REAGENT KIT AND MARKERS FOR DETECTING RENAL CELL CARCINOMA METHOD THEREOF

Abstract

Embodiments of the present disclosure provide a reagent kit and a method for detecting renal cell carcinoma. The reagent kit includes a set of primer-probe mixes for detecting miRNAs in exosomes. The set of primer-probe mixes includes a reverse transcription primer R-23b for a specific reverse transcription miR-23b-5p target, a universal forward primer Ge—F, a specific reverse primer 23b-R, and a specific probe 23b-P; a reverse transcription primer R-34c for a specific reverse transcription miR-34c-5p target, a universal forward primer Ge—F, a specific reverse primer 34c-R, and a specific probe 34c-P; a reverse transcription primer R-210 for a specific reverse transcription miR-210-3p target, a universal forward primers Ge—F f, a specific reverse primer 210-R, and a specific probe 210-P; and a reverse transcription primer R-508 for a specific reverse transcription miR-508-3p target, a universal forward primer Ge—F, a specific reverse primer 508-R, and a specific probe 508-P.

Claims

1. A reagent kit for detecting renal cell carcinoma, comprising: a set of primer-probe mixes for detecting miRNAs in exosomes, wherein: the set of primer-probe mixes includes a reverse transcription primer R-23b for a specific reverse transcription miR-23b-5p target, a universal forward primer Ge—F for fluorescent PCR (polymerase chain reaction) detection, a specific reverse primer 23b-R, and a specific probe 23b-P; a reverse transcription primer R-34c for a specific reverse transcription miR-34c-5p target, a universal forward primer Ge—F for fluorescent PCR detection, a specific reverse primer 34c-R, and a specific probe 34c-P; a reverse transcription primer R-210 for a specific reverse transcription miR-210-3p target, a universal forward primers Ge—F for fluorescent PCR detection, a specific reverse primer 210-R, and a specific probe 210-P; and a reverse transcription primer R-508 for a specific reverse transcription miR-508-3p target, a universal forward primer Ge—F for fluorescent PCR detection, a specific reverse primer 508-R, and a specific probe 508-P; wherein the set of primer-probe mixes includes: TABLE-US-00015 R-23b: 5′-GTGCTAAGCACAGCAGGGTCCGAGGTATTCGCTGTGCTTA GCACGTGGTA-3′; R-34c: 5′-CACGATTCGTGAGCAGGGTCCGAGGTATTCGCTCACGAAT CGTGGCAATC-3′; R-210: 5′-TGCATCAGATGTGCAGGGTCCGAGGTATTCGCACATCTGA TGCATCAGCC-3′; R-508 5-CGTACAGTCCAGGCAGGGTCCGAGGTATTCGCCTGGACTGT ACGTCTACTC-3′; Ge-F: 5′-GCAGGGTCCGAGGTATTC-3′; 23b-R: 5′-CATCACATTGCCAGGGAT-3′; 34c-R: 5′-GCAGGCAGTGTAGTTAGCT-3′; 210-R: 5′-GGCTGTGCGTGTGACAGC-3′; 508-R: 5′-GCTGATTGTAGCCTTTTG-3′; 23b-P: 5′FAM-ACCACGTGCTAAGCACAG-MGB 3′; 34c-P: 5′VIC-GATTGCCACGATTCGTGAGC-MGB3′; 210-P: 5′ROX-GCTGATGCATCAGATGTG-MGB 3′; and 508-P:  5′CY5-AGTAGACGTACAGTCCAGG-MGB 3′.

2. The reagent kit according to claim 1, wherein: a target detection of each of miR-23b-5p, miR-34c-5p, miR-210-3p, and miR-508-3p is combined by applying a logistic regression model to establish a combined detection measurement.

3. The reagent kit according to claim 2, wherein: the combined detection measurement includes a combined application determination formula for miR-23b-5p, miR-34c-5p, miR-210-3p and miR-508-3p as follows: Mi-Score=1/[1+exp(−z)], and a parameter z=52.3−0.18*A−0.33*B−0.79*C−0.76*D, wherein A is a Ct value of miR-23b-5p detection, B is a Ct value of miR-34c-5p detection, C is a Ct value of miR-210-3p detection, and D is a Ct value of miR-508-3p detection.

4. The reagent kit according to claim 3 wherein: the reagent kit includes a reverse transcription reagent, a primer-probe mix reagent containing the set of primer-probe mixes, a PCR reagent, a positive control, and a negative control for the combined detection measurement.

5. The reagent kit according to claim 4, wherein: each target detection is completed using a same-tube sample with only two steps including reverse transcription and PCR for a urine sample; an Mi-Score is obtained for the urine sample based on the combined application determination formula, identifying each of A as a Ct value of miR-23b-5p detection, B as a Ct value of miR-34c-5p detection, C as a Ct value of miR-210-3p detection, and D as a Ct value of miR-508-3p detection, for the urine sample; and when the Mi-Score is greater than 0.65, it indicates that a subject has a higher risk of renal cancer, and when the Mi-Score is less than 0.65, it indicates that a subject has a lower risk of renal cancer.

6. The reagent kit according to claim 4, wherein: a composition of the primer-probe mix reagent includes 1 μM Ge—F, 0.25 μM 23b-R, 0.25 μM 34c-R, 0.25 μM 210-R, 0.25 μM 508-R, 0.25 μM 23b-P, 0.25 μM 34c-P, 0.25 μM 210-P, and 0.25 μM 508-P.

7. A method for detecting renal cell carcinoma, comprising: preparing a reagent kit for detecting renal cell carcinoma, wherein the reagent kit includes: a set of primer-probe mixes for detecting miRNAs in exosomes, wherein: the set of primer-probe mixes includes a reverse transcription primer R-23b for a specific reverse transcription miR-23b-5p target, a universal forward primer Ge—F for fluorescent PCR (polymerase chain reaction) detection, a specific reverse primer 23b-R, and a specific probe 23b-P; a reverse transcription primer R-34c for a specific reverse transcription miR-34c-5p target, a universal forward primer Ge—F for fluorescent PCR detection, a specific reverse primer 34c-R, and a specific probe 34c-P; a reverse transcription primer R-210 for a specific reverse transcription miR-210-3p target, a universal forward primers Ge—F for fluorescent PCR detection, a specific reverse primer 210-R, and a specific probe 210-P; and a reverse transcription primer R-508 for a specific reverse transcription miR-508-3p target, a universal forward primer Ge—F for fluorescent PCR detection, a specific reverse primer 508-R, and a specific probe 508-P, wherein the set of primer-probe mixes includes: TABLE-US-00016 R-23b: 5′-GTGCTAAGCACAGCAGGGTCCGAGGTATTCGCTGTGCTTA GCACGTGGTA-3′; R-34c: 5′-CACGATTCGTGAGCAGGGTCCGAGGTATTCGCTCACGAAT CGTGGCAATC-3′; R-210: 5′-TGCATCAGATGTGCAGGGTCCGAGGTATTCGCACATCTGA TGCATCAGCC-3′; R-508 5-CGTACAGTCCAGGCAGGGTCCGAGGTATTCGCCTGGACTGT ACGTCTACTC-3′; Ge-F: 5′-GCAGGGTCCGAGGTATTC-3′; 23b-R: 5′-CATCACATTGCCAGGGAT-3′; 34c-R: 5′-GCAGGCAGTGTAGTTAGCT-3′; 210-R: 5′-GGCTGTGCGTGTGACAGC-3′; 508-R: 5′-GCTGATTGTAGCCTTTTG-3′; 23b-P: 5′FAM-ACCACGTGCTAAGCACAG-MGB 3′; 34c-P: 5′VIC-GATTGCCACGATTCGTGAGC-MGB3′; 210-P: 5′ROX-GCTGATGCATCAGATGTG-MGB 3′; and 508-P:  5′CY5-AGTAGACGTACAGTCCAGG-MGB 3′. obtaining a Mi-Score based on a combined application determination formula: Mi-Score=1/[1+exp(−z)], and a parameter z=52.3−0.18*A−0.33*B−0.79*C−0.76*D, wherein A is a Ct value of miR-23b-5p detection, B is a Ct value of miR-34c-5p detection, C is a Ct value of miR-210-3p detection, and D is a Ct value of miR-508-3p detection; and indicating that a subject has a higher risk of renal cancer when the Mi-Score is greater than 0.65 and indicating that a subject has a lower risk of renal cancer when the Mi-Score is less than 0.65.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated into a part of the specification, illustrate embodiments of the present disclosure and together with the description to explain the principles of the present disclosure.

[0012] FIG. 1 illustrates a miR-23b-5p target amplification plot of renal cancer exosomal miRNA detection;

[0013] FIG. 2 illustrates a miR-34c-5p target amplification plot of renal cancer exosomal miRNA detection;

[0014] FIG. 3 illustrates an miR-210-3p target amplification plot of renal cancer exosomal miRNA detection;

[0015] FIG. 4 illustrates an miR-508-3p target amplification plot of renal cancer exosomal miRNA detection; and

[0016] FIG. 5 illustrates a comparison of AUC value based on ROC (receiver operating characteristic) curves of multi-target-combination index detection and single target index detection.

DETAILED DESCRIPTION

[0017] Technical solutions of the present disclosure are further described in detail below through examples and in conjunction with accompanying drawings.

[0018] The present disclosure relates to the field of preparation of renal cell carcinoma markers and, more particularly, relates to an application of a set of primer-probe mixes in preparing renal cell carcinoma markers and a reagent kit. The combination of miR-23b-5p, miR-34c-5p, miR-210-3p, miR-508-3p is applied in exosomes in the preparation of diagnostic and predictive prostate cancer markers. The method uses the TaqMan fluorescent RT-PCR (reverse transcription-polymerase chain reaction) manner to detect exosomal miRNA targets including miR-23b-5p, miR-34c-5p, miR-210-3p and miR-508-3p in a single tube. According to Ct values obtained after detection, the probability calculation formula of renal cancer occurrence is obtained by binary Logistic linear regression fitting. The reagent kit has the advantages of simple sampling, simple operation, short time, high efficiency and practicality, and objective results; and its clinical performance is better than existing renal cancer diagnosis technology, thereby having a desirable application prospect.

Exemplary Embodiment 1: The Composition of the Reagent Kit

[0019] In one embodiment, the renal cancer exosomal miRNA detection reagent kit may include a reagent for preparing reverse transcription reaction system, a primer-probe mix reagent and a PCR reagent for preparing a PCR system, and positive and negative controls for quality control testing process.

[0020] 1) The reverse transcription reagent may be 5×AMV RT (avian myeloblastosis virus reverse transcriptase) buffer, which may include AMV RT (10 U/μl), 0.05 μM R-23b, 0.05 μM R-34c, 0.05 μM R-210, 0.05 μM R-508, 250 mM Tris-HCl (pH 8.3), 250 mM KCl, 50 mM MgCl.sub.2, 2.5 mM spermine, 50 mM DTT (dithiothreitol), and 2.5 mM dNTP (deoxynucleotide triphosphate).

[0021] 2) The composition of the primer-probe mix reagent may include 1 μM Ge—F, 0.25 μM 23b-R, 0.25 μM 34c-R, 0.25 μM 210-R, 0.25 μM 508-R, 0.25 μM 23b-P, 0.25 μM 34c-P, 0.25 μM 210-P, and 0.25 μM 508-P.

[0022] 3) The PCR reagent may be 2×PCR buffer, which may include Taq (T. aquaticus) DNA polymerase (2 U/μl), 200 mmol/L Tris-HCl (pH 8.3), 60 mmol/L MgCl.sub.2, 350 mmol/L KCl, 2.5 mmol/L DTT, and 5 mM dNTP.

[0023] 4) The positive control may be combination of synthetic miRNA mimic fragments. The composition, concentration and sequence of the positive control may include:

TABLE-US-00003 0.1 pg/mL miR-23b-5p mimics (5′-AUCACAUUGCCAGGGAUUACCAC-3′) 0.1 pg/mLmiR-34c-5p mimics (5′-AGGCAGUGUAGUUAGCUGAUUGC-3′), 0.1 pg/mLmiR-210-3p mimics (5′-CUGUGCGUGUGACAGCGGCUGA-3), and 0.1 pg/mLmiR-508-3p mimics (5′-UGAUUGUAGCCUUUUGGAGUAGA-3′)

[0024] 5) The negative control may be ultrapure water for sub-packaging.

Exemplary Embodiment 2: Standard Material Preparation and Linearity Verification

[0025] 1) The Composition, Concentration and Sequence of Synthetic miRNA Mimic Fragments May Include:

TABLE-US-00004 10 pg/mL miR-23b-5p mimics (5′-AUCACAUUGCCAGGGAUUACCAC-3′), 10 pg/mLmiR-34c-5p mimics (5′-AGGCAGUGUAGUUAGCUGAUUGC-3′), 10 pg/mLmiR-210-3p mimics (5′-CUGUGCGUGUGACAGCGGCUGA-3′), and 10 pg/mLmiR-508-3p mimics (5′-UGAUUGUAGCCUUUUGGAGUAGA-3′)

[0026] The high-concentration miRNA standard material E1 may be prepared according to above-mentioned components with thorough mixing. E1 may be taken as the stock solution to be diluted with ultrapure water for 10-fold to obtain E2. Similarly, a 10-fold dilution gradient may be used to perform a total of 6 dilutions, and total 7 concentrations of standard materials of E1, E2, E3, E4, E5, E6, and E7 may be obtained, respectively.

[0027] Reverse transcription may be performed using the following formula shown in Table 1.

TABLE-US-00005 TABLE 1 Component Volume Standard material 8 μL Reverse transcription reagent 2 μL

[0028] 2) Fluorescent PCR Detection

[0029] After the preparation is completed, incubation may be performed at a constant temperature of 16° C. for 30 min, and cDNA fragments with extended sequences may be obtained after reverse transcription. After reverse transcription is completed, fluorescent PCR amplification may be performed according to the following formula shown in Table 2.

TABLE-US-00006 TABLE 2 Component Volume cDNA 10 μL Primer-probe mix reagent  5 μL PCR reagent 15 μL

[0030] After the preparation is completed, PCR detection may be performed using the following procedure, where corresponding procedure parameters are shown in Table 3.

TABLE-US-00007 TABLE 3 Temperature Time Number of cycles 95° C. 10 min  1 cycle 95° C. 10 s    45 cycles 60° C. 30 s   (fluorescence collection)

[0031] As the results shown in FIGS. 1 to 4, the fluorescence curves of four channels of miR-23b-5p, miR-34c-5p, miR-210-3p, and miR-508-3p may all show standard S shapes, and the linear correlation coefficients r may be 0.999, 0.997, 0.987, 0.981, respectively, showing a desirable linear relationship. It demonstrates that multiplex reverse transcription quantitative PCR (RT-qPCR) established in the present disclosure may have desirable analysis and detection performance in detection of mimic miRNA samples.

Exemplary Embodiment 3: Renal Cancer Exosomal miRNA Detection Using Urine Samples

[0032] 1) Exosomal miRNA Extraction and Reverse Transcription of Urine Samples

[0033] About 5 mL of a subject's urine may be collected; the exoEasy Maxi Kit (QIAGEN, CAT: 76064), which may include 20 vesicle preps containing 20 exoEasy maxi spin columns, collection tubes (50 ml) and the like, may be used to extract exosomes in the urine; and the Universal microRNA Purification Kit (EZ Bioscience, CAT: EZB-miRN1) may be used to extract miRNA extract in exosomes. Reverse transcription may be performed using the following formula shown in Table 4.

TABLE-US-00008 TABLE 4 Component Volume Urine miRNA extract/control 8 μL Reverse transcription reagent 2 μL

[0034] 2) Fluorescent PCR Detection:

[0035] After the preparation is completed, incubation may be performed at a constant temperature of 16° C. for 30 min, and cDNA fragments with extended sequences may be obtained after reverse transcription. After reverse transcription is completed, fluorescent PCR amplification may be performed according to the following formula shown in Table 5.

TABLE-US-00009 TABLE 5 Component Volume cDNA 10 μL Primer-probe mix reagent  5 μL PCR reagent 15 μL

[0036] After the preparation is completed, PCR detection may be performed using the following procedure, where corresponding procedure parameters are shown in Table 6.

TABLE-US-00010 TABLE 6 Temperature Time Number of cycles 95° C. 10 min  1 cycle 95° C. 10 s   45 cycles 60° C. 30 s   (fluorescence collection)

[0037] 3) Substitution into Regression Formula and Result Interpretation

[0038] According to the detection result of fluorescent PCR, the result may be substituted into the following formula: Mi-Score=1/[1+exp(−z)], and a parameter z=52.3−0.18*A−0.33*B−0.79*C−0.76*D, where A is the Ct value of miR-23b-5p detection, B is the Ct value of miR-34c-5p detection, C is the Ct value of miR-210-3p detection, and D is the Ct value of miR-508-3p detection. When detected Mi-Score is greater than 0.65, it indicates that the subject may have a higher risk of developing renal cancer; and when detected Mi-Score is less than 0.65, it indicates that the subject may have a lower risk of developing renal cancer.

Exemplary Embodiment 4: Clinical Sample Experiment 1 (Training Set)

[0039] The urine samples of 247 patients who were ready to undergo renal biopsy were collected from the First Affiliated Hospital of Zhejiang University for implementation. Four miRNA targets of miR-23b-5p, miR-34c-5p, miR-210-3p and miR-508-3p in urinary exosomes were detected simultaneously, and Mi-Score was calculated. Taking the result of renal biopsy as the gold standard (113 cases of renal cancer and 134 cases of non-renal cancer in 247 samples), the logistic regression model may be used to calculate the combined determination formula of all miRNAs, and the ROC (receiver operating characteristic) curve may be drawn (as shown in FIG. 5). The final formula of the Mi-Score may be Mi-Score=1/[1+exp(−z)], and a parameter z=52.3−0.18*A−0.33*B−0.79*C−0.76*D, where A is the Ct value of miR-23b-5p detection, B is the Ct value of miR-34c-5p detection, C is the Ct value of miR-210-3p detection, and D is the Ct value of miR-508-3p detection. Meanwhile, using the result of renal biopsy as the gold standard and the Ct value detected by a single exosomal miRNA as a variable, the ROC curve may be drawn (as shown in FIG. 5). Comparing the performance of the multi-target (e.g., four-target) combination detection with the performance of the single target detection, it is found that, AUC (area under curve) values may be the following: combination detection (AUC=0.908)>miR-23b-5p (AUC=0.783)>miR-34c-5p (AUC=0.751)>miR-210-3p (AUC=0.732)>miR-508-3p (AUC=0.696). According to the ROC curves, when the sensitivity is near 90%, specificity and other data of each detection system are compared and summarized in Table 7 below.

TABLE-US-00011 TABLE 7 Target detection AUC Sensitivity Specificity Combination detection 0.908 92.3% 93.4% miR-23b-5p 0.783 90.2% 60.4% miR-34c-5p 0.751 91.1% 52.9% miR-210-3p 0.732 90.7% 50.8% miR-508-3p 0.696 88.0% 47.2%

Exemplary Embodiment 5: Clinical Sample Experiment 2 (Testing Set)

[0040] The urine samples of 132 patients who were ready to undergo renal biopsy were collected from the First Affiliated Hospital of Zhejiang University for implementation. Meanwhile, according to exemplary embodiment 3, targets of miR-23b-5p, miR-34c-5p, miR-210-3p, and miR-508-3p in the exosomes in urine were detected, and Mi-Score was calculated. Taking the result of renal biopsy as the gold standard (67 cases of renal cancer and 65 cases of non-renal cancer in 132 samples), a two-way table may be drawn; and the data of sensitivity, specificity, negative predictive value, positive predictive value and the like of exosomal miRNA for renal cancer detection (Table 8) may be calculated. Meanwhile, diagnostic information of CT scan images for a total of 109 cases was collected. Taking renal biopsy as the gold standard, the sensitivity, specificity, negative predictive value, and positive predictive value of CT detection of renal cancer (Table 9) may be calculated. Through detection performance comparison between renal cancer miRNA detection and CT detection, it is found that detection performance of renal cancer miRNA may be significantly desirable than detection performance of CT scan images (Table 9), which may indicate that renal cancer exosomal miRNA may have excellent detection performance. Combined with advantages of simple sampling, simple operation, short time and low cost, renal cancer exosomal miRNA detection may have broad application prospects.

TABLE-US-00012 TABLE 8 Renal cancer exosomal Renal biopsy miRNA detection Positive Negative Total Positive 63 3 66 Negative 4 62 66 Total 67 65 132

TABLE-US-00013 TABLE 9 Renal cancer CT scan Performance index miRNA detection image diagnosis Sensitivity 94.0% 82.1% Specificity 95.4% 76.9% Positive predictive value 95.5% 78.6% Negative predictive value 93.9% 80.6% Compliance rate 94.7% 79.5%

[0041] Sequence

[0042] Reverse transcription primers and probes may include:

TABLE-US-00014 R-23b: 5′-GTGCTAAGCACAGCAGGGTCCGAGGTATTCGCTGTGCTTA GCACGTGGTA-3′; R-34c: 5′-CACGATTCGTGAGCAGGGTCCGAGGTATTCGCTCACGAAT CGTGGCAATC-3′; R-210: 5′-TGCATCAGATGTGCAGGGTCCGAGGTATTCGCACATCTGA TGCATCAGCC-3′; R-508 5-CGTACAGTCCAGGCAGGGTCCGAGGTATTCGCCTGGACTGT ACGTCTACTC-3′; Ge-F: 5′-GCAGGGTCCGAGGTATTC-3′; 23b-R: 5′-CATCACATTGCCAGGGAT-3′; 34c-R: 5′-GCAGGCAGTGTAGTTAGCT-3′; 210-R: 5′-GGCTGTGCGTGTGACAGC-3′; 508-R: 5′-GCTGATTGTAGCCTTTTG-3′; 23b-P: 5′FAM-ACCACGTGCTAAGCACAG-MGB 3′; 34c-P: 5′VIC-GATTGCCACGATTCGTGAGC-MGB3′; 210-P: 5′ROX-GCTGATGCATCAGATGTG-MGB 3′; and 508-P:  5′CY5-AGTAGACGTACAGTCCAGG-MGB 3′.

[0043] From above-mentioned embodiments, it may be seen that the solutions provided by the present disclosure may achieve at least the following beneficial effects.

[0044] The present disclosure provides an exosomal miRNA detection kit which is fast, convenient, and high in accuracy, and realizes early auxiliary diagnosis of renal cancer, and provides the combined use of miR-23b-5p, miR-34c-5p, miR-210-3p, miR-508-3p in exosomes as a renal cancer molecular marker.

[0045] The present disclosure provides a kit for the combined detection of renal cancer with miR-23b-5p, miR-34c-5p, miR-210-3p, and miR-508-3p exosomes of urine. The kit uses multiplex qRT-PCR technology to simultaneously detect four markers of miR-23b-5p, miR-34c-5p, miR-210-3p and miR-508-3p in exosomes in one urine sample. Furthermore, miRNA positive and negative controls have been developed to control entire detection process of the kit. The positive control may be synthetic miRNA mixtures, and the negative control may be ultrapure water.

[0046] One objective of the present disclosure is to provide a combined application of miR-23b-5p, miR-34c-5p, miR-210-3p and miR-508-3p in exosomes in the preparation of markers for diagnosis and prediction of prostate cancer. The method may use the Taqman fluorescence RT-PCR manner to detect the exosomal miRNA targets miR-23b-5p, miR-34c-5p, miR-210-3p, miR-508-3p in a single tube; and according to the Ct values obtained after detection, the calculation formula of the probability of occurrence of renal cancer may be obtained by binary Logistic linear regression fitting.

[0047] The present disclosure provides a set of miRNA targets in renal cancer exosomes for combined detection. The targets may include miR-23b-5p, miR-34c-5p, miR-210-3p, and miR-508-3p, and a Logistic regression model may be established with such four targets. The model can obtain the risk index Mi-Score of renal cancer, which can be used as an indicator for early diagnosis of renal cancer, thereby improving assay performance for diagnosing and predicting kidney cancer.

[0048] Moreover, the present disclosure also provides a detection kit based on above-mentioned solutions and corresponding application. The kit may have the advantages of simple sampling, simple operation, short time, high efficiency and practicality, and objective results; and its clinical performance is better than existing renal cancer diagnosis technology, thereby having a desirable application prospect.

[0049] Although some embodiments of the present disclosure have been described in detail through examples, those skilled in the art should understand that above-mentioned examples are provided for illustration only and not for the purpose of limiting the scope of the disclosure. Those skilled in the art should understand that modifications may be made to above-mentioned embodiments without departing from the scope and spirit of the present disclosure. The scope of the present disclosure may be defined by appended claims.