CN112646891A - Digestive tract tumor marker combination, detection kit and application thereof - Google Patents
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Abstract
The invention belongs to the field of biomedicine, and particularly relates to a digestive tract tumor marker combination, a detection kit and application thereof, wherein the marker gene comprises methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; the sequences of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are respectively shown as SEQ ID NO. 34-SEQ ID NO. 39. The invention develops a non-invasive method capable of simultaneously detecting esophageal cancer, gastric cancer and colorectal cancer, a marker composition and a using method thereof, and provides a new technology for early prevention and control of digestive tract tumors.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a digestive tract tumor marker combination, a detection kit and application thereof.
Background
According to the latest statistics of the World Health Organization (WHO), the incidence rate and the mortality rate of the global malignant tumor are 12%, 11% and 7%, the second, third and sixth positions of the incidence rate of the colorectal cancer, the gastric cancer and the esophageal cancer respectively in 2018, and the combined incidence rate of the colorectal cancer, the gastric cancer and the esophageal cancer accounts for 30% of the incidence rate of the malignant tumor, so that the types of all cancers are the first position. In terms of mortality, gastric cancer accounts for 14%, esophageal cancer accounts for 10%, colorectal cancer accounts for 9%, the second, fourth and fifth positions respectively indicate the morbidity and mortality of Chinese malignant tumors, the combined mortality of the three cancers reaches 33%, and the mortality of all cancers is the first position at the same high level.
The major reasons for the high morbidity and mortality of digestive tract tumors in china are the lack of early screening, early diagnosis and early intervention, leading to metastasis already at the time of most cancer findings. Colorectal cancer has a total 10 year development cycle, while the best intervention period is advanced adenomas and early colorectal cancer (stages I-II). The initial treatment stage proportion of colorectal cancer in China at present is as follows: the I period accounts for 15 percent; the phase II accounts for 20-30%; stage III accounts for 30% -40%; the IV stage accounts for 20 to 25 percent. In terms of survival, the 5-year survival rate of advanced adenoma patients approaches 100%, the 5-year survival rate of stage I patients can reach over 90%, while the survival rate of stage IV patients is only slightly greater than 10%. The development process of gastric cancer is mostly from atrophic gastritis to precancerous lesion (intestinal metaplasia, dysplasia), then to early gastric cancer (stage I-II), then to late gastric cancer (stage III-IV) of lymphatic metastasis and organ metastasis. At present, the five-year survival rates of gastric cancer in China are respectively as follows: 85-89% of the phase I, 65-77% of the phase II, 31-44% of the phase III, and only 13% of the phase IV. The average survival rate of stomach cancer patients in China in five years is 36%, while the survival rate of stomach cancer patients in Japan and Korea in five years exceeds 60%. However, throughout the stage of the gastric cancer in our country: the phase I accounts for 4.1 percent, the phase II accounts for 21.8 percent, the phase III accounts for 31.7 percent, and the phase IV accounts for 42.4 percent, which is just in negative correlation with the five-year survival rate of gastric cancer in China. The five-year survival rate of esophageal cancer in China is about 30%, but the five-year survival rate of esophageal cancer in urban population is only 18%, which is far lower than 33.2% of rural population, and the five-year survival rate of esophageal cancer in China also shows a descending trend. Esophageal cancer does not have obvious early symptoms, so that the diagnosis and treatment time is usually middle and late, the optimal treatment and intervention window period is missed, and the prognosis is poor. In china, if esophageal cancer is diagnosed at the advanced stage, its five-year survival rate is less than 10%, and if found at an early stage, its five-year survival rate is as high as 85%. In conclusion, early diagnosis and early treatment of the malignant tumor of the digestive tract are not realized, and the method is an important reason for high morbidity and mortality of the malignant tumor of the digestive tract in China.
The gold standard for early screening and diagnosis of digestive tract tumors is endoscopic (enteroscope or gastroscope) detection. The endoscope detection has the advantages of high accuracy and strong specificity, and can directly observe the focus position and carry out biopsy. However, endoscopic detection methods have a number of drawbacks: 1. due to the traditional concept, insufficient cognition which brings benefits to endoscope screening by people, low consciousness of early diagnosis and early treatment and great worry about discomfort brought by endoscope examination, the willingness of residents to participate in endoscope screening is low; 2. at present, government-oriented endoscope free screening projects are tried in high-risk groups in regions with high incidence of malignant tumors in a small part of alimentary tracts in China, endoscope screening is not brought into the government medical insurance payment range in most regions, China is still a developing country, even in rural regions such as Jiangsu, developed provinces, endoscope screening still has high cost for ordinary families, and the positivity of residents for actively seeking endoscope screening is limited. 3. The endoscope screening needs a plurality of conditions such as endoscope doctors with abundant experience, expensive endoscope equipment, perfect endoscope cleaning and disinfecting facilities and the like, so that in a country with a large population, due to the lack of necessary medical and sanitary resources and manpower resources of the endoscope doctors, convenient endoscope screening cannot be realized, and the difficulty is particularly prominent in regions with laggard economic development level. Meanwhile, most of the hospital endoscopy needs to be reserved in advance, is time-consuming and labor-consuming, and also limits the endoscopy to become a first-line digestive tract malignant tumor screening means. 4. Endoscopic screening, as an invasive screening means, requires preparation in advance, such as taking cathartics or narcotics, is painful during the detection process, and is less acceptable for Chinese people who have a strong traditional concept and who invade privacy. However, some other existing protein tumor markers such as CEA and CA199 can only detect a small part of late-stage cancers, have a detection rate of less than 30% for early-stage cancers and canceration, and cannot be used for early-stage screening of digestive tract malignant tumors at all. However, in China, 8 billion of people who need early screening of the digestive tract are more than 40 years old, and even 5% of people who really participate in early screening are not, which means that early diagnosis and early treatment of digestive tract tumors in China still face huge challenges.
Abnormal DNA methylation is closely linked to the occurrence and maintenance of many diseases, and particularly recent research results show that DNA methylation plays an important role in the induction and maintenance of cancer, making it a good biomarker for many cancers. The DNA of cancer tissues is dropped into blood along with the apoptosis and metabolism of cells, so the blood extraction for detecting the free tumor DNA (ctDNA) is one of the ideal methods for realizing the noninvasive early diagnosis of cancer. At the same time, the digestive tract is derived from the same germ layer in human development, and therefore has certain similarities in function and molecular characteristics, and therefore also in biomarker selection. Therefore, if esophageal cancer, gastric cancer and colorectal cancer are detected simultaneously through one tube of blood, the screening cost is reduced to 1/3 compared with that of single cancer screening, the benefit of the testee is maximized, more screening people are included to the greatest extent, and the method has great significance for promoting early diagnosis and early diagnosis of the digestive tract tumor.
Disclosure of Invention
The invention aims to develop a non-invasive method, a marker composition and a using method for simultaneously detecting esophageal cancer, gastric cancer and colorectal cancer, and provides a new technology for early prevention and control of digestive tract tumors.
The technical scheme provided by the invention is as follows:
a gut tumor marker combination, said marker genes comprising methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; the sequences of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are respectively shown as SEQ ID NO. 34-SEQ ID NO. 39.
KCNQ5 sequence (SEQ ID NO.34)
GGTGGCGGCGGCGGTGGTTTGAGGGAGAGTCGTCGGGGTAAGTAGGGGGTTCGGATGAGTTTGTTGGGGAAGTCGTTTTTTTATACGAGTAGTTAGAGTTGTCGGCGTAACGTTAAGTATCGGCGGGTGTAGAATTATTTGTATAACGTGTTGGAGAGATTT
C9orf50 sequence (SEQ ID NO.35)
AGGAGTTTTTTTAGGAAGGCGTTTAAGAAGTCGGGGTTTTTTTTGGTTACGCGTTTTCGGGGGCGTTCGCGTTTTTTAGGTTTTGGTTGTTTGGGCGTCGATTTTCGGGACGCGTCGGTCGATAGTAGGGGAGGCGGTAGTAGGGATCGTAGTAGTTTTCGT
CLIP4 sequence (SEQ ID NO.36)
TTCGGTCGTTTGTATTGCGCGCGCGTTTATTTCGCGTGGGAGGTAGCGGGAGGGGTTCGGAGAGGTGTGGAGCGGCGCGGCGGGAGGTTTCGTGGGCGGTTACGGGAGATAGCGTCGGCGGGAGCGCGTTTTTCGGTTTTTTTTTCGCGTTTTCGCGTTTTT
ZNF582 sequence (SEQ ID NO.37)
TTTTTGGGGTTTGTGGTGTTGGGTGTGTTATTTGCGTGTGATTTTTTAGCGAGAGATTGTGGGCGAGTGATCGAGTGGGTAAGGGGTCGTTATTGTGTGTGCGTGATTTTGATAGTGTGTGGTGGTAGTTTTTGATTTCGCGTGGGTCGTTGAATGTATGATTGGGATCGTTTAGCGGTGGATATATAATTGTGTGCGGTTGTGGA
TFPI2 sequence (SEQ ID NO.38)
TTATTTTTTAGGTTTCGTTTCGGCGGGGGTCGGTCGGACGTTCGTTTCGTATAAAGCGGGTATTCGGGTCGTTTGGAGTAGAAAGTCGCGTATTTTTTTTCGTTAGGCGTTTTTTCGGACGTTTTGTTTAGCGGGTCGTTCGATTTTTTGTATTATGGATTTCGTTCGTTTTTTGGGGTT
ELMO1 sequence (SEQ ID NO.39)
TTCGTAGCGTTCGTTCGGGAGGAGAGTTCGCGGTTGATTTTCGGATGTTTTGTAGATGTAAAATGTGTTTTTGGTTAGTAGGAGGAAGGAAGAGGAAGTGAGAGTAGCGGTAGTCGGCGGTGTAGTAGTCGGTCGATTTAGAGTGTAAGTGCGTGTGTTGGGGCGAGCGGGAGCGGGCGAGGATGGGTATAGGATAGAGGTAGAGTTATTTACGTCGTCGCGGTTTTACGTTGGGCGATAGAGTTTTTAGTTTTTTTTTAATGGTGGCGGGTCGTCGGAGTTTTGATCGTCGGGAATTTT
Further, the amplified primer probe sequences used by the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are shown in SEQ ID NO. 1-SEQ ID NO. 30.
Further, the amplified fragments of methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 contain at least one CpG site.
Furthermore, the marker combination also comprises 1 internal reference gene, the internal reference gene is ACTB, and the primer probe sequence of the internal reference gene is shown in SEQ ID NO. 31-SEQ ID NO. 33.
The invention also provides application of the digestive tract tumor marker combination in preparing a reagent for detecting digestive tract tumors.
The invention also provides a primer group, and the sequence of the primer group is shown as SEQ ID NO. 1-SEQ ID NO. 33.
The invention also provides a detection kit, which comprises the primer group.
The invention also provides a combined use method of the digestive tract tumor marker, which comprises the steps of extracting and purifying nucleic acid from a sample, treating with bisulfite, converting unmethylated cytosine into uracil, and performing methylation specific fluorescent quantitative PCR detection by adopting KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1;
when any gene of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 reacts positively, the detection range is judged to be positive.
Further, the method also comprises a positive external quality control and a negative external quality control, wherein the positive external quality control is a genome sequence methylated to be completely positive, and the negative external quality control is any one of deionized water, 1xTE buffer or 10mM Tris-HCl (pH 8.0).
Further, the tumors of the digestive tract comprise esophageal cancer, gastric cancer and colorectal cancer.
Advantageous effects
The invention provides a digestive tract tumor marker combination, wherein the marker genes comprise methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; the reason for selecting these genes is: KCNQ5, C9orf50, CLIP4 and TFPI2 show hypermethylation in colorectal cancer, KCNQ5, C9orf50, CLIP4 and ELMO1 show hypermethylation in gastric cancer, and KCNQ5, C9orf50, ZNF582 and TFPI2 show hypermethylation in esophageal cancer. However, the sensitivity of single gene methylation detection in blood is low, so that the combination detection of the compositions is performed, the sensitivity is improved, and 3 tumors can be well covered.
The detection sensitivity of the composition is obviously higher than that of four tumor markers including AFP, CEA, CA125 and CA199 and the combined detection sensitivity thereof, which shows that the tumor marker composition has higher sensitivity than that of the traditional serum marker, and the sensitivity difference between the three tumors is obviously smaller than that of the traditional serum marker, so that the composition has no obvious preference on the three tumors in early diagnosis and screening of the digestive tract tumor, has low omission factor and is more suitable for early diagnosis of the digestive tract tumor.
Drawings
FIG. 1 is a concentration gradient reaction amplification curve of methylated KCNQ 5;
FIG. 2 is a concentration gradient reaction amplification curve for methylated C9orf 50;
FIG. 3 is a concentration gradient reaction amplification curve of methylated CLIP 4;
FIG. 4 is a concentration gradient reaction amplification curve for methylated ZNF 582;
FIG. 5 is a concentration gradient reaction amplification curve of methylated TFPI 2;
FIG. 6 is a concentration gradient reaction amplification curve of methylated ELMO 1;
FIG. 7 is a graph of the sensitivity and specificity of methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2, and ELMO1 and compositions thereof for detecting esophageal, gastric, and colorectal cancers;
FIG. 8 is a ROC curve of methylated KCNQ5 for detecting digestive tract tumors; AUC ═ 0.694 (95% CI: 0.615-0.772);
FIG. 9 is a ROC curve for detecting digestive tract tumors using methylated C9orf 50; AUC 0.720 (95% CI: 0.643-0.796);
FIG. 10 is a ROC curve for detecting digestive tract tumors by methylated CLIP 4; AUC 0.682 (95% CI: 0.602-0.763);
FIG. 11 is a ROC curve for detecting digestive tract tumors using ZNF582 methylated; AUC ═ 0.574 (95% CI: 0.487-0.661);
FIG. 12 is a ROC curve for detecting digestive tract tumors using methylated TPFI 2; AUC 0.595 (95% CI: 0.509-0.680);
FIG. 13 is a ROC curve for methylated ELMO1 detection of tumors of the digestive tract; AUC 0.640 (95% CI: 0.558-0.723);
FIG. 14 is a ROC curve for detecting digestive tract tumors using a combination of methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; AUC ═ 0.821 (95% CI: 0.753-0.888).
FIG. 15 is a graph of the sensitivity of methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2, and ELMO1 combinations of the present invention versus AFP, CEA, CA125, and CA19-9 for the detection of digestive tract tumors.
Detailed Description
Example 1
Taking the methylation positive genome DNA solution after bisulfite conversion as a detection object, respectively setting 4 concentration extractions, 10 copies/reaction, 100 copies/reaction, 1000 copies/reaction and 10000 copies/reaction. Preparing a KCNQ5, C9orf50, CLIP4 and ACTB mixed detection fluorescent quantitative PCR reaction system by using SEQ ID 1-3, SEQ ID 7-12 and SEQ ID 31-33, wherein the fluorescent quantitative PCR reaction system is as follows: primer concentration 0.4mM, probe concentration 0.1mM,1 XPCR buffer, 6mM MgCl20.1U/ul DNA polymerase, 15ul of PCR reaction mix volume, 15ul of DNA template volume, and reaction conditions of 95 ℃ for 20 minutes, (95 ℃ for 10 seconds, 58 ℃ for 30 seconds, 72 ℃ for 10 seconds) x 50 cycles, 40 ℃ for 1 minute.
As shown in FIGS. 1-3, the methylated KCNQ5, C9orf50 and CLIP4 were amplified well, and the minimum reaction concentration was 10 copies/reaction.
Example 2
Taking the methylation positive genome DNA solution after bisulfite conversion as a detection object, respectively setting 4 concentration extractions, 10 copies/reaction, 100 copies/reaction, 1000 copies/reaction and 10000 copies/reaction. Preparing a ZNF582, TFPI2, ELMO1 and ACTB mixed detection fluorescent quantitative PCR reaction system by using SEQ ID 16-18, SEQ ID 22-23 and SEQ ID 28-33, wherein the fluorescent quantitative PCR reaction system is as follows: primer concentration 0.2mM, probe concentration 0.1mM,1 XPCR buffer, 6mM MgCl2Solution, 0.12U/ulDNA polymerase, PCR reaction mixing volume of 15ul, DNA template volume of 15ul, reaction conditions of 95 degrees C20 minutes, (95 degrees C10 seconds, 58 degrees C30 seconds, 72 degrees C10 seconds) x 50 cycles, 40 degrees C1 minutes.
As shown in FIGS. 4-6, the methylated ZNF582, TFPI2 and ELMO1 were all amplified well and the minimum reaction concentration was 10 copies/reaction.
Example 3
Blood of 10 cases of esophageal cancer, 54 cases of gastric cancer, 34 cases of colorectal cancer and 67 normal control samples are taken as detection objects, 10mL of blood is respectively extracted, 3.5mL of blood plasma is separated, cfDNA is extracted by a free nucleic acid extraction kit of Suzhou Wei Biotechnology Co., Ltd, and then conversion and purification are carried out by a bisulfite rapid conversion kit of Suzhou Wei Biotechnology Co., Ltd. Preparing KCNQ5, C9orf50, CLIP4 and ACTB from SEQ ID 1-7, SEQ ID 13-15 and SEQ ID 31-33 to prepare a fluorescence quantitative PCR reaction system 1, and preparing ZNF582, TFPI2, ELMO1 and ACTB from SEQ ID 19-21 and SEQ ID 25-33 to prepare a fluorescence quantitative PCR reaction system 2; the rest components of the 2 groups of fluorescent quantitative PCR reaction systems are as follows: primer concentration 0.4mM, probe concentration 0.1mM,1 XPCR buffer, 6mM MgCl20.12U/ul DNA polymerase, 15ul of PCR reaction mix volume, 15ul of DNA template volume, and reaction conditions of 95 ℃ for 20 minutes, (95 ℃ for 10 seconds, 60 ℃ for 30 seconds, 72 ℃ for 15 seconds) x 50 cycles, 40 ℃ for 30 seconds.
The detection result is shown in fig. 7, the sensitivity of the single marker for detecting esophageal cancer is 10% -40%, the sensitivity is improved to 80% by combining a plurality of markers, the sensitivity of the single marker for detecting colorectal cancer is 13% -43%, the sensitivity is improved to 72% by combining a plurality of markers, the sensitivity of the single marker for detecting colorectal cancer is 24-68%, and the sensitivity is improved to 85% by combining a plurality of markers. The sensitivity of detecting three digestive tract tumors by a single target is 16-48%, the sensitivity can reach 78% after the combination of a plurality of markers, and the specificity is 87%.
The ROC curves for the single marker and the multiple marker combination to detect three digestive tract tumors are shown in FIGS. 8-14, and the AUC area for the single target to detect the digestive tract tumors ranges from 0.574 to 0.720, which is significantly increased to 0.821 by the multiple marker combination.
The data fully prove that the composition and the detection method can effectively detect the digestive tract tumor, have higher sensitivity and specificity and provide an effective method for early diagnosis of the digestive tract tumor.
Example 4
Blood samples of 10 cases of esophageal cancer, 42 cases of gastric cancer and 24 cases of colorectal cancer are taken as detection objects, 10mL of blood is respectively extracted, 3.5mL of blood plasma is separated, cfDNA is extracted by a free nucleic acid extraction kit of Suzhou Wei Biotechnology Co., Ltd, and then conversion and purification are carried out by a bisulfite rapid conversion kit of Suzhou Wei Biotechnology Co., Ltd. Preparing KCNQ5, C9orf50, CLIP4 and ACTB from SEQ ID 1-7, SEQ ID 13-15 and SEQ ID 31-33 to prepare a fluorescence quantitative PCR reaction system 1, and preparing ZNF582, TFPI2, ELMO1 and ACTB from SEQ ID 19-21 and SEQ ID 25-33 to prepare a fluorescence quantitative PCR reaction system 2; the rest components of the 2 groups of fluorescent quantitative PCR reaction systems are as follows: primer concentration 0.4mM, probe concentration 0.1mM,1 XPCR buffer, 6mM MgCl20.12U/ul DNA polymerase, 15ul of PCR reaction mix volume, 15ul of DNA template volume, and reaction conditions of 95 ℃ for 20 minutes, (95 ℃ for 10 seconds, 60 ℃ for 30 seconds, 72 ℃ for 15 seconds) x 50 cycles, 40 ℃ for 30 seconds. Meanwhile, 3mL of blood of the sample is extracted, four tumor markers including AFP, CEA, CA125 and CA199 in the blood are detected, and the sensitivity difference between the detection sensitivity of the AFP, CEA, CA125 and CA199 in the blood and the detection sensitivity of the digestive tract tumor in the invention are compared.
The detection result is shown in fig. 15, the detection sensitivity of the composition is obviously higher than that of four tumor markers including AFP, CEA, CA125 and CA199 and the combined detection sensitivity thereof, and the tumor marker composition is proved to have higher sensitivity than that of the traditional serum tumor marker and to be more suitable for early diagnosis of digestive tract tumors.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 25
ttttttaggt ttcgtttcgg c 21
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gcctaacgaa aaaaaatacg cg 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tataaagcgg gtattcgggt cg 22
<210> 28
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cgacgacgta aataactcta cctc 24
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ggtgtagtag tcggtcgatt taga 24
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tatacccatc ctcgcccgct cccg 24
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gtgatggagg aggtttagta agtt 24
<210> 32
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ccaataaaac ctactcctcc cttaa 25
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
accaccaccc aacacacaat aacaaacaca 30
<210> 34
<211> 162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ggtggcggcg gcggtggttt gagggagagt cgtcggggta agtagggggt tcggatgagt 60
ttgttgggga agtcgttttt ttatacgagt agttagagtt gtcggcgtaa cgttaagtat 120
cggcgggtgt agaattattt gtataacgtg ttggagagat tt 162
<210> 35
<211> 162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aggagttttt ttaggaaggc gtttaagaag tcggggtttt ttttggttac gcgttttcgg 60
gggcgttcgc gttttttagg ttttggttgt ttgggcgtcg attttcggga cgcgtcggtc 120
gatagtaggg gaggcggtag tagggatcgt agtagttttc gt 162
<210> 36
<211> 162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttcggtcgtt tgtattgcgc gcgcgtttat ttcgcgtggg aggtagcggg aggggttcgg 60
agaggtgtgg agcggcgcgg cgggaggttt cgtgggcggt tacgggagat agcgtcggcg 120
ggagcgcgtt tttcggtttt tttttcgcgt tttcgcgttt tt 162
<210> 37
<211> 206
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tttttggggt ttgtggtgtt gggtgtgtta tttgcgtgtg attttttagc gagagattgt 60
gggcgagtga tcgagtgggt aaggggtcgt tattgtgtgt gcgtgatttt gatagtgtgt 120
ggtggtagtt tttgatttcg cgtgggtcgt tgaatgtatg attgggatcg tttagcggtg 180
gatatataat tgtgtgcggt tgtgga 206
<210> 38
<211> 180
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ttatttttta ggtttcgttt cggcgggggt cggtcggacg ttcgtttcgt ataaagcggg 60
tattcgggtc gtttggagta gaaagtcgcg tatttttttt cgttaggcgt tttttcggac 120
gttttgttta gcgggtcgtt cgattttttg tattatggat ttcgttcgtt ttttggggtt 180
<210> 39
<211> 300
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ttcgtagcgt tcgttcggga ggagagttcg cggttgattt tcggatgttt tgtagatgta 60
aaatgtgttt ttggttagta ggaggaagga agaggaagtg agagtagcgg tagtcggcgg 120
tgtagtagtc ggtcgattta gagtgtaagt gcgtgtgttg gggcgagcgg gagcgggcga 180
ggatgggtat aggatagagg tagagttatt tacgtcgtcg cggttttacg ttgggcgata 240
gagtttttag ttttttttta atggtggcgg gtcgtcggag ttttgatcgt cgggaatttt 300
Claims (10)
1. A gut tumor marker combination comprising methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; the sequences of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are respectively shown as SEQ ID NO. 34-SEQ ID NO. 39.
2. The gut tumor marker combination of claim 1, wherein the amplified primer probe sequences used for methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are shown in SEQ ID No. 1-SEQ ID No. 30.
3. The gut tumor marker combination of claim 1, wherein the amplified fragment of methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 comprises at least one CpG site.
4. The gut tumor marker combination of claim 1, further comprising 1 internal reference gene, wherein the internal reference gene is ACTB and the primer probe sequence is shown in SEQ ID No. 31-SEQ ID No. 33.
5. Use of the gut tumor marker combination of any one of claims 1 to 4 in the preparation of a reagent for the detection of gut tumors.
6. A primer group is characterized in that the sequence of the primer group is shown as SEQ ID NO. 1-SEQ ID NO. 33.
7. A detection kit comprising the primer set according to claim 6.
8. The method for using the combination of tumor markers of the digestive tract according to any one of claims 1 to 4, wherein after nucleic acid extraction and purification, the sample is treated with bisulfite, unmethylated cytosine is converted into uracil, and methylation-specific fluorescent quantitative PCR detection is performed using KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO 1; when any gene of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 reacts positively, the detection range is judged to be positive.
9. The method for using the combination of digestive tract tumor markers in claim 8, wherein the method further comprises a positive external quality control and a negative external quality control, the positive external quality control is a genome sequence which is methylated to be completely positive, and the negative external quality control is any one of deionized water, 1xTE buffer or 10mM Tris-HCl (pH 8.0).
10. The combination of gut tumor markers according to any one of claims 1 to 4, wherein the gut tumor comprises esophageal cancer, gastric cancer and colorectal cancer.
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| CN113355415A (en) * | 2021-06-04 | 2021-09-07 | 武汉艾米森生命科技有限公司 | Detection reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer |
| WO2022143226A1 (en) * | 2020-12-30 | 2022-07-07 | 苏州唯善生物科技有限公司 | Digestive track tumor marker combination, detection reagent kit, and uses thereof |
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| WO2024086373A1 (en) * | 2022-10-21 | 2024-04-25 | The Johns Hopkins University | Biomarkers for detection and treatment of barrett's esophagus |
| CN116083581B (en) * | 2022-12-27 | 2023-12-08 | 广州优泽生物技术有限公司 | Kit for detecting early digestive tract tumor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110423814A (en) * | 2019-07-19 | 2019-11-08 | 江苏元丞生物科技有限公司 | ELMO1 gene methylation detection kit and its application |
| CN110551818A (en) * | 2019-08-14 | 2019-12-10 | 苏州唯善生物科技有限公司 | Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumor and application of multi-gene detection primer probe set and kit |
| CN110938694A (en) * | 2019-12-05 | 2020-03-31 | 苏州唯善生物科技有限公司 | Esophageal squamous carcinoma detection kit and detection device |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014062218A1 (en) * | 2012-10-16 | 2014-04-24 | University Of Southern California | Colorectal cancer dna methylation markers |
| CN106232833A (en) * | 2014-01-30 | 2016-12-14 | 加利福尼亚大学董事会 | The haplotyping that methylates (MONOD) for non-invasive diagnostic |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
| CN110616263A (en) * | 2019-09-27 | 2019-12-27 | 江苏元丞生物科技有限公司 | Multi-site colorectal cancer methylation detection kit |
| CN111560434A (en) * | 2020-05-18 | 2020-08-21 | 苏州唯善生物科技有限公司 | Primer probe set, kit, detection method and application for detecting digestive tract tumor marker |
| CN111793688B (en) * | 2020-07-10 | 2024-01-30 | 苏州唯善生物科技有限公司 | Primer probe group and kit for detecting digestive tract tumor markers and application of primer probe group and kit |
| CN112646891B (en) * | 2020-12-30 | 2023-09-05 | 苏州唯善生物科技有限公司 | Digestive tract tumor marker combination, detection kit and application thereof |
-
2020
- 2020-12-30 CN CN202011620171.6A patent/CN112646891B/en active Active
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110423814A (en) * | 2019-07-19 | 2019-11-08 | 江苏元丞生物科技有限公司 | ELMO1 gene methylation detection kit and its application |
| CN110551818A (en) * | 2019-08-14 | 2019-12-10 | 苏州唯善生物科技有限公司 | Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumor and application of multi-gene detection primer probe set and kit |
| CN110938694A (en) * | 2019-12-05 | 2020-03-31 | 苏州唯善生物科技有限公司 | Esophageal squamous carcinoma detection kit and detection device |
Non-Patent Citations (2)
| Title |
|---|
| FRANCESCA PIRINI等: "Early detection of gastric cancer using global, genome-wide and IRF4, ELMO1, CLIP4 and MSC DNA methylation in endoscopic biopsies", 《ONCOTARGET》 * |
| SARAH ØSTRUP JENSEN 等: "Novel DNA methylation biomarkers show high sensitivity and specificity for bloodbased detection of colorectal cancer—a clinical biomarker discovery and validation study", 《CLINICAL EPIGENETICS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022143226A1 (en) * | 2020-12-30 | 2022-07-07 | 苏州唯善生物科技有限公司 | Digestive track tumor marker combination, detection reagent kit, and uses thereof |
| CN113355415A (en) * | 2021-06-04 | 2021-09-07 | 武汉艾米森生命科技有限公司 | Detection reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer |
| CN113355415B (en) * | 2021-06-04 | 2022-06-10 | 武汉艾米森生命科技有限公司 | Detection reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer |
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