CN111187842A - Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer - Google Patents
Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer Download PDFInfo
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Abstract
The invention relates to a primer, a probe and a kit for early screening and auxiliary diagnosis of bladder cancer, wherein the primer for early screening and auxiliary diagnosis of bladder cancer comprises a primer for detecting PCDH17 gene, and the nucleotide sequence of the primer is shown as SEQ ID NO. 4-5; the nucleotide sequence of the primer for detecting the POU4F2 gene is shown as SEQ ID NO. 7-8; the nucleotide sequence of the primer for detecting the PENK gene is shown in SEQ ID NO. 10-11. The kit for early screening and auxiliary diagnosis of bladder cancer has the advantages of no wound, convenience, high sensitivity and strong specificity.
Description
Technical Field
The invention relates to a gene detection technology, in particular to a primer, a probe and a kit for early screening and auxiliary diagnosis of bladder cancer.
Background
Bladder cancer is one of the most common malignancies of the urinary system. According to the data published by the cancer center of China in 2018, the bladder cancer position is the 7 th position of the onset of male cancer. 2012-2015 years, the 5-year relative survival rate of bladder cancer is 72.9%. The recurrence rate of bladder cancer is 60-70%, which is the first of all solid tumors, 11% of patients with recurrence develop invasive bladder cancer, and the patients usually need to undergo cystoscopy 3-4 times per year after surgery. Effective treatment relies heavily on early detection and treatment of bladder cancer.
The FDA has approved various methods for clinical urine diagnosis of bladder cancer, such as exfoliative cytology, analysis of bladder cancer antigen (BTA), analysis of nuclear matrix protein 22(NMP22), fluorescence in situ hybridization (Fish), and the like. Microscopic examination is used as a gold standard for clinically diagnosing bladder cancer, has the characteristics of invasiveness and high price, and the urine examination based on exfoliative cytology has low specificity and poor sensitivity, and compared with microscopic examination, the early low-differentiation tumor sensitivity is only 4% -31%.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: based on the defects of the prior art, the primers, the probes and the kit for early screening and auxiliary diagnosis of bladder cancer are provided, so that the detection specificity and sensitivity can be improved.
In order to solve the technical problems, the invention adopts the technical scheme that:
provides a primer for early screening and auxiliary diagnosis of bladder cancer, which comprises a primer for detecting PCDH17 gene, the nucleotide sequence of which is shown as SEQ ID NO. 4-5; the nucleotide sequence of the primer for detecting the POU4F2 gene is shown as SEQ ID NO. 7-8; the nucleotide sequence of the primer for detecting the PENK gene is shown in SEQ ID NO. 10-11.
Provides a probe for early screening and auxiliary diagnosis of bladder cancer, which comprises a probe for detecting PCDH17 gene, the nucleotide sequence of which is shown in SEQ ID NO. 6; the nucleotide sequence of the probe for detecting the POU4F2 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the probe for detecting the PENK gene is shown in SEQ ID NO. 12.
The kit for early screening and aided diagnosis of the bladder cancer comprises a reaction solution, wherein the reaction solution comprises the primer for early screening and aided diagnosis of the bladder cancer and the probe for early screening and aided diagnosis of the bladder cancer.
The invention has the beneficial effects that:
(1) the primers and the probes are designed aiming at three target genes, namely PCDH17, POU4F2 and PENK, the designed primer probe sequence has strong specificity, and the performance is superior to other target genes; the method can be used for detecting the urine sample of a suspected bladder cancer patient by adopting a multiple qMSP technology so as to screen the bladder cancer, and has the advantages of high detection sensitivity and high specificity.
(2) The kit can be used for the auxiliary diagnosis of early bladder cancer and the tumor prognosis monitoring of patients with definite bladder cancer so as to reduce the frequency of bladder cancer microscopic examination.
(3) Can realize complete noninvasive detection, can finish detection only by 45mL of urine, is simple and convenient to operate, and is suitable for popularization and application of clinical detection work.
Drawings
FIG. 1 is a ROC curve analysis chart of the Δ Ct values of the kit for early screening and auxiliary diagnosis of bladder cancer according to example 2 of the present invention for detection of three target genes.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: the primers and the probes are designed aiming at three target genes of PCDH17, POU4F2 and PENK, and early screening and auxiliary diagnosis of bladder cancer are realized based on methylation specific fluorescence quantitative PCR technology of urine sediment cells.
The primer for early screening and auxiliary diagnosis of bladder cancer provided by the invention comprises a primer for detecting PCDH17 gene, and the nucleotide sequence of the primer is shown as SEQ ID NO. 4-5; the nucleotide sequence of the primer for detecting the POU4F2 gene is shown as SEQ ID NO. 7-8; the nucleotide sequence of the primer for detecting the PENK gene is shown in SEQ ID NO. 10-11.
Further, the kit also comprises a primer for detecting β -Actin gene, and the nucleotide sequence of the primer is shown as SEQ ID NO. 1-2.
The probe for early screening and auxiliary diagnosis of bladder cancer provided by the invention comprises a probe for detecting PCDH17 gene, the nucleotide sequence of which is shown as SEQ ID NO. 6; the nucleotide sequence of the probe for detecting the POU4F2 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the probe for detecting the PENK gene is shown in SEQ ID NO. 12.
Furthermore, the kit also comprises a probe for detecting β -Actin gene, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
The kit for early screening and auxiliary diagnosis of bladder cancer provided by the invention comprises a reaction solution, wherein the reaction solution comprises the primer for early screening and auxiliary diagnosis of bladder cancer and the probe for early screening and auxiliary diagnosis of bladder cancer.
From the above description, the beneficial effects of the present invention are:
(1) the primers and the probes are designed aiming at three target genes, namely PCDH17, POU4F2 and PENK, the designed primer and probe have strong sequence specificity, and the performance is superior to that of other target genes.
(2) The kit of the invention can not only carry out the auxiliary diagnosis of early bladder cancer, but also monitor the tumor prognosis of patients with definite bladder cancer so as to reduce the frequency of bladder cancer microscopic examination.
(3) Can realize complete noninvasive detection, can finish detection only by 45mL of urine, is simple and convenient to operate, and is suitable for popularization and application of clinical detection work.
Example 1
This example provides a set of specific primers and probes for detecting bladder cancer, including β -Actin gene amplification primers and probes, PCDH17 gene amplification primers and probes, POU4F2 gene amplification primers and probes, and PENK gene amplification primers and probes.
β -Actin gene amplification primer sequence is shown in SEQ ID NO. 1-2;
SEQ ID.1：
5’-GGACGTAGGGAGTATATAGGTTG-3’；
SEQ ID.2：
5’-CACACAGTAACAAACACAAATTCAC-3’。
the PCDH17 gene amplification primer sequence is shown in SEQ ID NO. 4-5;
SEQ ID.4：
5’-CGGGTCTTGGAGAATTTCG-3’；
SEQ ID.5：
5’-CGCGATCGATACGCTACTTA-3’。
the POU4F2 gene amplification primer sequence is shown as SEQ ID NO. 7-8;
SEQ ID.7：
5’-AAGGGTTCTGCGAAGTTG-3’；
SEQ ID.8：
5’-AACGCGTAAGGGAAATCA-3’。
the sequence of the PENK gene amplification primer is shown in SEQ ID NO. 10-11;
SEQ ID.10：
5’-GGTTGTTGTTGTTGCGTTTC-3’；
SEQ ID.11：
5’-CGACCGAACGCACTAAAC-3’。
wherein, the detection probes are all fluorescence labels, the 5 'end of the detection probes is labeled with a report group, and the 3' end of the detection probes is labeled with a non-fluorescence quenching group.
The detection probes in this example are as follows:
β -Actin detection probe SEQ ID.3:
5’-Texas Red-AAACTTACTAAACCTCCTCCATCACCACCC-BHQ2-3’；
PCDH17 detection probe SEQ id.6:
5’-FAM-CCGCTATGTACGTCCACGTCCAACA-BQ1-3’；
POU4F2 detection probe SEQ ID.9:
5’-VIC-CGTACAAAATCCGAAAACGACGACGAA-BQ1-3’；
PENK detection probe SEQ id.12:
5’-CY5-AAGTACACGTCGCGCAATCCTAACTACAT-BQ3-3’。
the embodiment also provides a detection kit for assisting the diagnosis of bladder cancer, which comprises the series of specific primers and probes for detecting bladder cancer, and also comprises a positive quality control product, a PCR mix reaction solution and deionized water.
Example 2:
establishment of a bladder cancer detection method:
the test kit of example 1 was used to test the exfoliated cells in urine of 32 patients and non-bladder cancer patients diagnosed with bladder cancer by clinical cystoscopy and in urine of 14 healthy persons (the test was informed and agreed by the subjects).
The specific detection method comprises the following steps:
1. extracting DNA of urine sediment cell, and performing quality inspection of purity and concentration of the extracted DNA
(1) Collecting and centrifuging a urine sample: collecting 45mL of second morning urine of each volunteer into a 50mL centrifuge tube containing 5mL of urine preservation solution, mixing, centrifuging at 2200g for 20min, and removing supernatant.
(2) Extracting DNA of urine sediment cells: urine sediment DNA was extracted according to the protocol of a micro-sample genomic DNA extraction kit (manufacturer: Tiangen, cat # DP316), and the DNA concentration was quantitatively determined by Qubit.
2. DNA methylation modification
(1) The DNA extracted in the above step was diluted to 1 ng/. mu.L with nuclease-free water for use.
(2) 20 mu L of the diluted DNA is put into a 0.2mL centrifuge tube, 55 mu L of bisulfite solution is added, the mixture is slightly shaken and mixed evenly and then is centrifuged briefly, and the following procedures are operated on a PCR instrument: [ 98 ℃ for 10 min; 2.5h at 64 ℃; infinity at 4 ℃.
(3) Placing the Spin column into a collection tube and washing in Zymo-SpinTM600. mu.L of M-BindingBuffer was added to the column. The reaction product is transferred into a centrifugal column, and is inverted and mixed for several times.
(4) Centrifuging at high speed (>10000 Xg) for 30 seconds, and discarding the waste liquid; add 100 u L M-Wash Buffer, full speed centrifugation for 30 seconds, discard waste liquid.
(5) 200 mu L M-depletion Buffer was added, and after standing at room temperature for 15 minutes, the mixture was centrifuged at full speed for 30 seconds, and the waste liquid was discarded.
(6) 200 mu L M-Wash Buffer was added, centrifuged at full speed for 30 seconds, and the waste was discarded. And repeating the steps once.
(7) The column was transferred to a clean 1.5mL centrifuge tube and 10. mu. L M-elute Buffer was added and the filtrate was collected after centrifugation at 10000 Xg for 30 s.
3. qMSP reaction
(1) The PCR reaction system was prepared as follows:
TABLE 1
| Reagent composition | Dosage (mu L) |
| PCR Master Mix | 10 |
| Primer and probe | 4 |
| H2O | 4 |
| DNA input | 2 |
| Total | 20 |
And preparing the system according to the table, subpackaging the system in a PCR eight-connected tube, marking, and placing the tube in ABI 7500 for reaction.
(2) Selecting instrument detection channels:
Reporter Dye1(β-Actin)：Texas Red，Quencher Dye1：BQ2；
Reporter Dye2(PCDH17)：FAM，Quencher Dye2：BQ1；
Reporter Dye2(POU4F2)：VIC，Quencher Dye2：BQ1；
Reporter Dye2(PENK)：CY5，Quencher Dye2：BQ3。
(3) the reaction procedure is as follows in table 2:
TABLE 2
4. The results are shown in Table 3 below. Table 3 shows Ct values of reference genes and delta Ct values of target genes in 46 samples.
TABLE 3
Note: 1. "HV" represents health volume, a Healthy human sample; "BC" means the bladder cancer, i.e., bladder cancer patient; 2. "/" indicates that no Ct value was detected.
ROC curve analysis (see fig. 1) was performed on the Δ Ct values detected for each target gene, and AUC (PCDH17) was 0.9244, AUC (POU4F2) was 0.9773, and AUC (penk) was 0.9917.
According to the statistics of the results, the reference gene meets the Ct value of 23.5-25.5, and the positive judgment standard of bladder cancer is set as follows:
the following conditions are simultaneously satisfied:
a. the Ct value of the target gene PCDH17/POU4F2/PENK is less than 35;
b.ΔCtPCDH17<7.70；ΔCtPOU4F2<9.55；ΔCtPENK<9.35。
based on this criterion, the sensitivity and specificity were calculated according to the following formulas, and the results are shown in Table 4. Table 4 shows the sensitivity and specificity of the kit of the invention for detecting bladder cancer.
The sensitivity is the number of bladder cancer samples with positive detection results/total bladder cancer samples;
the specificity is the number of non-bladder cancer samples with negative detection results/total number of non-bladder cancer samples.
TABLE 4
| Target gene combination | Sensitivity of the probe | Specificity of |
| PCDH17 | 100％ | 52.17 |
| POU4F2 | ||
| 100％ | 86.95 | |
| PENK | ||
| 100％ | 91.30％ | |
| PCDH17+ |
100％ | 86.95％ |
| PCDH17+ |
100％ | 100％ |
| POU4F2+ |
100％ | 91.30％ |
| PCDH17+POU4F2+ |
100％ | 95.65％ |
As can be seen from Table 4, the single-gene evaluation standard has large specificity difference, the optimal detection performance of the double-gene combined use is PCDH17+ PENK, and the specificity and the sensitivity are both 100%. The specificity of the three-gene combined detection performance is 95.65 percent, and the sensitivity can reach 100 percent
Example 3:
detection of clinical samples:
based on the results of example 2, the detection performance of the multiple targets of the present invention was further verified in this example. The method comprises the following steps: collecting 33 samples of bladder cancer and urine of healthy people in Beijing tumor hospital, collecting centrifugal cells, and performing DNA extraction, sulfite conversion and quantitative detection. The reference gene Ct and the target gene delta Ct of each sample are obtained and are shown in Table 5.
TABLE 5
Note: "HV" represents health volume, a Healthy human sample; "J-P" refers to urine samples from bladder cancer patients in the Beijing tumor Hospital.
The performance of this test is shown in Table 6, based on the criteria of example 2.
TABLE 6
| Target gene combination | Sensitivity of the probe | Specificity of |
| PCDH17 | 100％ | 63.63 |
| POU4F2 | ||
| 100％ | 90.90 | |
| PENK | ||
| 100％ | 90.90％ | |
| PCDH17+ |
100％ | 90.90％ |
| PCDH17+ |
100％ | 90.90％ |
| POU4F2+ |
100％ | 90.90％ |
| PCDH17+POU4F2+ |
100％ | 90.90％ |
According to the table 6, clinical samples verify that the single gene detection performance has larger difference, the sensitivity of the multi-gene combination can reach 100%, and the specificity is consistent to 90.90%. When ROC analysis is carried out on the sample result delta Ct, the target gene POU4F2 is better detected, and the specificity of the detection method under the condition of larger sample amount is improved by a target gene combination mode on the premise of higher sensitivity of each target gene.
In conclusion, the primer, the probe and the kit for early screening and auxiliary diagnosis of bladder cancer, provided by the invention, have the advantages of non-invasive convenience, high sensitivity and strong specificity, can be used for monitoring low-grade bladder cancer tumors to improve the life quality of patients, and can be used for early finding the recurrence of tumors to improve the survival rate of patients in high-grade diseases.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Yaenergetic Biotechnology (Shenzhen) Limited
<120> primers, probes and kit for early screening and auxiliary diagnosis of bladder cancer
<130>2020
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<170>PatentIn version 3.5
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Claims (9)
1. A primer for early screening and auxiliary diagnosis of bladder cancer is characterized by comprising a primer for detecting PCDH17 gene, the nucleotide sequence of which is shown as SEQ ID NO. 4-5; the nucleotide sequence of the primer for detecting the POU4F2 gene is shown as SEQ ID NO. 7-8; the nucleotide sequence of the primer for detecting the PENK gene is shown in SEQ ID NO. 10-11.
2. The primer for early screening and aided diagnosis of bladder cancer according to claim 1, further comprising a primer for detecting β -Actin gene, the nucleotide sequence of which is shown in SEQ ID NO. 1-2.
3. A probe for early screening and auxiliary diagnosis of bladder cancer is characterized by comprising a probe for detecting PCDH17 gene, the nucleotide sequence of which is shown in SEQ ID NO. 6; the nucleotide sequence of the probe for detecting the POU4F2 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the probe for detecting the PENK gene is shown in SEQ ID NO. 12.
4. The probe for early screening and aided diagnosis of bladder cancer according to claim 3, further comprising a probe for detecting β -Actin gene, the nucleotide sequence of which is shown in SEQ ID NO. 3.
5. The probe for early screening and aided diagnosis of bladder cancer according to claim 3 or 4, wherein each probe is labeled with a reporter group at the 5 'end and a non-fluorescent quencher group at the 3' end.
6. A kit for early screening and aided diagnosis of bladder cancer, which comprises a reaction solution, and is characterized in that the reaction solution comprises the primer for early screening and aided diagnosis of bladder cancer as claimed in any one of claims 1 to 2 and the probe for early screening and aided diagnosis of bladder cancer as claimed in any one of claims 3 to 5.
7. The kit for early screening and aided diagnosis of bladder cancer according to claim 6, wherein the reaction solution further comprises PCR Master mix, positive quality control substances and water.
8. The kit for early screening and aided diagnosis of bladder cancer according to claim 6, wherein the detection sample adopted by the kit is urine sediment cell DNA and the detection sample is methylated.
9. The kit for early screening and aided diagnosis of bladder cancer according to claim 6, wherein the kit employs methylation-specific fluorescent quantitative PCR detection technology.
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| CN202010907482.4A CN111826446A (en) | 2020-03-24 | 2020-09-02 | Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer |
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| CN117363733B (en) * | 2023-12-07 | 2024-02-23 | 湖南宏雅基因技术有限公司 | Application of detection primer probe group for PER1 and LOX double-gene methylation joint diagnosis in preparation of bladder cancer diagnosis reagent |
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| EP2390371A1 (en) * | 2007-11-30 | 2011-11-30 | Genomictree, Inc. | Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene |
| US9546403B1 (en) * | 2011-12-14 | 2017-01-17 | University Of Utah Research Foundation | Substrate for methylated DNA testing |
| CN108531594A (en) * | 2018-04-19 | 2018-09-14 | 安徽达健医学科技有限公司 | A kind of polygene combined non-invasive detection methods and its kit for carcinoma of urinary bladder early screening |
| CN110343762A (en) * | 2019-06-06 | 2019-10-18 | 宽盈医疗科技(上海)有限公司 | Bladder carcinoma marker group and its application |
| CN110387417A (en) * | 2019-06-06 | 2019-10-29 | 宽盈医疗科技(上海)有限公司 | The method of target gene methylation level in Diagnosis of Bladder system, reagent combination and detection urine |
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