Primer probe combination and kit for detecting chromosome deletion in bladder cancer and its application

Abstract

The present invention relates to the field of biological detection and analysis technology, and discloses a primer probe composition and a kit and application for detecting chromosome deletion in bladder cancer. The method for detecting chromosome deletion in bladder cancer comprises: collecting 50 mL of morning urine from the test subject, centrifuging and collecting the precipitate as a sample; extracting sample DNA; preparing a digital PCR reaction solution using a kit for detecting chromosome deletion in bladder cancer to obtain a detection solution; performing chip-type digital PCR amplification on the detection solution, detecting the fluorescent signal and determining the result. The present invention detects the copy number of 9 chromosomes in the DNA of bladder tumor tissue or urine cells of patients through a chip-type digital PCR system, takes the average value of the concentration of chromosomes 2 and 12 as a reference value, calculates the relative concentration of other chromosomes, and determines whether each chromosome has a copy number abnormality through the relative value, which can be used to screen for early bladder cancer and monitor the prognosis and recurrence of bladder cancer.

Description

Technical Field

[0001] The invention relates to a primer-probe combination and a kit and application for detecting chromosome deletion in bladder cancer, belonging to the technical field of biological detection and analysis. Background Art

[0002] Bladder cancer (BLCA) is one of the most common malignant tumors of the urinary system, ranking 7th in the incidence of male systemic tumors and 10th in females. Bladder cancer most often originates from the cells covering the lining of the bladder, so its research is focused on urothelial carcinoma (UC), which accounts for the majority (90%) of tumors diagnosed in the country. UC, like other malignant tumors, has complex cytogenetic and molecular genetic changes.

[0003] More than 70% of UC are diagnosed as non-invasive (Ta) or superficial invasive papillary lesions (T1). Although they frequently relapse, progression to muscle invasive disease is rare (10% to 20%) and carries a favorable prognosis. In contrast, tumors diagnosed as muscle invasive (≥T2) carry a poor prognosis (5-year survival less than 50%). Carcinoma in situ (CIS) is a high-grade lesion that is strictly “superficial” but carries a poor prognosis. More than 50% of UC of all grades and stages show loss of heterozygosity (LOH) on chromosome 9, many with deletions of entire homologous regions. A critical region has been identified on chr9p21, and the candidate gene within this region is CDKN2A on 9p21.3. In bladder cancer, in addition to chromosome 9, chromosomes 1, 3, 4, 7, 8, 10, 11, and 17 also undergo numerical changes.

[0004] Urine cytology and cystoscopy are the first-line methods for diagnosing bladder cancer. Cystoscopy may cause complications such as urogenital infection, urethral and bladder bleeding. Therefore, non-invasive detection technology with high specificity and sensitivity is urgently needed.

[0005] Liquid biopsy technology based on urine is non-invasive, real-time and dynamic. However, liquid biopsy with urine as a carrier faces the problem that urogenital tumors release few signals, and the corresponding exfoliated cells may not be able to detect driver gene mutations. At present, the methods for using urine exfoliated cells to examine chromosomal abnormalities include fluorescence in situ hybridization (FISH), real-time fluorescence quantitative PCR (real-time PCR), microarray comparative genomic hybridization (a-CGH), etc. Although these methods are non-invasive methods, they have low sensitivity, low throughput, and poor efficiency, and their diagnostic value for early and low-grade tumors is limited. Digital PCR (dPCR) is a quantitative analysis of the reaction system by dividing it into a large number of independent micro-reaction units for PCR amplification, and calculating the number of nucleic acid copies based on Poisson distribution and positive ratio. Compared with traditional PCR, digital PCR technology does not rely on standard curves, has higher sensitivity, accuracy and high tolerance, and is simpler and faster to operate than gene sequencing technology, and can achieve absolute quantitative analysis of samples.

[0006] Patent publication number CN112538530A discloses a bladder cancer detection kit, which provides a modified fluorescent subtraction detection probe for detecting chromosomal and genetic abnormalities that are closely related to the occurrence and development of bladder cancer, and uses FISH technology to detect the probe. Although this method has rapid hybridization and high detection efficiency, the operation is complicated, and medical technicians need to observe each cell under a fluorescent microscope and interpret the results with the naked eye, which is prone to subjective interpretation errors. Summary of the invention

[0007] The present invention overcomes the shortcomings of the existing technology and innovatively uses digital PCR technology to detect the gene copy number in the target area, providing a highly sensitive and specific diagnostic method for bladder cancer, which can be used to screen early bladder cancer and monitor the prognosis and recurrence of bladder cancer.

[0008] To achieve the above object, the present invention provides a primer-probe combination for detecting chromosome deletion in bladder cancer, comprising:

[0009] A primer-probe composition for detecting chromosome 1, comprising:

[0010] Upstream primer C1 F: 5'-ACACTGGGGATCAGGAGGAAA-3',

[0011] Downstream primer C1 R: 5'-CAGTCAGCTTGAGGTGAAGGATA-3',

[0012] Probe C1 Pb: 5′-CTCTGCCCAACGAGCAGCCCC-3′;

[0013] A primer-probe composition for detecting chromosome 2, comprising:

[0014] Upstream primer C2 F: 5'-GCACTTGAGGAGTGTCAGGAAA-3',

[0015] Downstream primer C2 R: 5'-TCCGACTTCTTTGTATGGCAAA-3',

[0016] Probe C2 Pb: 5′-CCCTCCGCAGCAGGTTGGGC-3′;

[0017] A primer-probe composition for detecting chromosome 3, comprising:

[0018] Upstream primer C3 F: 5'-CGCGCCTGGACGAGTCTA-3',

[0019] Downstream primer C3 R: 5'-GGGACACCCTGGGAATGAA-3',

[0020] Probe C3 Pb: 5′-TCCCAGGCCGCAAGGGATGC-3′;

[0021] A primer-probe composition for detecting chromosome 7, comprising:

[0022] Upstream primer C7 F: 5'-CCGAGGGGTAACCCTCATGT-3',

[0023] Downstream primer C7 R: 5'-GCTGCCCTGAGGCACTCTTC-3',

[0024] Probe C7 Pb: 5'-CCGGGAGACAGTCTCCACTCACCC-3';

[0025] A primer-probe composition for detecting chromosome 8, comprising:

[0026] Upstream primer C8 F: 5'-CAGCCTCCTGAGTAGCTGAGATTA-3',

[0027] Downstream primer C8 R: 5'-TGGTGAGACCCCGTCTCTACTAAA-3',

[0028] Probe C8 Pb: 5′-CAGGTGCCCACCACCATGCCC-3′;

[0029] A primer-probe composition for detecting chromosome 9, comprising:

[0030] Upstream primer C9 F: 5'-CAAATGGGAAATAATCCCGAAA-3',

[0031] Downstream primer C9 R: 5'-TGGTGGTATGCTTTGGGAAGTATAA-3',

[0032] Probe C9 Pb: 5′-CACGTGAAAGCCC-3′;

[0033] A primer-probe composition for detecting chromosome 11, comprising:

[0034] Upstream primer C11 F: 5'-GGGAGCAGCCGAGCAAA-3',

[0035] Downstream primer C11 R: 5'-GTTGGCCTCCTTGCTGTTG-3',

[0036] Probe C11 Pb: 5′-CACGCGCCCCGCTGGGG-3′;

[0037] A primer-probe composition for detecting chromosome 12, comprising:

[0038] Upstream primer C12 F: 5'-CACTGGTGCCTCTGCTGTCAT-3',

[0039] Downstream primer C12 R: 5'-CCTCTGCCTCATGCTTGTCAT-3',

[0040] Probe C12 Pb: 5′-CTCCCTGGGCACTGAG-3′;

[0041] A primer-probe composition for detecting chromosome 17, comprising:

[0042] Upstream primer C17 F: 5'-GGGACAATCACGGCTCATTTC-3',

[0043] Downstream primer C17 R: 5'-GGCTGAGGTGGGAGGATCA-3',

[0044] Probe C17 Pb: 5′-CAGCCTCTGCCTCCTGGGCTCA-3′.

[0045] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0046] Preferably, the fluorescent reporter group is any one of ROX, HEX, Cy5, Cy5.5, ATTO425, and FAM; and the fluorescent quencher group is any one of MGB, BHQ1, and BHQ3.

[0047] Preferably, the 5' end of the probe C1 Pb is labeled with a fluorescent reporter group ROX, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C2 Pb is labeled with a fluorescent reporter group HEX, and the 3' end is labeled with a fluorescent quencher group BHQ1; the 5' end of the probe C3 Pb is labeled with a fluorescent reporter group Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ3; the 5' end of the probe C7Pb is labeled with a fluorescent reporter group ATTO425, and the 3' end is labeled with a fluorescent quencher group BHQ1; the 5' end of the probe C8Pb is labeled with a fluorescent reporter group Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ3; the 5' end of the probe C9 Pb is labeled with a fluorescent reporter group ROX, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C11 Pb is labeled with a fluorescent reporter group ATTO425, and the 3' end is labeled with a fluorescent quencher group BHQ1; The 5' end of Pb is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C17 Pb is labeled with a fluorescent reporter group Cy5.5, and the 3' end is labeled with a fluorescent quencher group BHQ3.

[0048] The present invention also provides a kit for detecting chromosome deletion in bladder cancer, comprising a primer-probe combination for detecting bladder cancer, 0.05-0.15 U / μL Taq enzyme, 1.5-5 mmol / LMg 2+ , 10mmol / LTris-HCl, 50mmol / LKCl, 0.05-0.5mmol / L dNTPs, 0.02% Tween 20, and the concentration of the primer-probe combination is 0.5-5pmol / μL.

[0049] The present invention also provides an application of a primer-probe combination in preparing a reagent for detecting chromosome deletion in bladder cancer.

[0050] The present invention provides a method for detecting chromosome deletion in bladder cancer, comprising:

[0051] Collect 50 mL of the test subject's morning urine, centrifuge it at 500-1000 rpm for 5-15 minutes, and collect the precipitate as the sample;

[0052] Extract DNA from samples;

[0053] Using the above-mentioned bladder cancer chromosome deletion detection kit to prepare a digital PCR reaction solution to obtain a detection solution;

[0054] The test solution is amplified by chip-based digital PCR, the fluorescence signal is detected and the result is determined.

[0055] Preferably, the digital PCR reaction solution includes 2.5 pmol / μL primer-probe combination, 0.05-0.15 U / μL Taq enzyme, 1.5-5 mmol / LMg 2+ , 10mmol / L Tris-HCl, 50mmol / L KCl, 0.05-0.5mmol / L dNTPs, 0.02% Tween20, 10mmol / L ROX reference dye, 2.5ng / μL sample DNA and water.

[0056] Preferably, the reaction procedure of the chip-based digital PCR is: 50°C, 2 min; 95°C, 10 min; 95°C, 15 s, 60°C, 40 s, 45 cycles; 10°C, 5 min.

[0057] The beneficial effects achieved by the present invention are:

[0058] 1. The present invention uses a chip-based digital PCR system to detect the copy numbers of 9 chromosomes in the DNA of bladder tumor tissue or urine cells of patients. Because chromosomes 2 and 12 are relatively stable in bladder cancer, they are not prone to amplification and / or deletion or have extremely low frequencies. Therefore, the present invention uses the average value of the concentration of chromosomes 2 and 12 as a reference value, calculates the relative concentrations of other chromosomes, and determines whether each chromosome has a copy number abnormality through the relative value, thereby providing doctors with a basis for diagnosis and prognosis related to bladder cancer.

[0059] 2. The kit for detecting chromosome deletion in bladder cancer provided by the present invention is based on digital PCR technology, which can efficiently and sensitively complete the PCR amplification of the target nucleic acid fragment in the micro-reaction unit, obtain the fluorescent signal for statistical analysis, and can directly give the copy number of the target sequence without relying on the standard curve and standard substance, thereby improving the stability of the experimental results within and between batches, achieving absolute quantification of the starting sample, and at the same time improving the sensitivity, accuracy and high tolerance, effectively reducing the occurrence of false negatives, and the reagents and instruments required for digital PCR are low in cost, which is easy to promote. In addition, digital PCR can automatically interpret the results, while the FISH method requires medical technicians to interpret the results with the naked eye, so subjective interpretation errors can be effectively avoided. This method can not only detect with high throughput, but also has high speed and low personnel burden. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the chip-based digital PCR fluorescence imaging result of Sample 2300125 in the example. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0062] Example

[0063] A method for detecting chromosome deletion in bladder cancer comprises the following steps:

[0064] (1) Sample nucleic acid extraction

[0065] 50 mL of morning urine was collected from 50 bladder cancer patients who were undergoing postoperative treatment, and the urine was centrifuged at 1000 rpm for 15 min. The precipitate was collected as the sample.

[0066] The sample DNA was extracted using a commercial DNA extraction kit (Qiagen DNA extraction kit, 51304). The specific steps are as follows:

[0067] a) Add 180 μL digestion solution and 20 μL proteinase K solution to the sample, vortex to mix, and digest at 56°C for 30 min.

[0068] b) After digestion, add 200 μL of binding solution and 200 μL of anhydrous ethanol, shake to mix, add to the extraction column, and centrifuge at 6000 g for 1 min.

[0069] c) Place the extraction column in a new waste liquid collection tube, add 500 μL of washing solution 1, and centrifuge at 6000 g for 1 min.

[0070] d) Place the extraction column in a new waste liquid collection tube, add 500 μL of washing solution 2, and centrifuge at 20,000 g for 3 min.

[0071] e) Pour away the washing liquid in the waste liquid collection tube and centrifuge at 20,000 g for 3 min.

[0072] f) Place the extraction column in a 1.5 mL centrifuge tube, add 100 μL of elution solution, centrifuge at 6000 g for 1 min to elute the DNA, and collect the solution in a centrifuge tube. The obtained solution is the sample DNA.

[0073] (2) Preparation of digital PCR reaction solution

[0074] A kit for detecting chromosome deletion in bladder cancer (as shown in Table 1) was used. Various reagents were taken out from the kit, melted at room temperature, oscillated evenly, and then centrifuged briefly for 5 seconds. Each digital PCR reaction system I and II was prepared according to Tables 2 and 3. The specific primers and probes used are shown in Table 4.

[0075] Table 1 Composition of the kit

[0076]

[0077] Table 2 Digital PCR reaction system I

[0078]

[0079]

[0080] Table 3 Digital PCR reaction system II

[0081]

[0082]

[0083] Table 4 Primers and probes

[0084]

[0085]

[0086] (3) Amplification and detection

[0087] a) Use the AccuONE digital PCR system (Zhenzhun Biotechnology) to perform digital PCR, turn on the sample preparation instrument for preheating, and load the chip and scraper into the designated position. Inject the digital PCR reaction solutions I and II prepared in step (2) into the scraper collection hole in sequence, turn on the sample preparation instrument, and evenly coat the reaction solution on the chip. Use a capping device to fix the chip cover on the chip to complete the chip sealing process. Mark the chip (two chips are required for each sample, corresponding to reaction solutions I and II respectively).

[0088] b) Turn on the thermal amplification instrument and set the amplification program, which is shown in Table 5. Place the chip on the thermal amplification instrument plate and start the amplification program. After the amplification is completed, place the chips in the chip rack in sequence.

[0089] c) Open the chip reader, switch to the "Read" window, and create a reading plan. Click "Eject / Retract Tray" to eject the tray. Place the chip rack on the reader's tray, click "Eject / Retract Tray" again, retract it, set parameters, and click "Start Reading".

[0090] d) Result analysis: After the reading and analysis is completed, enter the project to be viewed and check the results.

[0091] Sample testing and data analysis: After the 50 samples were tested according to the above steps, the test results were exported and summarized. The chip-based digital PCR fluorescence imaging results of sample 2300125 are as follows: Figure 1As shown in Figure 6, there are about 20,000 hexagonal microwells on the chip. The chip preparation instrument spreads the digital PCR reaction solution in the microwells, seals them and moves them to the amplifier for amplification. After amplification, they are placed in the reader for reading and analysis. The software automatically identifies the valid wells (quality control), and counts the number of negative wells and positive wells according to the brightness of the wells, and calculates the concentration of the DNA target in the reaction solution. The overall test results are shown in Table 6. The average value of the concentration of chromosomes 12 and 2 (in copies / μL) is used as the reference value to calculate the relative concentration of chromosomes 1, 3, 7, 8, 9, 11, and 17. Relative concentrations below 0.8 or above 1.2 are abnormal values. A relative concentration below 0.8 indicates the presence of chromosome deletion (complete deletion or heterozygous deletion), and a value above 1.2 indicates that some cells have chromosome polyploidy (triploid or quadruple). Values ​​between 0.8 and 1.2 are normal or below the detection limit.

[0092] Table 5 Amplification procedures

[0093]

[0094] Table 6 Sample test results

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Note: A positive result indicates suspected bladder cancer recurrence or metastasis, while a negative result indicates no genomic abnormality was detected.

[0102] As can be seen from Table 6, there are 36 positive samples and 14 negative samples among the 50 samples (bladder cancer patients who have undergone postoperative treatment), and the positive sample represents the suspected recurrence or metastasis of bladder cancer, and the negative sample represents that no genomic abnormality is detected. Based on the test results, further tests can be performed on positive patients to determine whether bladder cancer has recurred or metastasized in patients who have undergone postoperative treatment.

[0103] The method of the present invention is simple and easy to operate. Even if the number of samples is large, high-throughput detection can be achieved in a short time. It is not only fast but also has a low burden on personnel. If the same number of samples is tested using a FISH detection kit (such as Abbott UROVYSIONBLADDER CANCER KIT), the workload is large; in addition, because the detection method requires medical technicians to interpret the results with the naked eye, subjective errors are prone to occur, which is also very likely to affect the detection results of bladder cancer recurrence or metastasis, and the efficiency is also low.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe composition for detecting chromosome deletion in bladder cancer, characterized in that: include: A primer-probe composition for detecting chromosome 1, comprising: Upstream primer C1 F: 5'-ACACTGGGGATCAGGAGGAAA-3', Downstream primer C1 R: 5'-CAGTCAGCTTGAGGTGAAGGATA-3', Probe C1 Pb: 5′-CTCTGCCCAACGAGCAGCCCC-3′; A primer-probe composition for detecting chromosome 2, comprising: Upstream primer C2 F: 5'-GCACTTGAGGAGTGTCAGGAAA-3', Downstream primer C2 R: 5'-TCCGACTTCTTTGTATGGCAAA-3', Probe C2 Pb: 5′-CCCTCCGCAGCAGGTTGGGC-3′; A primer-probe composition for detecting chromosome 3, comprising: Upstream primer C3 F: 5'-CGCGCCTGGACGAGTCTA-3', Downstream primer C3 R: 5'-GGGACACCCTGGGAATGAA-3', Probe C3 Pb: 5′-TCCCAGGCCGCAAGGGATGC-3′; A primer-probe composition for detecting chromosome 7, comprising: Upstream primer C7 F: 5'-CCGAGGGGTAACCCTCATGT-3', Downstream primer C7 R: 5'-GCTGCCCTGAGGCACTCTTC-3', Probe C7 Pb: 5'-CCGGGAGACAGTCTCCACTCACCC-3'; A primer-probe composition for detecting chromosome 8, comprising: Upstream primer C8 F: 5'-CAGCCTCCTGAGTAGCTGAGATTA-3', Downstream primer C8 R: 5'-TGGTGAGACCCCGTCTCTACTAAA-3', Probe C8 Pb: 5′-CAGGTGCCCACCACCATGCCC-3′; A primer-probe composition for detecting chromosome 9, comprising: Upstream primer C9 F: 5'-CAAATGGGAAATAATCCCGAAA-3', Downstream primer C9 R: 5'-TGGTGGTATGCTTTGGGAAGTATAA-3', Probe C9 Pb: 5′-CACGTGAAAGCCC-3′; A primer-probe composition for detecting chromosome 11, comprising: Upstream primer C11 F: 5'-GGGAGCAGCCGAGCAAA-3', Downstream primer C11 R: 5'-GTTGGCCTCCTTGCTGTTG-3', Probe C11 Pb: 5′-CACGCGCCCCGCTGGGG-3′; A primer-probe composition for detecting chromosome 12, comprising: Upstream primer C12 F: 5'-CACTGGTGCCTCTGCTGTCAT-3', Downstream primer C12 R: 5'-CCTCTGCCTCATGCTTGTCAT-3', Probe C12 Pb: 5'-CTCCCTGGGCACTGAG-3'; and A primer-probe composition for detecting chromosome 17, comprising: Upstream primer C17 F: 5'-GGGACAATCACGGCTCATTTC-3', Downstream primer C17 R: 5'-GGCTGAGGTGGGAGGATCA-3', Probe C17 Pb: 5′-CAGCCTCTGCCTCCTGGGCTCA-3′; The 5' end of each probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; the fluorescent reporter group is any one of ROX, HEX, Cy5, Cy5.5, ATTO425, and FAM; the fluorescent quencher group is any one of MGB, BHQ1, and BHQ3.

2. The primer-probe composition for detecting chromosome deletion in bladder cancer according to claim 1, characterized in that: The 5' end of the probe C1 Pb is labeled with a fluorescent reporter group ROX, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C2 Pb is labeled with a fluorescent reporter group HEX, and the 3' end is labeled with a fluorescent quencher group BHQ1; the 5' end of the probe C3 Pb is labeled with a fluorescent reporter group Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ3; the 5' end of the probe C7 Pb is labeled with a fluorescent reporter group ATTO425, and the 3' end is labeled with a fluorescent quencher group BHQ1; the 5' end of the probe C8 Pb is labeled with a fluorescent reporter group Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ3; the 5' end of the probe C9 Pb is labeled with a fluorescent reporter group ROX, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C11 Pb is labeled with a fluorescent reporter group ATTO425, and the 3' end is labeled with a fluorescent quencher group BHQ1; the probe C12 The 5' end of Pb is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group MGB; the 5' end of the probe C17 Pb is labeled with a fluorescent reporter group Cy5.5, and the 3' end is labeled with a fluorescent quencher group BHQ3.

3. A kit for detecting chromosome deletion in bladder cancer, characterized in that: The method comprises the primer-probe combination according to any one of claims 1 to 2, 0.05 to 0.15 U / μL Taq enzyme, 1.5 to 5 mmol / L Mg 2+ , 10 mmol / L Tris-HCl, 50 mmol / L KCl, 0.05-0.5 mmol / L dNTPs and 0.02% Tween 20, and the concentration of the primer-probe combination is 0.5-5 pmol / μL.

4. Use of the primer-probe combination according to any one of claims 1 to 2 in the preparation of a reagent for detecting chromosome deletion in bladder cancer.

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