CN113528672B - Primer and probe combination for early screening of bladder cancer, kit and application - Google Patents
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Abstract
The invention relates to the technical field of molecular biology, in particular to a primer and probe combination, a kit and application for early screening of bladder cancer, and the primer and probe combination for early screening of bladder cancer comprises 4 pairs of target specific primers related to early methylation sites of a population with bladder cancer, and a corresponding reference primer and a corresponding specific primer probe. Compared with the prior art, the method is simple and feasible, has objective interpretation based on the detection of DNA methylation, avoids the subjectivity of artificial interpretation results, and improves the accuracy rate; meanwhile, the detection method disclosed by the invention is non-invasive, can avoid complications caused by cystoscopy, improves the patient compliance, and is used for early screening, early diagnosis, curative effect evaluation and tracking and prognosis evaluation of tumors.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer and probe combination, a kit and application for early screening of bladder cancer.
Background
Bladder cancer is one of the most common malignant tumors of the urinary system, the number of the bladder cancer patients is 7.8 thousands per year in 2014 in China, and the incidence rate tends to rise year by year. Bladder cancer has the characteristics of high morbidity and easy recurrence. Hematuria is a common clinical symptom of bladder cancer, with about 17% of patients with hematuria detecting bladder cancer. Currently, the main diagnostic methods for bladder cancer include cystoscopy, urine cast cytology, urine FISH test, and tumor marker test. Among them, cystoscopy combines biopsy histopathology as a gold standard for cystoscopy diagnosis, but this examination method is invasive, is likely to cause complications, and has low patient compliance. The diagnostic capability of the imaging examination on thousands of tiny lesions is limited, the sensitivity of the urine cast-off cytology examination is low, the urine FISH detection operation is complex, and the interpretation result is subjective. The existing tumor marker detection is mainly based on the existence of specific protein in urine for detection, but the content of the protein in the urine for determination is low, and the sensitivity and specificity are still limited.
Disclosure of Invention
Aiming at the defects of the background technology, the invention provides a primer and probe combination, a kit and application for early screening of bladder cancer, and solves the problems of low detection sensitivity and poor specificity of bladder cancer and avoids complications caused by cystoscopy.
The technical scheme adopted by the invention is as follows: the combination of primers and probes for early screening of bladder cancer is characterized in that: comprises 4 pairs of target specific primers related to early methylation sites of the population with bladder cancer, and corresponding reference primers and specific primer probes; wherein the sequences of the 4 pairs of target specific primers are as follows:
Seq ID NO.1：F-CCGCGCTCACGTCGGTTCC，
Seq ID NO.2：R-CCCCCTACTTCCCAGCTCACGA；
Seq ID NO.3：F-TCCTATCCTCATCGGTTCTCGTACT，
Seq ID NO.4：R-AATTGTCCAACGAATGCACCTG；
Seq ID NO.5：F-CCCGCACACTACTCCAAATCAA，
Seq ID NO.6：R-GCAAAATCGAGGGCAGCTGAAG；
Seq ID NO.7：F-CAGCGCGTGTCCTTCTTCGCCTG，
Seq ID NO.8：R-AGTTCTCGAGCGTGCCACCGTTG；
the sequences of the internal reference primers are as follows:
Seq ID NO.9：F-GACCCGCGCTTCGACACCG，
Seq ID NO.10：R-CCAGCGTCGCGTGCTTCTC。
preferably, the specific primer probe sequence is as follows:
Seq ID NO.11：FAM- ATGACCCCTCTCCAAACGGCGCAG-BHQ1，
Seq ID NO.12：FAM- AAAAGTCTGCGCGTGAGCCT-BHQ1；
Seq ID NO.13：FAM- CCACTAAGAGTTCCTCCCGCGCAGA-BHQ1，
Seq ID NO.14：FAM- CCTCGCCGCATCCGCTCAGCC-BHQ1；
the probe sequence of the internal reference primer is as follows:
Seq ID NO.15：VIC-CCAGGCTCTGCTTGTGCGTCACCA-BHQ1。
the kit for early screening of bladder cancer is characterized in that: the primer and probe combination and methylation sensitive endonuclease system for early bladder cancer screening in the scheme are included.
Preferably, the methylation sensitive endonuclease system comprises HinP1I, HpaII, AciI, HpyCH4 IV.
The application of the kit in preparing the early-stage bladder cancer screening reagent is characterized in that: the screening method comprises the following steps:
s1, extracting free cfDNA in urine;
s2, incubating the cfDNA and a methylation sensitive endonuclease system at 37 ℃;
s3, taking the incubation product as a template, adding 4 pairs of target specific primers and internal reference primers, and performing multiple PCR amplification;
s4, taking the amplification product as a template, adding 4 pairs of target specific primers, 4 specific primer probes, an internal reference primer probe and an internal reference primer, and carrying out qPCR detection reaction;
and S5, evaluating the bladder cancer risk according to the difference delta Ct between the Ct values of the 4 pairs of target specific primers and the internal reference primer.
Preferably, the S1 is specifically: extracting cfDNA in urine by using a Qiagen urine free DNA extraction kit.
Preferably, the amplification procedure of S3 is: 98 ℃/45 s, 1 cycle; 98 ℃/15 s, 55 ℃/30 s, 72 ℃/30 s, 12 cycles; 72 ℃/1 min, 1 cycle; 4 ℃/hold.
Preferably, the qPCR detection reaction procedure of S4 is: 95 ℃/3min, 1 cycle; 98 ℃/15 s, 60 ℃/60 s, 45 cycles.
Preferably, the evaluation manner of S5 is:
delta Ct = reference primer amplification Ct value-4 pair primer specific amplification Ct value;
Δ Ct > -2.7 high risk of bladder cancer;
the delta Ct is less than or equal to-2.7 of low risk of bladder cancer.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, through deep excavation of CGAC projects and public database resources, hypermethylated regions of early genomes of bladder cancer of Chinese people are screened and compared with corresponding regions of genomes of white blood cells, regions which are hypomethylated in the white blood cells and contain at least two sites in CCGC, CCGG, GCGC, ACGT and GCGG are selected as target points, and more than 100 pairs of primers are designed. Screening primers with high PCR efficiency, strong specificity and good stability to finally obtain 4 pairs of primers and corresponding probe sequences which can be used for distinguishing the risk of early bladder cancer;
2. the detection of thousands of DNA methylation sites is carried out through the combination of the bladder cancer specific DNA methylation biomarkers, the cfDNA from non-tumor sources is degraded by utilizing the characteristic that methylation sensitive incision enzymes cannot cut the methylation sites, the detection sensitivity is greatly improved by specifically amplifying the ctDNA from the tumor sources, the problem of low single DNA methylation signals is solved, and the detection sensitivity and specificity are improved;
3. the detection based on DNA methylation is simple and feasible, and the interpretation is objective, so that the subjectivity of an artificial interpretation result is avoided, and the accuracy is improved; meanwhile, the detection is non-invasive, can avoid complications caused by cystoscopy, improves the patient compliance, and is used for early screening, early diagnosis, curative effect evaluation and tracking and prognosis evaluation of tumors.
Drawings
FIG. 1 is a graph of risk determination in accordance with the present invention;
fig. 2 is a graph of classification performance AUC based on the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1 kit for early screening of bladder cancer
Comprises a Qiagen urine free DNA extraction kit, HinP1I, HpaII, AciI, HpyCH4IV methylation sensitive endonuclease system, 4 pairs of target specific primers and internal reference primers, 4 pairs of specific primer probes, internal reference primer probes and internal reference primer probes;
wherein the sequences of the 4 pairs of target specific primers are as follows:
Seq ID NO.1：F-CCGCGCTCACGTCGGTTCC，
Seq ID NO.2：R-CCCCCTACTTCCCAGCTCACGA；
Seq ID NO.3：F-TCCTATCCTCATCGGTTCTCGTACT，
Seq ID NO.4：R-AATTGTCCAACGAATGCACCTG；
Seq ID NO.5：F-CCCGCACACTACTCCAAATCAA，
Seq ID NO.6：R-GCAAAATCGAGGGCAGCTGAAG；
Seq ID NO.7：F-CAGCGCGTGTCCTTCTTCGCCTG，
Seq ID NO.8：R-AGTTCTCGAGCGTGCCACCGTTG；
the sequences of the internal reference primers are as follows:
Seq ID NO.9：F-GACCCGCGCTTCGACACCG，
Seq ID NO.10：R-CCAGCGTCGCGTGCTTCTC；
the specific primer probe sequence is as follows:
Seq ID NO.11：FAM- ATGACCCCTCTCCAAACGGCGCAG-BHQ1，
Seq ID NO.12：FAM- AAAAGTCTGCGCGTGAGCCT-BHQ1；
Seq ID NO.13：FAM- CCACTAAGAGTTCCTCCCGCGCAGA-BHQ1，
Seq ID NO.14：FAM- CCTCGCCGCATCCGCTCAGCC-BHQ1；
the probe sequence of the internal reference primer is as follows:
Seq ID NO.15：VIC-CCAGGCTCTGCTTGTGCGTCACCA-BHQ1。
example 2 method for detecting changes in the methylation level of ctDNA
1. Taking 10 mL of urine, and extracting free cfDNA by using a Qiagen urine free DNA extraction kit (Cat: 954154);
2. taking 20 ng cfDNA and 20 mu LHinP1I, HpaII, AciI and HpyCH4IV four methylation sensitive endonuclease systems (the final concentration is 10U/. mu.L), incubating for 16 h at 37 ℃, and inactivating the enzymes at 80 ℃ for 20 min;
3. taking all incubated products as templates, configuring a system by adding 4 pairs of target specific primers (each 50 nM) and 1 internal reference primer (10 nM), and setting the following set program of the table 1 to perform an amplification program in a common PCR instrument;
TABLE 1
4. Taking 1 mu L of the amplification product in the previous step as a template, adding 4 pairs of target specific primers (each 0.25 mu M), 4 specific primer probes (each 0.1 mu M), 1 internal reference primer (0.05 mu M) and internal reference primer probe (0.02 mu M), setting the following setting program of the following table 2 to carry out qPCR reaction in an ABI 7500 fluorescent PCR instrument, and collecting FAM and VIC channel signals before the end of each cycle;
TABLE 2
5. Evaluating bladder cancer risk according to the difference delta Ct between the Ct values of the internal reference primer and the 4 pairs of target specific primers, as shown in figure 1, wherein delta Ct = Ct value amplified by the internal reference primer-Ct value amplified by the 4 pairs of primers specifically amplified Ct value;
delta Ct > -2.7 bladder cancer high risk, suggesting imaging examination;
the delta Ct is less than or equal to-2.7 of low risk of bladder cancer.
Urine samples from 95 healthy subjects and 71 stage i-iii bladder cancer patients were also evaluated for bladder cancer risk performance using the present invention, with the sample information and test results shown in table 3 below:
TABLE 3
The data in table 3 are imported into Graphpadprism 6.0 software, an ROC curve is automatically generated, the AUC value of the area under the curve is counted (figure 2), the abscissa is 1-specificity, the ordinate is sensitivity, the software statistics result takes delta Ct > -2.7 as a threshold, the classifier sensitivity is 90.91%, and the specificity is 95.24%.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Sequence listing
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Claims (7)
1. A primer and probe combination for early screening of bladder cancer, characterized by: comprises 4 pairs of target specific primers and 4 specific primer probes related to early methylation sites of the population with bladder cancer, and corresponding internal reference primers and internal reference primer probes; wherein the sequences of the 4 pairs of target specific primers are as follows:
Seq ID NO.1：F-CCGCGCTCACGTCGGTTCC，
Seq ID NO.2：R-CCCCCTACTTCCCAGCTCACGA；
Seq ID NO.3：F-TCCTATCCTCATCGGTTCTCGTACT，
Seq ID NO.4：R-AATTGTCCAACGAATGCACCTG；
Seq ID NO.5：F-CCCGCACACTACTCCAAATCAA，
Seq ID NO.6：R-GCAAAATCGAGGGCAGCTGAAG；
Seq ID NO.7：F-CAGCGCGTGTCCTTCTTCGCCTG，
Seq ID NO.8：R-AGTTCTCGAGCGTGCCACCGTTG；
the sequences of the internal reference primers are as follows:
Seq ID NO.9：F-GACCCGCGCTTCGACACCG，
Seq ID NO.10：R-CCAGCGTCGCGTGCTTCTC；
the specific primer probe sequence is as follows:
Seq ID NO.11：FAM- ATGACCCCTCTCCAAACGGCGCAG-BHQ1，
Seq ID NO.12：FAM- AAAAGTCTGCGCGTGAGCCT-BHQ1；
Seq ID NO.13：FAM- CCACTAAGAGTTCCTCCCGCGCAGA-BHQ1，
Seq ID NO.14：FAM- CCTCGCCGCATCCGCTCAGCC-BHQ1；
the probe sequence of the internal reference primer is as follows:
Seq ID NO.15：VIC-CCAGGCTCTGCTTGTGCGTCACCA-BHQ1。
2. a kit for early screening of bladder cancer, characterized by: the primer and probe combination for early screening of bladder cancer and the methylation sensitive endonuclease system, which are disclosed by the claim 1, are included.
3. The kit for early screening of bladder cancer according to claim 2, wherein: the methylation sensitive endonuclease system comprises HinP1I, HpaII, AciI and HpyCH4 IV.
4. Use of a kit according to claim 3 for the preparation of a reagent for early screening of bladder cancer, characterized in that the screening method comprises the following steps:
s1, extracting free cfDNA in urine;
s2, incubating the cfDNA and a methylation sensitive endonuclease system at 37 ℃;
s3, taking the incubation product as a template, adding 4 pairs of target specific primers and internal reference primers in the kit according to claim 3, and performing multiple PCR amplification;
s4, adding 4 pairs of target specific primers, 4 specific primer probes, an internal reference primer probe and an internal reference primer in the kit according to claim 3 by taking the amplification product as a template to perform qPCR detection reaction;
s5, evaluating the bladder cancer risk according to the difference delta Ct between the Ct values of the 4 pairs of target specific primers and the internal reference primer; the evaluation mode of S5 is as follows:
delta Ct = reference primer amplification Ct value-4 pair primer specific amplification Ct value;
Δ Ct > -2.7 high risk of bladder cancer;
the delta Ct is less than or equal to-2.7 of low risk of bladder cancer.
5. The use according to claim 4, wherein said S1 is specifically: extracting cfDNA in urine by using a Qiagen urine free DNA extraction kit.
6. The use according to claim 4, wherein the amplification procedure of S3 is: 98 ℃/45 s, 1 cycle; 98 ℃/15 s, 55 ℃/30 s, 72 ℃/30 s, 12 cycles; 72 ℃/1 min, 1 cycle; 4 ℃/hold.
7. The use according to claim 4, characterized in that the qPCR detection reaction program of S4 is: 95 ℃/3min, 1 cycle; 98 ℃/15 s, 60 ℃/60 s, 45 cycles.
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