Markers, primer sets, high-sensitivity and high-specificity kits and detection methods for detecting urothelial carcinoma

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a marker, a primer set, a highly sensitive and highly specific kit and a detection method for detecting urothelial carcinoma. The present invention avoids nonspecific amplification by reselecting markers, designing primers and probes; then, by adjusting the concentration ratio of primers and probes and the PCR reaction conditions, by setting a positive threshold and three repeated experiments based on the minimum detection limit study, the developed detection kit is tested in 80 UC and 130 non-cancer individuals. The results show that the sensitivity is as high as 97.5% and the specificity is as high as 96.9%. Compared with existing products, the sensitivity and specificity are significantly improved, and it is more suitable for clinical auxiliary diagnosis and population screening than existing products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a marker, a primer set, a highly sensitive and highly specific kit and a detection method for detecting urothelial carcinoma. Background Art

[0002] Urothelial carcinoma (UC) is one of the most common malignant tumors of the urinary system, including bladder and urethral malignancies in the lower urinary tract and renal pelvis and ureteral malignancies in the upper urinary tract, of which about 90% are bladder UC. According to the World Health Organization, there are nearly 800,000 new cases of bladder UC worldwide each year, while there are only nearly 100,000 new cases of bladder UC in my country each year. The number of people with bladder UC in my country has reached 1.3 million, ranking sixth in the world. The incidence rate in men is 4-5 times that in women. Men over 45 years old are the most susceptible group.

[0003] The preoperative qualitative diagnosis of UC mainly relies on ureteroscopy and pathological biopsy. The current non-invasive urine diagnostic techniques such as urine exfoliative cytology and fluorescence in situ hybridization (FISH) have low sensitivities (40-50% and 60-70%, respectively), which are difficult to meet clinical needs. Recently, Guangzhou Benchmark Medical Co., Ltd. (hereinafter referred to as Benchmark Medical) developed a urine DNA methylation qPCR detection kit based on human ONECUT2 and VIM genes (patent number: CN110872631B). It was tested in two groups of suspected bladder UC patients (one group of 192 people and the other group of 98 people) (Clin Epigenet. 2021, 13: 91), with sensitivities of 88.1% and 91.2%, respectively, and specificities of 89.7% and 85.7%, respectively. Next, the kit was tested in two groups of suspected upper urinary tract UC (one group of 153 people and the other group of 75 people) (Chinese Journal of Urology, 2023, 44(10): 725-730), with sensitivities of 83.0% and 85.2%, and specificities of 92.0% and 85.7%, respectively. In summary, the sensitivity and specificity of the kit for detecting UC are both 80-90%, which is much higher than traditional non-invasive techniques (exfoliative cytology and FISH). It is easy to operate and inexpensive, and is expected to be used in clinical practice.

[0004] However, the sensitivity and specificity of the test kit need to be improved, only 80-90%, which is prone to misdiagnosis. The characteristic of UC is that the onset is hidden; even if patients have hematuria, 80-90% of them do not have UC. A large number of misdiagnoses will burden patients and hospitals, and they have to perform a large number of endoscopic examinations, which are both invasive and expensive. Summary of the invention

[0005] In response to the above problems, the present invention improves the defects of the existing detection system (non-specific amplification and system deviation) and provides a highly sensitive and specific kit and detection method for urothelial carcinoma detection, with a sensitivity of up to 97.5% and a specificity of up to 96.9%. Compared with existing products, both significance and sensitivity are significantly improved, and it is more suitable for clinical auxiliary diagnosis and population screening.

[0006] The first aspect of the present invention provides a marker for detecting urothelial carcinoma, which is a combination of a nucleotide sequence of a co-methylated region indicated by CG, such as a target gene 1 of SEQ ID NO.1, and a nucleotide sequence of a target gene 2 of SEQ ID NO.2, or a combination of their fully complementary sequences.

[0007] A second aspect of the present invention provides a primer set for detecting urothelial carcinoma, the primer set comprising:

[0008] Primer set 5584-1216-2 for target gene 1 SEQ ID NO.1, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.3, a reverse primer with a nucleotide sequence such as SEQ ID NO.4, and a probe with a nucleotide sequence such as SEQ ID NO.5;

[0009] The primer set 4092-0204 for the target gene 2 SEQ ID NO.2 consists of a forward primer with a nucleotide sequence such as SEQ ID NO.6, a reverse primer with a nucleotide sequence such as SEQ ID NO.7, and a probe with a nucleotide sequence such as SEQ ID NO.8.

[0010] In one or more embodiments, the primer set also includes an internal reference ACTB primer set for the internal reference gene ACTB, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10, and a probe with a nucleotide sequence such as SEQ ID NO.11.

[0011] The third aspect of the present invention provides a highly sensitive and specific kit for detecting urothelial carcinoma, comprising the above-mentioned primer set 5584-1216-2 and primer set 4092-0204, or comprising the above-mentioned primer set 5584-1216-2 and primer set 4092-0204 and an internal reference primer set. Preferably, the internal reference primer set is the above-mentioned internal reference ACTB primer set.

[0012] In one or more embodiments, the kit further comprises at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

[0013] In one or more embodiments, the kit is a qPCR kit.

[0014] In one or more embodiments, the reaction volume of the qPCR kit is 25 μl.

[0015] In one or more embodiments, in the qPCR kit, the forward primer and reverse primer concentrations in the 5584-1216-2 primer set are both 0.2 μM, and the probe concentration is 0.1 μM;

[0016] The forward primer and reverse primer concentrations in the 4092-0204 primer set were both 0.4 μM, and the probe concentration was 0.2 μM;

[0017] The forward primer and reverse primer concentrations in the internal reference ACTB primer set were both 0.4 μM, and the probe concentration was 0.2 μM.

[0018] The fourth aspect of the present invention provides a highly sensitive and specific qPCR detection method for urothelial carcinoma, comprising the following steps:

[0019] (1) Extract DNA from urine samples;

[0020] (2) performing bisulfite conversion on the extracted DNA samples;

[0021] (3) Sample DNA detection: using primer set 5584-1216-2 and primer set 4092-0204 or the above-mentioned detection kit containing primer set 5584-1216-2 and primer set 4092-0204, and ACTB as the internal reference gene, the converted DNA sample was tested by triple qPCR, and the test results were determined based on the test data;

[0022] The judgment criteria are as follows: the minimum detection limit is 1.25%, ① when the Ct value of the internal reference gene Ct ACTB≤35, the test result is valid; Ct ACTB>35, the test result is invalid; ② when the Ct difference ΔCt between the marker target gene 1 or 2 and the internal reference gene ACTB≤8; the sample is judged to be positive; ③ when the Ct difference ΔCt between the marker target gene 1 or 2 and the internal reference gene ACTB is both>8, the sample is judged to be negative; in order to avoid population bias and random noise.

[0023] In one or more embodiments, in step (3), the qPCR detection reaction procedure is: 95°C for 30 seconds; 95°C for 15 seconds, 62°C for 15 seconds, collecting fluorescence signals at 62°C, and 45 cycles.

[0024] In one or more embodiments, three replicate experiments are performed for each sample to further avoid population bias and random noise.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention avoids nonspecific amplification by redesigning primers and probes, and then avoids group bias and random noise by adjusting the concentration ratio of primers and probes and PCR reaction conditions, setting the positive threshold and repeating the experiment three times based on the minimum detection limit, and developing a detection kit for 80 UC and 130 non-cancer individuals. The results showed that the sensitivity was as high as 97.5% and the specificity was as high as 96.9%. Compared with existing products, the sensitivity and specificity were significantly improved, and the product was more suitable for clinical auxiliary diagnosis and group screening than existing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The first generation sequencing results of human VIM gene (cg12874092 site) and ONECUT2 gene (cg10835584 site) in positive control and negative control; the blue peak represents the cytosine signal, i.e., the methylation level;

[0028] Figure 2 Schematic diagram of blank, negative and positive control experimental study results for the benchmark medical treatment and the primer set of the present invention;

[0029] Figure 3 Figure 1 is a schematic diagram of the design of primer probes of the present invention, Figure A is a schematic diagram of the design of the 4092-0204 primer set, and Figure B is a schematic diagram of the design of the 5584-1216-2 primer set;

[0030] Figure 4 The clinical sample test results were studied for the minimum detection limit of the present invention;

[0031] Figure 5 Schematic diagram of performance comparison between the kit of the present invention and the kit in the reference medical treatment, * represents P<=0.05. DETAILED DESCRIPTION

[0032] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0033] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0034] In the description of the present invention, the description with reference to the terms "some embodiments", "examples", etc. means that the specific methods and materials described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific methods and materials described can be combined in any one or more embodiments or examples in a suitable manner.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] the term:

[0037] As used in the present invention, "marker" refers to a biological molecule or a biological molecule fragment, whose change and / or detection can be associated with a specific physical condition or state. The terms "marker", "molecular marker" or "biomarker" are used interchangeably throughout the disclosure of the invention. In the present invention, unless otherwise stated, the "marker" refers to a "cancer (tumor) marker". In the present invention, unless otherwise stated, the cancer is urothelial carcinoma.

[0038] As used in the present invention, the "detection" includes "assessment", "determination", "analysis", "prediction" and "evaluation"; the "evaluation" or "assessment" also includes "scoring".

[0039] As used in the present invention, the "patient", "subject" or "individual" may refer to an organism, and in some aspects, the subject may be a human. The subject providing the sample may include a population at potential risk of the disease or a population diagnosed with the disease. The disease in the present invention refers to urothelial carcinoma.

[0040] As used in the present invention, the "sample" and "specimen" are used interchangeably, which include substances obtained from individual or separated tissues, cells (such as epithelial cells) or body fluids (such as urine, serum, plasma) that are suitable for tumor marker detection.

[0041] As used in the present invention, the "urothelial carcinoma" refers to a cancer that occurs in the epithelial cells of the urinary tract, including the bladder, renal pelvis, ureter, and urethra. Urothelial carcinoma is multiple in nature or synchrony, so the "urothelial carcinoma" of the present invention includes primary cancer and cancer that has metastasized in the tissue. In some specific embodiments, the urothelial carcinoma is classified according to the site of tumor occurrence, and includes but is not limited to: renal pelvic cancer, ureteral cancer, bladder cancer, and urethral cancer. It should be understood that the urothelial carcinoma includes high-grade urothelial carcinoma and low-grade urothelial carcinoma.

[0042] As used herein, "complementary" and "complementarity" refer to a nucleotide (e.g., 1 nucleotide) or polynucleotide (e.g., a sequence of nucleotides) associated with the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial", in which only some of the nucleic acid bases are matched according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains affects the efficiency and intensity of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0043] As used herein, "methylation" refers to methylation of cytosine at cytosine position C5 or N4, N6 site of adenine or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated, because in vitro DNA amplification methods generally do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA that is unmethylated or methylated from the original template, respectively.

[0044] The methylation state can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, ratio, percentage, etc.). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification spectra after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Therefore, a value such as a methylation value represents the methylation state and can therefore be used as a quantitative indicator of the methylation state in multiple copies of a locus. The degree of co-methylation is represented or indicated by the methylation state of more than one methylation site, and co-methylation is defined as when the methylation state of more than one methylation site is methylated within a methylated region.

[0045] Co-methylation detection methods include: methylation-specific PCR (MSP), DNA methylation chip, targeted DNA methylation sequencing, digital PCR quantification and fluorescence quantitative PCR.

[0046] In some embodiments, DNA (eg, genomic DNA, such as extracted genomic DNA or processed genomic DNA) is isolated by any standard means in the art, including use of commercially available kits.

[0047] As used herein, "methylation assay" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0048] Unless otherwise specified, the reagents, materials, and equipment used in the examples of the present invention are all commercially available; the test methods, unless otherwise specified, are all conventional test methods in the art.

[0049] Materials and Methods

[0050] 1. Sample collection

[0051] 210 urine samples (80 UC and 130 non-UC) were collected from Zhujiang Hospital of Southern Medical University and tested at Guangzhou Zhongxin Gene Medical Technology Co., Ltd. Each sample was 50-100 ml of urine, all of which were suspected UC cases and untreated. The procedures followed in this study were in accordance with the requirements of the World Medical Association's Declaration of Helsinki, revised in 2013.

[0052] 2. DNA extraction from urine exfoliated cells

[0053] In the examples, HiPure Tissue DNA MicroKits (column method) produced by Guangzhou Meiji Biotechnology Co., Ltd. was used to extract DNA from bladder exfoliated cells. The recovered DNA fragments were tested for concentration using qubit. Other commercially available reagents can also be used for DNA extraction.

[0054] 3. DNA transformation

[0055] Generally, DNA converted with bisulfite is used for whole genome amplification and then MSP sequencing. In the present embodiment, the bisulfite conversion kit of Lightning Conversion Reagent of zymo research is used to convert DNA for downstream methylation qPCR MSP detection (detection of methylated fragments). Bisulfite conversion is performed according to the instructions.

[0056] 4. First-generation sequencing analysis

[0057] BSP primers were designed using olig7 or methyl express software. The amplified products were sent to Guangzhou Aiki Biotechnology Co., Ltd. for first-generation sequencing. The sequencing results were analyzed using SnapGene 4.3.6 software.

[0058] 5. Statistical analysis:

[0059] Data were processed using R language 4.3.1 statistical software. The efficacy of the two examination methods was expressed as a rate, and the inter-group comparison was performed using the χ2 test. Statistical bar graph data were expressed as mean and 95% CI, and the 95% CI was calculated using the Wald method. P < 0.05 was considered statistically significant.

[0060] Example 1 Reproduction and sequencing analysis of prior art CN110872631B

[0061] Benchmark Medical's patent (patent number: CN110872631B) targets markers (sequences as shown in Table 1), including human VIM gene SEQ ID NO.2 (cg12874092 site) and ONECUT2 gene SEQ ID NO.1 (cg10835584 site), and designs three sets of primers and probes, which are as follows:

[0062] (1) SEQ ID NO. 23 and SEQ ID NO. 45 for SEQ ID NO. 1, and SEQ ID NO. 67;

[0063] SEQ ID NO.24 and SEQ ID NO.46 for SEQ ID NO.2, and SEQ ID NO.68;

[0064] (2) SEQ ID NO.89 and SEQ ID NO.111 for SEQ ID NO.1, and SEQ ID NO.133;

[0065] SEQ ID NO.90 and SEQ ID NO.112 for SEQ ID NO.2, and SEQ ID NO.134;

[0066] (3) SEQ ID NO.155 and SEQ ID NO.177 for SEQ ID NO.1, and SEQ ID NO.199;

[0067] SEQ ID NO. 156 and SEQ ID NO. 178 for SEQ ID NO. 2, and SEQ ID NO. 200.

[0068] At the same time, in Example 6 of the specification, the corresponding experimental conditions are given (reaction volume 25 μl, final primer / probe concentration is 0.24 μM / 0.12 μM, qPCR reaction program is: 95°C for 5 minutes; 95°C for 15 seconds, 62°C for 40 seconds, collecting fluorescence signals at 62°C, 60 cycles.

[0069] The inventors first used the above-mentioned system with the benchmark medical treatment to conduct qPCR experiments: blank control, negative control (healthy people) and positive control (UC patients), and then performed first-generation sequencing analysis. The BSP primers were designed as shown in Table 1.

[0070] Table 1 BSP sequencing primers

[0071]

[0072] The first generation sequencing results are as follows Figure 1 As shown, it can be seen that the positive control has methylation at both sites, while the negative control has no methylation at both sites.

[0073] qPCR results Figure 2 As shown, the results show that the three sets of primer probes of Benchmark Medical all have signals in the blank control or negative control target gene ( Figure 2 AC), indicating that there are different degrees of non-specific amplification. Among them, the second set of primer probes of the benchmark medical treatment has the least non-specific amplification signal. Therefore, the inventors then selected this group for clinical experiments and made improvements to overcome the non-specific amplification problem of the prior art benchmark medical experimental system.

[0074] Example 2 Design of new markers and primer sets

[0075] On the basis of Example 1, in order to overcome the non-specific amplification problem of the prior art benchmark medical experimental system, the detection area was redesigned, and a new marker for urothelial carcinoma detection was proposed, and the marker sequence is shown in Table 2. Although the new detection area overlaps with the marker detection area in the benchmark medical patent CN110872631B, it is lower than the patent requirement of benchmark medical>70%.

[0076] Table 2 Marker sequences

[0077]

[0078] Primers and probes were redesigned for the marker sequences in Table 2 ( Figure 3). The primer set designed for human ONECUT2 gene (cg10835584 site) is named 5584-1216-2. Its similarity with SEQ ID NO.155 and SEQ ID NO.177 of SEQ ID NO.1 of benchmark medical, and SEQ ID NO.199 is 100%, 84%, 54% and 74% respectively. The primer set designed for human VIM gene (cg12874092 site) is named 4092-0204, and its similarity with SEQ ID NO.90 and SEQ ID NO.112 of SEQ ID NO.2 of benchmark medical, and SEQ ID NO.134 is 62%, 0%, 0% and 67% respectively. It is not the same as the primer probe set sequence in the benchmark medical patent. The sequences of the redesigned primer sets 5584-1216-2 and 4092-0204 are shown in Table 3.

[0079] Table 3 Primer set sequences

[0080]

[0081] Example 3 qPCR detection

[0082] According to the primer set established in Example 2, ACTB was used as the internal reference gene, and the newly designed primer set 5584-1216-2 and 4092-0204 and the primer probe of the internal reference ACTB were combined to establish a triple qPCR system. The reagent used was FastStar Probe Kit from Cowin, and the converted urine DNA sample was subjected to qPCR test.

[0083] The qPCR reaction volume was 25 μl. The primer / probe concentration of the primer set 5584-1216-2 was 0.2 μM / 0.1 μM. The primer / probe concentration of the internal reference ACTB and 4092-0204 primer sets was twice that of the 5584-1216-2 primer set (0.4 μM / 0.2 μM).

[0084] The qPCR reaction program was: 95°C for 30 seconds; 95°C for 15 seconds, 62°C for 15 seconds, collecting fluorescence signals at 62°C, for 45 cycles.

[0085] Blank control, negative control and positive control experiments were carried out, and the results showed that non-specific amplification did not occur and did not affect the accuracy. Figure 2 As shown in D.

[0086] Example 4 Improved determination method

[0087] The evaluation system of Benchmark Medical's patent (patent number: CN110872631B) adopts a method of training an evaluation model based on a clearly diagnosed population (116 UC and 76 non-cancer individuals) (cited from Clin Epigenet. 2021, 13: 91 and Example 7 of patent CN110872631B). The principle of training is to find a threshold that can best distinguish cancer from non-cancer. The result evaluation system (cited from Chinese Journal of Urology, 2023, 44 (10): 725-730.) is as follows:

[0088] According to the Ct difference (ΔCt value) between ONECUT2 and the internal reference gene ACTB and the Ct value of the VIM gene, the methylation status of the sample was obtained by comparing with the critical value. Result judgment: ① When ACTB≤32, the result can be judged, otherwise the sample test result is invalid; ② When the ONECUT2ΔCt value ≤5.7 or the VIM Ct value ≤38.6, that is, when any one of the results of ONECUT2 and VIM is positive, the sample is judged to be positive; ③ When the ONECUT2ΔCt value>5.7 (or the result is not detected) and the VIM Ct value>38.6 (or the result is not detected), that is, when the results of ONECUT2 and VIM are both negative, the sample is judged to be negative.

[0089] Using this evaluation system, they tested two groups of patients suspected of bladder UC (192 people in one group and 98 people in the other group) (Clin Epigenet. 2021, 13: 91), with sensitivities of 88.1% and 91.2%, and specificities of 89.7% and 85.7%, respectively. Then, they tested two groups of patients suspected of upper urinary tract UC (153 people in one group and 75 people in the other group) (Chinese Journal of Urology, 2023, 44 (10): 725-730), with sensitivities of 83.0% and 85.2%, and specificities of 92.0% and 85.7%, respectively.

[0090] After analysis, we believe that there will be systematic deviations in the patent evaluation system of Benchmark Medical. The reasons are as follows:

[0091] Benchmark Medical trained its model based on a sample set of less than 200 people; with more than 100,000 UC cases each year, this sample set is small and cannot represent the entire population, so the trained evaluation model must have systematic bias. This is also the reason why their sensitivity and specificity decreased in other test populations.

[0092] Existing DNA conversion systems are prone to false positives. Before the existing methylation qPCR test, the DNA must be converted with bisulfite to convert the unmethylated C bases into T bases, while the methylated C bases remain unchanged. However, the conversion efficiency is usually around 99%, and nearly 1% of the C bases cannot be converted normally, especially in high GC areas where the conversion rate is poor, and the background noise is even as high as 10% (Nat Biotechnol.2024.PMID:38168991). The two areas detected by the benchmark medical patent system happen to fall in the high GC area (CpG island), so false positives are more likely to occur.

[0093] In order to overcome the problem of systematic bias in the benchmark medical patent evaluation system, this application makes the following improvements:

[0094] 1) Set the positive judgment threshold based on the minimum detection limit study.

[0095] Urine DNA from three UC patients was collected and mixed with urine DNA from healthy people at a ratio of 1:19, i.e., the patient urine DNA accounted for 5%; then gradient dilution was performed to obtain template DNA with concentrations of 2.5%, 1.25%, and 0.625%, for transformation and qPCR detection ( Figure 4 ). Currently, the only urine test kit for bladder UC that has obtained the national Class III medical device certificate on the market - Anhui Dajian Medical Technology Co., Ltd. (Dajian for short) has a minimum detection limit of 5% for its "Human Twist1 Gene Methylation Detection Kit (Fluorescence PCR Method)". Figure 4 As shown, based on the stability of the test results, we set the minimum detection limit to 1.25%, and the detection accuracy is 4 times higher than that of Dajian; the ΔCt value threshold here is 8. Therefore, the criteria for the result judgment of this application evaluation system are as follows:

[0096] ① When the Ct value of the internal reference gene Ct ACTB≤35, the test result is valid; the result can be judged, otherwise the sample test result is invalid; ② When the Ct difference ΔCt between the marker target gene 1 or 2 and the internal reference gene ACTB is ≤8, that is, when the difference result between any one of the target genes and the internal reference gene is positive, the sample is judged to be positive; ③ When the Ct difference between the marker target gene 1 or 2 and the internal reference gene ACTB is negative, the sample is judged to be negative.

[0097] 2) A positive result can only be determined as positive if it occurs repeatedly.

[0098] Since the detection area (marker sequence) of the present application falls on the CpG island position, and the conversion efficiency of the CpG island is low, about 10% of the C bases cannot be effectively converted into T bases, so false positives are prone to occur. In order to avoid false positives, repeated experiments are used. Two experimenters perform conversion and qPCR on the same sample respectively. If the judgment results of the two people are consistent, the results are reported directly. If the judgment results of the two people are inconsistent, a third experiment needs to be done and two consistent results are reported.

[0099] Example 5

[0100] A kit comprises primer sets 4092-0204 and 5584-1216-2, a probe, and an internal reference ACTB primer probe and matching reagents.

[0101] The kit is then used to perform urothelial carcinoma qPCR detection, which includes the following steps:

[0102] (1) Extract DNA from urine samples;

[0103] (2) performing bisulfite conversion on the extracted DNA samples;

[0104] (3) Sample DNA testing: Using the assembled test kit and ACTB as the internal reference gene, the converted DNA samples were tested by triple qPCR, and the test results were determined based on the test data;

[0105] The qPCR reaction volume was 25 μl. The primer / probe concentration of the primer set 5584-1216-2 was 0.2 μM / 0.1 μM. The primer / probe concentration of the internal reference ACTB and 4092-0204 primer sets was twice that of the 5584-1216-2 primer set (0.4 μM / 0.2 μM).

[0106] The qPCR detection reaction program was: 95°C for 30 seconds; 95°C for 15 seconds, 62°C for 15 seconds, collecting fluorescence signals at 62°C, for 45 cycles.

[0107] The judgment criteria are as follows: the minimum detection limit is 1.25%, ① when the Ct value of the internal reference gene Ct ACTB≤35, the test result is valid; Ct ACTB>35, the test result is invalid; ② when the Ct difference ΔCt between the marker target gene 1 or 2 and the internal reference gene ACTB≤8; the sample is judged to be positive; ③ when the Ct difference ΔCt between the marker target gene 1 or 2 and the internal reference gene ACTB is both>8, the sample is judged to be negative, in order to avoid population bias and random noise.

[0108] Two experimenters perform transformation and qPCR on the same sample. If the results of the two people are consistent, the results are reported directly. If the results of the two people are inconsistent, a third experiment is required to report the two consistent results.

[0109] 210 suspected UC cases (80 UC cases and 130 non-UC cases) were tested, and the results were as follows Figure 5 shown.

[0110] Comparative Example 1

[0111] According to the second set of primer probes in Example 2 of the patent specification of Benchmark Medical and the experimental conditions of Example 6, its kit was reproduced, wherein the primer probes are SEQ ID NO.155 and SEQ ID NO.177 for SEQ ID NO.1 in CN110872631B; and SEQ ID NO.90 and SEQ ID NO.112, and SEQ ID NO.134 for SEQ ID NO.2; and internal reference primers and probes SEQ ID NO.221 and SEQ ID NO.222, and SEQ ID NO.223.

[0112] The same 210 suspected UCs (80 UCs and 130 non-UCs) were tested and compared with the results of Example 5. The results are as follows: Figure 5 As shown. The sensitivity of the benchmark medical treatment is 87.5%, and the specificity is 90%, which is consistent with the results reported previously; while the sensitivity of the kit of the embodiment of the present invention is 97.5%, and the specificity is as high as 96.9%, both of which are significantly higher than the benchmark medical treatment. Therefore, the detection performance of the kit of the present invention is significantly better than the kit of the benchmark medical patent CN110872631B, and is more suitable for clinical auxiliary diagnosis and population screening.

[0113] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0114] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A marker for detecting urothelial carcinoma, characterized in that: The marker is a combination of a nucleotide sequence of a co-methylated region indicated by CG, such as a target gene 1 of SEQ ID NO. 1, and a nucleotide sequence of a target gene 2 of SEQ ID NO. 2, or a combination of their completely complementary sequences.

2. A primer set for detecting urothelial carcinoma, characterized in that: The primer set comprises: Primer set 5584-1216-2 for target gene 1, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.3, a reverse primer with a nucleotide sequence such as SEQ ID NO.4, and a probe with a nucleotide sequence such as SEQ ID NO.5; The primer set 4092-0204 for target gene 2 consists of a forward primer with a nucleotide sequence such as SEQ ID NO.6, a reverse primer with a nucleotide sequence such as SEQ ID NO.7, and a probe with a nucleotide sequence such as SEQ ID NO.

8.

3. The primer set according to claim 2, further comprising an internal reference ACTB primer set for an internal reference gene ACTB, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10, and a probe with a nucleotide sequence such as SEQ ID NO.

11.

4. A highly sensitive and specific kit for detecting urothelial carcinoma, characterized in that: Comprising the primer set as claimed in claim 2.

5. A highly sensitive and specific kit for detecting urothelial carcinoma, characterized in that: Comprising the primer set as claimed in claim 3.

6. The kit according to claim 4 or 5, characterized in that It also includes at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

7. The kit according to claim 4 or 5, characterized in that The kit is a qPCR kit.

8. The kit according to claim 5, characterized in that The forward primer and reverse primer concentrations in the 5584-1216-2 primer set were both 0.2 μM, and the probe concentration was 0.1 μM; The forward primer and reverse primer concentrations in the 4092-0204 primer set were both 0.4 μM, and the probe concentration was 0.2 μM; The forward primer and reverse primer concentrations in the internal reference ACTB primer set were both 0.4 μM, and the probe concentration was 0.2 μM.

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