-857pmol/mL of the target detection probe; optionally, the components of the detection solution b further comprise 143-857pmol/mL of the first internal standard primer and 143-857pmol/mL of the internal standard detection probe;
(4) The SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
The nucleotide sequence of the exogenous internal standard can be shown as SEQ ID NO. 61, the nucleotide sequences of the internal standard capture probe, the first internal standard primer, the second internal standard primer and the internal standard detection probe for specifically detecting the exogenous internal standard are respectively shown as SEQ ID NO. 62-65, and the two ends of the nucleotide sequence of the internal standard detection probe are respectively provided with a fluorescent reporter group and a quenching group.
For convenience and/or accuracy of detection, the kit provided in this embodiment further comprises one or more of the following components (5.2) - (5.8):
(5.2) washing solution: it contains NaCl and SDS, optionally 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA.
(5.3) mineral oil: the method is used for cleaning the magnetic bead organic phase.
(5.4) positive control: a system for in vitro transcription of RNA comprising the following genetic nucleic acids: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and/or KLK3, as prepared in example 1.
(5.5) negative control: a system that does not contain the following genetic nucleic acids: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR, and MALAT1, such as deionized water or sample-holding fluid (which contains high concentrations of detergent and physiological saline).
(5.6) positive standard: the concentration gradients were 10 respectively 2 -10 7 Copy/. Mu.L of in vitro transcribed RNA system comprising the following gene nucleic acids: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and/or KLK3, as prepared in example 1.
(5.7) standard curve: standard curves for the ordinate of the following genes as target dt value/internal standard dt value and abscissa as log value of concentration, respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and/or KLK3, as drawn in example 1.
(5.8) result determination Specification
If SPDEF is used as a detection reference gene, when the combined comprehensive score of 8 genes obtained by using the kit provided by the invention to detect clinical urine samples is larger than 0.21605, judging that the kit is a positive sample of the prostate cancer; and when the comprehensive score is less than or equal to 0.21605, judging that the prostate cancer is a negative sample.
If KLK3 is used as a reference gene, when the combined comprehensive score of 8 genes obtained by using the kit to detect clinical urine samples is larger than 0.56704, judging that the kit is a positive sample of the prostate cancer; and when the comprehensive score is less than or equal to 0.56704, judging that the prostate cancer is a negative sample.
Example 6: clinical sample validation
By using the kit (SPDEF is used as a detection reference gene) and the judgment standard provided in the above example 5, real-time fluorescent nucleic acid isothermal amplification detection is performed on 21 blood PSA gray zone patient clinical urine samples (clinical prostate puncture biopsy shows that the samples comprise 5 positive samples and 16 negative samples) respectively, so as to verify the reliability of the kit provided by the invention. Specific detection method referring to example 1, each urine sample was divided into 9 parts, each biomarker gene was detected separately, the detection results are shown in table 6 below, and table 7 is the statistical result of the detection results of table 6. The sensitivity of the kit provided by the invention to the detection of the prostate cancer is 100% (5/5), the specificity is 62.5% (10/16), the negative predictive value is 100% (10/10), the positive predictive value is 45% (5/11), and the kit provided by the invention has extremely high sensitivity and negative predictive value in the aspect of detecting the prostate cancer, so that unnecessary puncture biopsy of a prostate cancer negative object can be greatly reduced.
Table 6: detection result of clinical urine sample of 21 blood PSA gray area patients
Table 7: clinical sample validation results
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Use of a reagent for detecting a biomarker combination in the preparation of a kit for detecting prostate cancer, wherein the biomarker combination is a combination of the following genes: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3, wherein the SPDEF and/or KLK3 are used as detection reference genes.
2. The use according to claim 1, wherein the kit comprises reagents for the respective specific detection of each gene in the biomarker combination based on a real-time fluorescent isothermal nucleic acid amplification method.
3. The use according to claim 2, wherein the specific detection reagent for each gene comprises a specific binding reagent for that gene:
(1) Nucleic acid extract: comprising a solid support comprising a specific capture probe for capturing a gene sequence and a first primer for specifically binding to a target sequence in the gene sequence;
(2) Detection liquid a: comprising a second primer that cooperates with the first primer for amplifying a target sequence;
(3) Detection liquid b: comprising a first primer and a target detection probe, wherein the target detection probe specifically binds to an amplified product RNA copy of a target.
4. The use according to claim 3, wherein the kit further comprises:
(4) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase.
5. The use according to claim 3, characterized in that,
the nucleotide sequences of specific capture probes for specifically detecting the following genes are shown in SEQ ID NOs 1-10 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of the first primers for specifically detecting the following genes are shown in SEQ ID NOs 11-20 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of the second primers for specifically detecting the following genes are shown in SEQ ID NOs 21-30 respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3;
the nucleotide sequences of target detection probes for specifically detecting the following genes are respectively shown in SEQ ID NO. 31-40: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR, MALAT1, SPDEF and KLK3, and a fluorescent reporter group and a quenching group are respectively carried at two ends of the nucleotide sequence of the target detection probe.
6. The use according to any one of claims 3 to 5, wherein the kit further comprises an exogenous internal standard having a nucleotide sequence as set forth in SEQ ID No. 61;
The kit also comprises an internal standard capture probe, a first internal standard primer, a second internal standard primer and an internal standard detection probe for specifically detecting the exogenous internal standard, wherein the nucleotide sequences of the internal standard capture probe, the first internal standard primer, the second internal standard primer and the internal standard detection probe are respectively shown as SEQ ID NO. 62-65, and fluorescent reporter groups and quenching groups are respectively carried at two ends of the nucleotide sequence of the internal standard detection probe.
7. The use according to any one of claims 3-5, wherein the kit further comprises:
(5) Washing liquid: it contains 5-50 mM HEPES, 50-500 mM NaCl, 0.5-1.5% SDS, 1-10 mM EDTA; and/or
(6) Mineral oil; and/or
(7) Positive control: a system for in vitro transcription of RNA comprising the following gene nucleic acids, respectively: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3; and/or
(8) Negative control: a system that does not contain the following genetic nucleic acids: PCA3, ERG, SPDEF, KLK3, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1; and/or
(9) Positive standard: the concentration gradients were 10 respectively 2 -10 7 Copy/. Mu.L of in vitro transcribed RNA system comprising the following gene nucleic acids: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3; and/or
(10) Standard curve: standard curves with the ordinate of the following genes as target dt value/internal standard dt value and the abscissa as concentration log value: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3.
8. The use according to any one of claims 3 to 5, characterized in that,
the nucleic acid extraction solution comprises the following components: 250-800 mM HEPES, 4-10% lithium dodecyl sulfate, 1-50 μΜ of the specific capture probe, 50-500 mg/L magnetic beads, 25-150 pmol/mL of the first primer;
the components of the detection liquid a comprise: 10-50 mM Tris, 5-40 mM KCl, 10-40 mM MgCl 2 1-20 mM NTP, 0.1-10 mM dNTPs, 1-10% PVP40, 250-750 pmol/mL of the second primer;
the components of the detection liquid b comprise: 10-50 mM Tris, 5-40 mM KCl, 10-40 mM MgCl 2 1-20 mM NTP, 0.1-10 mM dNTPs, 1-10% PVP40, 143-857 pmol/mL of the first primer, 143-857 pmol/mL of the target detection probe;
the SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10 mM HEPES pH7.5, 10-100 mM of N-acetyl-L-cysteine, 0.04-0.4 mM zinc acetate, 10-100 mM trehalose, 40-200 mM of Tris-HCl pH 8.0, 40-200 mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% of Triton X-100 v/v, and 20-50% of glycerol v/v.
9. The use according to claim 8, wherein the components of the nucleic acid extraction solution further comprise: an exogenous internal standard, 1-50 mu m internal standard capture probe and 25-150 pmol/mL first internal standard primer;
the components of the detection liquid a further comprise: 250-750 pmol/mL of a second internal standard primer;
the components of the detection liquid b further comprise: 143-857 pmol/mL of first internal standard primer and 143-857 pmol/mL of internal standard detection probe;
wherein the exogenous internal standard, internal standard capture probe, first internal standard primer, second internal standard primer and internal standard detection probe are all as described in claim 6.
10. A biomarker combination for detecting prostate cancer based on a real-time fluorescent nucleic acid isothermal amplification detection principle, characterized in that the biomarker combination is a combination of the following genes: PCA3, ERG, DLX1, HOXC6, HOXC4, TDRD1, AMACR and MALAT1, and SPDEF and/or KLK3, wherein the SPDEF and/or KLK3 are used as detection reference genes.
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* Cited by examiner, † Cited by third party
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CN111518908A (en) * 2020-05-18 2020-08-11 奥尔文泰生物科技(杭州)有限公司 Urine prostate cancer marker combination and application thereof in preparation of accurate diagnostic reagent
CN113512597A (en) * 2020-12-02 2021-10-19 上海仁度生物科技股份有限公司 Real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis and special primer and probe thereof
CN113528660A (en) * 2020-09-18 2021-10-22 深圳汇芯生物医疗科技有限公司 Risk assessment device for prostate cancer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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WO2017085553A1 (en) * 2015-11-20 2017-05-26 Mdxhealth Research B.V. Method for predicting and treating clinically significant prostate cancer
US11913076B2 (en) * 2017-05-12 2024-02-27 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Prostate cancer gene profiles and methods of using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111518908A (en) * 2020-05-18 2020-08-11 奥尔文泰生物科技(杭州)有限公司 Urine prostate cancer marker combination and application thereof in preparation of accurate diagnostic reagent
CN113528660A (en) * 2020-09-18 2021-10-22 深圳汇芯生物医疗科技有限公司 Risk assessment device for prostate cancer
CN113512597A (en) * 2020-12-02 2021-10-19 上海仁度生物科技股份有限公司 Real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis and special primer and probe thereof

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