CN116445619A - Primer and fluorescent probe for detecting endometrial cancer - Google Patents
Primer and fluorescent probe for detecting endometrial cancer Download PDFInfo
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- CN116445619A CN116445619A CN202310481725.6A CN202310481725A CN116445619A CN 116445619 A CN116445619 A CN 116445619A CN 202310481725 A CN202310481725 A CN 202310481725A CN 116445619 A CN116445619 A CN 116445619A
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- 206010014759 Endometrial neoplasm Diseases 0.000 title claims abstract description 73
- 206010014733 Endometrial cancer Diseases 0.000 title claims abstract description 72
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 27
- 101000724725 Homo sapiens 5-hydroxytryptamine receptor 1B Proteins 0.000 claims abstract description 19
- 230000011987 methylation Effects 0.000 claims abstract description 19
- 238000007069 methylation reaction Methods 0.000 claims abstract description 19
- 101000914306 Homo sapiens CUGBP Elav-like family member 4 Proteins 0.000 claims abstract description 17
- 102100025488 CUGBP Elav-like family member 4 Human genes 0.000 claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims abstract description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 6
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 238000003745 diagnosis Methods 0.000 claims description 15
- 102100027499 5-hydroxytryptamine receptor 1B Human genes 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 8
- 102100024755 T-box transcription factor TBX5 Human genes 0.000 claims description 8
- 108010014480 T-box transcription factor 5 Proteins 0.000 claims description 7
- 230000002357 endometrial effect Effects 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 101150087690 ACTB gene Proteins 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 29
- 230000035945 sensitivity Effects 0.000 abstract description 19
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- 238000012216 screening Methods 0.000 abstract description 11
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- 210000004027 cell Anatomy 0.000 description 9
- 230000003321 amplification Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 108060004795 Methyltransferase Proteins 0.000 description 5
- 102000016397 Methyltransferase Human genes 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 206010046798 Uterine leiomyoma Diseases 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 101150067711 CELF4 gene Proteins 0.000 description 4
- 102100035342 Cysteine dioxygenase type 1 Human genes 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 3
- 101000737778 Homo sapiens Cysteine dioxygenase type 1 Proteins 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 210000004696 endometrium Anatomy 0.000 description 3
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- 239000003550 marker Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 101150004658 BHLHE22 gene Proteins 0.000 description 2
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- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 102000043362 human HTR1B Human genes 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 101150115978 tbx5 gene Proteins 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 102100024154 Cadherin-13 Human genes 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 102100026204 Class E basic helix-loop-helix protein 22 Human genes 0.000 description 1
- 102100023934 Heparan sulfate glucosamine 3-O-sulfotransferase 2 Human genes 0.000 description 1
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- 101001048053 Homo sapiens Heparan sulfate glucosamine 3-O-sulfotransferase 2 Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
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- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 210000005168 endometrial cell Anatomy 0.000 description 1
- 210000004996 female reproductive system Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 239000013642 negative control Substances 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 238000005070 sampling Methods 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 208000010579 uterine corpus leiomyoma Diseases 0.000 description 1
- 201000007954 uterine fibroid Diseases 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a primer and a fluorescent probe for detecting endometrial cancer. The nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO.6, and the nucleic acid sequence of the fluorescent probe comprises sequences shown in SEQ ID NO.7-SEQ ID NO. 9. The invention creatively designs the primer and the corresponding fluorescent probe for detecting the endometrial cancer, can specifically detect the new methylation target genes HTR1B, TBX and CELF4 in the DNA of the endometrial cancer, has good detection effect on the precancerous lesions of the endometrial cancer, can be used for early screening and result judgment of the endometrial cancer, and has high sensitivity and accuracy and easy operation.
Description
Technical Field
The invention belongs to the technical field of medical molecular biology, and relates to a primer and a fluorescent probe for detecting endometrial cancer.
Background
Endometrial cancer (endometrial cancer, EC) is one of the most common malignant tumors of the female reproductive system, is well developed in perimenopausal and postmenopausal women, has an increasing trend in morbidity and mortality worldwide, and has a tendency to jump to the first place of gynecological cancer in developed areas at home and abroad. The number of new cases of endometrial cancer of China female in 2020 is 8 ten thousand, and the incidence rate of malignant tumor of China female in the first line is ninth. Early (stage I, stage II) endometrial cancer patients are diagnosed with tumors confined to the uterus, with five-year survival rates greater than 70%, local spread (stage III) or distant metastasis (stage IV) occurring as the disease progresses, with five-year survival rates of about 40-50% for stage III patients and only 15-20% for stage IV patients. Thus, achieving early diagnosis of endometrial cancer is of great importance in cancer prevention, improving patient prognosis and increasing survival.
At present, an early detection method of endometrial cancer mainly adopts imaging examination, serum tumor marker CA125, cytological detection based on an endometrial cell sampler and finally definite gold standard method for diagnostic uterine curettage and hysteroscopic biopsy clinically, but has certain problems. The imaging examination can not effectively detect early cancer, the sensitivity is poor, the positive rate of the serum tumor markers such as CA125 to endometrial cancer and benign uterine lesion patients is only 24% and 20%, the accuracy is poor, the diagnosis and treatment guide does not recommend the diagnosis and treatment guide to be used as an independent screening means of endometrial cancer, and the final diagnosis still depends on pathological examination. The micro-tissue pathological examination of endometrium and the cytology examination of endometrium have certain requirements on materials, and have higher requirements on the pathologists. Diagnostic curettage and hysteroscopic biopsy are necessary methods for clear diagnosis of endometrial cancer, endometrial lesions have multifocal properties, biopsy may have about 10% of false negatives, examination is easy to miss, examination has certain invasiveness, sampling satisfaction is low, uterine cavity infection probability is increased, ascites cells are positive due to cervical adhesion and uterine distension pressure, and the method is not applicable to early screening of endometrial cancer and can only be used for final pathological diagnosis. Because of certain defects in the diagnosis method, early detection of endometrial cancer is difficult to achieve, and a method with high sensitivity and specificity and simple interpretation is needed for early screening and diagnosis of endometrial cancer.
CN113755603B discloses a marker, a primer probe and a kit for early screening and diagnosis of endometrial cancer, wherein the marker is a part of methylation regions in four genes of CDO1, CELF4, band 2 and HS3ST2, and a detection primer and a probe are designed for the methylation regions, and are all in a snap ring structure. The kit comprises the primer and the probe. The invention screens and combines the methylation areas, determines the most suitable methylation position for joint diagnosis, and can obviously improve the sensitivity and specificity of early endometrial cancer detection.
CN115184619B discloses a marker for pre-operation screening and diagnosis of endometrial cancer and application thereof, and uses a combination of A4GS, A2SG, A4FGS, A4FE, A2F0SG and MHy for diagnosis of endometrial cancer, so that healthy people and endometrial cancer patients can be distinguished very accurately, and basis is provided for clinical traditional Chinese medicine diagnosis of endometrial cancer.
In summary, the existing method for early detection of endometrial cancer has the problems of poor sensitivity, poor accuracy and easy missed diagnosis, certain invasiveness and increased infection, and the like, and the development of a primer and a fluorescent probe for early detection of endometrial cancer is necessary.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the primer and the fluorescent probe for detecting the endometrial cancer, solves the problems of poor sensitivity, poor accuracy, easy missed diagnosis, increased infection and the like of an early detection method of the endometrial cancer, has good detection effect on the precancerous lesions of the endometrial cancer, can be used for early screening and result judgment of the endometrial cancer, and has high sensitivity and accuracy and easy operation.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer for detecting endometrial cancer and a fluorescent probe, wherein the nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO.6, and the nucleic acid sequence of the fluorescent probe comprises sequences shown in SEQ ID NO.7-SEQ ID NO. 9.
The invention creatively designs the primer and the corresponding fluorescent probe for detecting the endometrial cancer, can specifically detect the new methylation target genes HTR1B, TBX and CELF4 in the DNA of the endometrial cancer, has good detection effect on the precancerous lesions of the endometrial cancer, can be used for early screening and result judgment of the endometrial cancer, and has high sensitivity and accuracy and easy operation.
SEQ ID NO.1：TGCGATCGTTACGGTTTTTTC。
SEQ ID NO.2：CGAACGACGATAAAACGCACT。
SEQ ID NO.3：CGGGGAGTTTCGAGTCGC。
SEQ ID NO.4：GACCGAATCGACTAAAACGACG。
SEQ ID NO.5：GGGTCGTTCGGATTTTGTTTTC。
SEQ ID NO.6：GAACCGCCCGATCCGAA。
SEQ ID NO.7：CGCCCCAACTCCGAAACGCAA。
SEQ ID NO.8：AACGAATTCCAACCCGACTTTACA。
SEQ ID NO.9：CGCCGAACTCCCCGCCAAA。
Preferably, the fluorescent probe comprises a fluorescent group at the 5 'end and a quenching group at the 3' end.
Preferably, the fluorescent group comprises any one or a combination of at least two of FAM, VIC or ROX.
Preferably, the quenching group comprises any one or a combination of at least two of BHQ1, BHQ2 or MGB.
In a second aspect, the invention provides the use of the primer and fluorescent probe for detecting endometrial cancer according to the first aspect for the preparation of a product for detecting endometrial cancer.
In a third aspect, the invention provides a kit for detecting endometrial cancer, comprising the primer for detecting endometrial cancer of the first aspect and a fluorescent probe.
In a fourth aspect, the invention provides the primer for detecting endometrial cancer and the application of the fluorescent probe in detecting endometrial cancer.
In a fifth aspect, the present invention provides a method for detecting endometrial cancer for the purpose of non-disease diagnosis and/or treatment, said method comprising:
extracting DNA from a sample to be detected, performing sulfite conversion, performing fluorescent PCR amplification by using the DNA after sulfite conversion and purification as a template and using the primer and the fluorescent probe for detecting endometrial cancer according to the first aspect, and judging according to a fluorescent PCR amplification result.
Preferably, the sample to be tested comprises any one or a combination of at least two of endometrial tissue, cervical exfoliated cells, uterine cavity exfoliated cells or plasma.
Preferably, the amplification targets of the primers include endometrial cancer methylation positive genes and internal reference genes.
Preferably, the endometrial cancer methylation positive genes include HTR1B, TBX and CELF4 and the reference genes include ACTB genes.
Preferably, the criterion for the determination is that any two or three of HTR1B, TBX and CELF4 are positive, i.e. positive for the test sample.
Compared with the prior art, the invention has the following beneficial effects:
the methylation target combination and the kit of the endometrial cancer have excellent comprehensive performance in detecting the sensitivity, the specificity and the precancerous lesion detection rate of the endometrial cancer, can be used for early screening and result judgment of the endometrial cancer, and also have good differentiation for benign lesions such as uterine fibroids.
Drawings
FIG. 1 is a diagram of DNA amplification of CpG methyltransferase treated HeLa cells with addition of 10ng, 1000pg and 100pg of HTR1B gene at the selection region of example 1;
FIG. 2 is a schematic diagram showing DNA amplification of CpG methyltransferase-treated HeLa cells with the addition of 10ng, 1000pg and 100pg of TBX5 gene at the selected region of example 1;
FIG. 3 is a chart of DNA amplification of Hela cells treated with CpG methyltransferase with addition of 10ng, 1000pg and 100pg for the CELF4 gene in the selection region of example 1.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
Primer probe amplification efficiency detection and target and segment selection.
Human cervical cancer cell Hela cell DNA was extracted and treated with CpG methyltransferase. DNA from endometrial cancer and normal endometrial samples is extracted. 10ng, 1000pg and 100pg of CpG methyltransferase treated Hela cell DNA and 28 cases of endometrial cancer and normal endometrial sample DNA were added respectively, sulfite conversion was performed using a Zymo sulfite conversion kit, sample detection was performed, and the reaction system is shown in Table 1.
TABLE 1
The sequences and detection segments of the primers and probes are shown in Table 2.
TABLE 2
The PCR amplification procedure was as follows:
95 ℃ for 5min;94 ℃ for 10s,64 ℃ for 30s,10 cycles; fluorescence was collected in 35 cycles of 94℃for 10s and 60℃for 31 s.
The results of the tests are shown in Table 3, "-" is negative and "+" is positive.
TABLE 3 Table 3
The sensitivity and specificity test results are shown in Table 4.
TABLE 4 Table 4
Results: the amplification efficiencies of the primer probes of the 9 sections all meet the detection requirement, wherein the amplification patterns of the HTR1B section 1, the TBX5 section 2 and the CELF4 section 1 are shown in the accompanying drawings 1-3, and the amplification curves of the target HTR1B section 1, the TBX5 section 2 and the CELF4 section 1 are good and all detected according to the results of the drawings 1-3. According to the sample detection results, the sensitivity and the specificity of the HTR1B section 1, the TBX5 section 2 and the CELF4 section 1 are above 70%, which indicates that the primer and the corresponding fluorescent probe for detecting endometrial cancer designed by the invention can specifically detect the novel methylation target genes HTR1B, TBX and CELF4 in endometrial cancer DNA.
Example 2
High volume FFPE sample validation
Samples of 56 endometrial cancers, 21 normal endometrium, 19 precancerous lesions, and 7 uterine fibroids were collected from An Linjian for a number of sample validations. Meanwhile, the methylation results of CDO1 and BHLHE22 genes are compared and analyzed.
The experimental process comprises the following steps: 103 samples of DNA were extracted, 500ng of DNA was added, sulfite conversion was performed using a Zymo sulfite conversion kit, and sample detection was performed, and the reaction system was as shown in Table 5.
TABLE 5
| Concentration of | Dosage of | |
| PCR reaction solution | - | 7.2μL |
| HTR1B-MF1 | 10μM | 1μL |
| HTR1B-MR1 | 10μM | 1μL |
| HTR1B-MP1 | 10μM | 1μL |
| TBX5-MF2 | 10μM | 1μL |
| TBX5-MR2 | 10μM | 1μL |
| TBX5-MP2 | 10μM | 1μL |
| CELF4-MF1 | 10μM | 1μL |
| CELF4-MR1 | 10μM | 1μL |
| CELF4-MP1 | 10μM | 1μL |
| ACTB-MF1 | 10μM | 1μL |
| ACTB-MR1 | 10μM | 1μL |
| ACTB-MP1 | 10μM | 1μL |
| Template | - | 5μL |
| Water and its preparation method | Up to 50μL | |
| Total system | 50μL |
The PCR amplification procedure was as follows:
95 ℃ for 5min;94 ℃ for 10s,64 ℃ for 30s,10 cycles; fluorescence was collected in 35 cycles of 94℃for 10s and 60℃for 31 s.
Interpretation of the results: and judging the gene methylation condition of the detection sample according to the fluorescent quantitative PCR amplification result.
(1) The positive control reaction tube HTR1B, TBX, CELF4 and ACTB genes all have signals corresponding to fluorescent channels; the negative control ACTB gene should have amplified signal corresponding to the fluorescent channel, and HTR1B, TBX5 and CELF4 genes should not have signal corresponding to the fluorescent channel.
(2) The amplification curve of the ACTB gene corresponding to the fluorescent channel is raised, so that the experimental result of the sample is reliable.
(3) The HTR1B, TBX5 or CELF4 gene has an amplification curve corresponding to a fluorescent channel, and the Ct value is less than or equal to 29, which indicates that the methylation of the human HTR1B, TBX or CELF4 gene is positive; if Ct value > 29, it indicates that the methylation negative or methylation degree of the human HTR1B, TBX or CELF4 gene is lower than the minimum detection limit.
(5) In contrast to the histological examination results, the sensitivity and specificity of the targets were calculated and the results are shown in table 6.
TABLE 6
The results show that in the performance comparison of single targets, the sensitivity, the specificity and the precancerous lesion detection rate of HTR1B are better, but the uterine myoma specificity is poorer. The sensitivity and the precancerous lesion detection rate of the target combination of HTR1B, TBX5 and CELF4 are close to those of the single target, but the specificity and the uterine myoma specificity are better than those of the single target. Compared with the existing single-target CDO1 and BHLHE22, the target combination of HTR1B, TBX and CELF4 has better comprehensive performance of sensitivity, specificity, precancerous lesion detection rate and uterine fibroid specificity. The sensitivity of the double target detection of the CN113755603B patent is 53.3-66.7%, and the sensitivity of the double target detection of the CN113755603B patent is 76.79-80.36%, which is superior to the detection effect. Compared with the joint inspection data of CDH13 segment 1+TBX5 segment 3, the specificity is 47.62%, which indicates that simply selecting 2 segments for combination cannot improve the detection effect, and indicates that the selection of positive targets and segments selected by the patent is a significant progress. The primer for detecting endometrial cancer and the corresponding fluorescent probe designed by the invention can specifically detect the new methylation target genes HTR1B, TBX and CELF4 in endometrial cancer DNA, have good detection effect on endometrial precancerous lesions, can be used for early screening and result judgment of endometrial cancer, and have high sensitivity and accuracy.
In conclusion, the invention creatively designs the primer and the corresponding fluorescent probe for detecting the endometrial cancer, can specifically detect the novel methylation target genes HTR1B, TBX and CELF4 in the DNA for detecting the endometrial cancer, has good detection effect on the precancerous lesions of the endometrial cancer, can be used for early screening and result judgment of the endometrial cancer, and has high sensitivity and accuracy and easy operation.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A primer and a fluorescent probe for detecting endometrial cancer, which are characterized in that the nucleic acid sequence of the primer comprises sequences shown in SEQ ID NO.1-SEQ ID NO. 6; the nucleic acid sequence of the fluorescent probe comprises sequences shown in SEQ ID NO.7-SEQ ID NO. 9.
2. The fluorescent probe of claim 1, wherein the fluorescent probe comprises a fluorescent group at the 5 'end and a quenching group at the 3' end;
preferably, the fluorophore comprises any one or a combination of at least two of FAM, VIC or ROX;
preferably, the quenching group comprises any one or a combination of at least two of BHQ1, BHQ2 or MGB.
3. Use of a primer for detecting endometrial cancer as claimed in claim 1 or 2, and a fluorescent probe for the manufacture of a product for detecting endometrial cancer.
4. A kit for detecting endometrial cancer, comprising the primer for detecting endometrial cancer of claim 1 or 2 and a fluorescent probe.
5. Use of the primer for detecting endometrial cancer as claimed in claim 1 or 2, and a fluorescent probe for detecting endometrial cancer.
6. A method for detecting endometrial cancer for non-disease diagnosis and/or treatment purposes, said method comprising:
extracting DNA from a sample to be detected, performing sulfite conversion, performing fluorescent PCR amplification by using the DNA obtained after sulfite conversion and purification as a template and using the primer and the fluorescent probe for detecting endometrial cancer according to claim 1 or 2, and judging according to a fluorescent PCR amplification result.
7. The method of claim 6, wherein the sample to be tested comprises any one or a combination of at least two of endometrial tissue, cervical exfoliated cells, uterine cavity exfoliated cells, or plasma.
8. The method of claim 6 or 7, wherein the amplified targets of the primer comprise endometrial cancer methylation positive genes and reference genes.
9. The method of claim 8, wherein the endometrial cancer methylation positive genes comprise HTR1B, TBX and CELF4 and the reference gene comprises ACTB gene.
10. The method of any one of claims 6-9, wherein the criteria for the determination are any two or three positives, i.e., the determination is positive for the test sample, of HTR1B, TBX5 and CELF4.
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| CN202310481725.6A CN116445619A (en) | 2023-04-28 | 2023-04-28 | Primer and fluorescent probe for detecting endometrial cancer |
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| CN202310481725.6A CN116445619A (en) | 2023-04-28 | 2023-04-28 | Primer and fluorescent probe for detecting endometrial cancer |
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