CN115927608B - Biomarkers, methods and diagnostic devices for predicting pancreatic cancer risk - Google Patents
Biomarkers, methods and diagnostic devices for predicting pancreatic cancer risk Download PDFInfo
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Abstract
The present application provides biomarkers, methods, and diagnostic devices for predicting the risk of pancreatic carcinogenesis. In particular, the present application provides the use of genes, mRNA, cDNA, proteins or detection reagents thereof for markers of risk of pancreatic carcinogenesis for the preparation/establishment of diagnostic reagents or kits/models for determining risk of occurrence. Research shows that the pancreatic cancer risk marker can be used as a marker for judging pancreatic cancer occurrence, and has high sensitivity and specificity.
Description
Technical Field
The present application relates to the field of clinical medicine, in particular to biomarkers, methods and diagnostic devices for predicting the risk of pancreatic carcinogenesis.
Background
Pancreatic cancer is called the king of cancers, and is one of the most fatal cancers. Most pancreatic cancer patients do not have any special clinical symptoms at an early stage, and therefore most pancreatic cancer patients miss an optimal treatment period. During the last decades, no method has been found that can significantly increase patient survival, pancreatic cancer patients have a5 year survival rate of only 5% -15%, an overall survival rate of about 6%, and only 20% of patients can undergo surgical treatment at the time of discovery. Thus, timely and effective diagnosis plays an important role in the pancreatic cancer prevention and treatment process.
Currently, the main diagnosis methods of pancreatic cancer mainly include ultrasonic examination, electronic computed tomography (Computed Tomography, CT), and nuclear magnetic resonance imaging technologies, but these imaging technologies cannot meet the requirements of early pancreatic cancer screening. The blood tumor marker detection only needs a small amount of blood samples, has the advantages of being minimally invasive and safe, and is an ideal way for tumor screening diagnosis. Some tumor antigens are used as related indexes for pancreatic cancer diagnosis, and saccharide antigen 199 (Carbohydrate antigen, 199, CA199) is the most widely used, but the sensitivity and the specificity of the markers are low, so that the application of the markers in pancreatic cancer screening diagnosis is limited. There is currently no effective and accurate biomarker for pancreatic cancer screening and diagnosis.
In view of the above, it is urgent to find new pancreatic cancer markers with diagnostic or combined diagnostic value, and to develop targeted drugs with targeting. Thus, there is an urgent need in the art to develop pancreatic cancer specific markers with high sensitivity and specificity for early diagnosis or effective treatment of pancreatic cancer, or to evaluate the prognostic effect of disease.
Disclosure of Invention
The application aims to provide a pancreatic cancer marker with high sensitivity and high specificity and application thereof in clinical diagnosis and treatment.
In a first aspect of the present application, there is provided the use of a gene, mRNA, cDNA, protein, or a detection reagent thereof, for the manufacture of a diagnostic reagent or kit for determining the risk of pancreatic cancer;
wherein the pancreatic cancer risk marker is selected from the group consisting of:
(A) Any one of the markers selected from A1 to A7, or a combination thereof: (A1) hx2; (A2) CYYR1; (A3) GJA4; (A4) GSDME; (A5) LYNX1; (A6) RARP10; (A7) ZNF250.
In another preferred embodiment, the pancreatic cancer risk marker further comprises any one marker selected from the following group B, or a combination thereof: (B1) COL10A1; (B2) COL17A1; (B3) CDH3; (B4) CUZD1; (B5) GPT; (B6) SLC45A4; (B7) SQLE.
In another preferred embodiment, the pancreatic cancer risk marker is mRNA or cDNA.
In another preferred embodiment, the pancreatic cancer risk marker further comprises at least 2 selected from A1 to A7.
In another preferred embodiment, the pancreatic cancer risk marker is selected from the group consisting of one or more markers of A1 to A7 and one or more markers of B1 to B7.
In another preferred embodiment, the A1-A7 markers are selected from Table A:
table A
In another preferred embodiment, the B1-B7 marker is selected from table B:
table B
In another preferred embodiment, the pancreatic cancer risk marker combination is: (A1) hx2; (A2) CYYR1; (B1) COL10A1; (B2) COL17A1.
In another preferred embodiment, the pancreatic cancer risk marker combination is: (A2) CYYR1; (B1) COL10A1; (B2) COL17A1.
In another preferred embodiment, the pancreatic cancer risk marker combination is: (A1) hx2; (B1) COL10A1.
In a second aspect of the application, there is provided a kit comprising a detection reagent for detecting a gene, mRNA, cDNA, protein, or a combination thereof, which is a marker for pancreatic cancer,
wherein the pancreatic cancer risk marker is selected from the group consisting of:
(A) A combination of two or more markers selected from A1 to A7;
(B) A combination of one or more markers selected from A1 to A7 and one or more markers selected from B1 to B7.
In another preferred embodiment, the detection reagent comprises:
(a) A specific antibody, a specific binding molecule, directed against the pancreatic cancer risk marker; and/or
(b) Primers or primer pairs, probes or chips (e.g., nucleic acid chips or protein chips) that specifically amplify the mRNA or cDNA of the pancreatic cancer risk marker.
In another preferred embodiment, the gene, mRNA, cDNA, or protein of any one of the markers set forth in table a and/or table B is of human origin.
In another preferred embodiment, the subject is a human.
In another preferred embodiment, the subject is a non-tumor patient, a tumor patient.
In another preferred embodiment, the tumor patient comprises pancreatic cancer.
In another preferred embodiment, the gene, mRNA, cDNA, or protein of the pancreatic cancer risk marker is of human origin.
In another preferred embodiment, the detection is for an ex vivo sample.
In another preferred embodiment, the ex vivo sample comprises: tissue samples, cell samples, blood samples, serum samples.
In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.
In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.
In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.
In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to polynucleotides (mRNA or cDNA) of any of the pancreatic cancer risk markers.
In another preferred embodiment, the protein chip comprises a substrate and specific antibodies spotted on the substrate, wherein the specific antibodies comprise specific antibodies against the pancreatic cancer risk marker.
In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
In another preferred embodiment, the kit contains genes, mRNAs, cDNAs and/or proteins of pancreatic cancer risk markers as a control or quality control.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used to (a) determine the risk of developing pancreatic cancer, and/or (b) evaluate the effectiveness of pancreatic cancer treatment.
In another preferred embodiment, the reagents comprise primers, probes, gRNA or a combination thereof, more preferably a primer pair or probe for PCR, qPCR, RT-PCR.
In another preferred embodiment, the pancreatic cancer risk marker is detected by the following method: sequencing, PCR, or a combination thereof.
In another preferred embodiment, the detection of the pancreatic cancer risk marker is quantitatively detectable.
In a third aspect of the present application, there is provided a detection method comprising the steps of:
(a) Providing a test sample, wherein the test sample is selected from blood samples;
(b) Detecting the expression level of a pancreatic cancer risk marker gene in the detection sample, and marking the expression level as C1; and
(c) Comparing the pancreatic cancer risk marker concentration C1 with a control reference value C0;
wherein the pancreatic cancer risk marker is selected from the group consisting of:
(A) Any one of the markers selected from A1 to A7, or a combination thereof: (A1) hx2; (A2) CYYR1; (A3) GJA4; (A4) GSDME; (A5) LYNX1; (A6) RARP10; (A7) ZNF250;
a subject is prompted to have a high risk of pancreatic carcinogenesis if the detection result of pancreatic cancer risk in the subject meets the following conditions:
(1) When a certain marker is an up-regulated marker in the table a in a detection subject and the expression level of the marker is higher than a reference value or a standard value C0, the detection subject has high pancreatic cancer occurrence risk;
(2) When a certain marker is a marker down-regulated in table a in a test subject and the expression level of the marker is lower than a reference value or standard value C0, the test subject is at high risk of developing pancreatic cancer.
In a fourth aspect of the present application, there is provided a pancreatic cancer diagnosis apparatus comprising:
(a) The input module is used for inputting pancreatic cancer risk marker data of a blood sample of a certain object;
wherein said risk marker gene comprises a gene selected from the group consisting of:
(A) Any one of the markers selected from A1 to A7, or a combination thereof: (A1) hx2; (A2) CYYR1; (A3) GJA4; (A4) GSDME; (A5) LYNX1; (A6) RARP10; (A7) ZNF250;
(b) The processing module calculates the input marker genes according to a preset scoring formula to obtain a risk score; and comparing the score with a Cut-off value (Cut-off) to obtain a discrimination result, wherein when the risk score is higher than the Cut-off value (Cut-off), the subject is prompted to be a pancreatic cancer patient; when the risk score is below the Cut-off value, then suggesting that the subject is a non-pancreatic cancer patient; and
(c) And the output module is used for outputting the diagnosis result.
In another preferred embodiment, the scoring formula is:
wherein Wi is the weight value of each gene; the Pi is the expression level of each gene.
In another preferred embodiment, the scoring formula may be calculated manually.
In another preferred embodiment, the scoring formula may be automatically calculated by designing a computer-aided program.
In a fifth aspect of the present application, there is provided a method for detecting the expression level of pancreatic cancer risk-marker combination, comprising the steps of:
(a) Providing a serum sample;
(b) Extracting total RNA of the serum sample;
(c) Performing reverse transcription on the product RNA obtained in step (b);
(d) Performing fluorescent quantitative PCR on the reverse transcription product obtained in the step (c), thereby obtaining the expression level of the risk marker gene;
wherein the risk marker is selected from the group consisting of:
(A) Any one of the markers selected from A1 to A7, or a combination thereof: (A1) hx2; (A2) CYYR1; (A3) GJA4; (A4) GSDME; (A5) LYNX1; (A6) RARP10; (A7) ZNF250.
In another preferred embodiment, the method is a non-diagnostic, non-therapeutic method;
in another preferred embodiment, the method is an in vitro method. In another preferred embodiment, in the step (d), the upstream and downstream specific primer sequences corresponding to the respective genes are respectively: SEQ ID NOS: 1-28.
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a ROC graph of the diagnostic effect of scheme 1 (COL 10A1, COL17A1, CYYR1, ZHX2 as a combination marker)
FIG. 2 shows a ROC graph of the diagnostic effect of scheme 2 (COL 10A1, COL17A1, CYYR1 as a combination marker)
FIG. 3 shows a ROC graph of the diagnostic effect of scheme 3 (COL 10A1, ZHX2 as a combined marker)
Detailed Description
The present inventors have made extensive and intensive studies, and for the first time developed a marker combination for diagnosis of pancreatic cancer with high sensitivity and high specificity. Specifically, through database study, mRNA expression profile levels of pancreatic cancer and normal pancreatic tissue samples were analyzed, from which 37 specific mRNA markers were screened for the first time by statistical methods. Serum level tests prove that 14 specific mRNA markers are selected from the kit, so that pancreatic cancer patients and healthy people can be very effectively distinguished, and corresponding auxiliary treatment or intervention treatment can be carried out on pancreatic cancer high-risk patients as early as possible. The present application has been completed on the basis of this finding.
Terminology
The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject.
As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density on immunoblots, such as Western blots, is also within the term "expression" based on biological molecules.
As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined based on mRNA expression and/or protein expression compared to pancreatic cancer risk markers and statistically analyzed. Some of these studies are shown in the examples section herein. However, the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are particularly relevant to the patient's population, medical history, genetics, age, and other factors.
Pancreatic cancer risk markers
As used herein, the term "pancreatic cancer risk marker of the present application" refers to one or more markers shown in table a and/or table B.
In the present application, the terms "pancreatic cancer risk marker protein of the present application", "polypeptide of the present application", or "marker shown in table a and/or table B" are used interchangeably, and refer to any one or more of the pancreatic cancer risk markers of the present application.
In the present application, the terms "pancreatic cancer risk marker gene", "pancreatic cancer risk marker polynucleotide" are used interchangeably and refer to the nucleotide sequence of any one of the pancreatic cancer risk markers shown in table a and/or table B.
It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.
In case that information on pancreatic cancer risk markers is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequence of pancreatic cancer risk marker disclosed in the present application, particularly the open reading frame sequence, and amplified to obtain the relevant sequence using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the application (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.
The polynucleotide sequences of the present application can be used to express or produce recombinant pancreatic cancer risk markers by conventional recombinant DNA techniques.
Specific antibodies
In the present application, the terms "antibody of the present application" and "specific antibody against pancreatic cancer risk marker" are used interchangeably to refer to an antibody that can be used to specifically bind to and detect the pancreatic cancer risk marker of the present application.
Antibodies of the application directed against pancreatic cancer risk markers (table a and/or table B) include polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, having specificity.
The application includes not only intact monoclonal or polyclonalAntibodies, and also include immunologically active antibody fragments, such as Fab' or (Fab) 2 Fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.
Antibodies of the application may be prepared by various techniques known to those skilled in the art. For example, a purified gene product of a human pancreatic cancer risk marker, or an antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing a human pancreatic cancer risk marker protein or an antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibodies of the application may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology.
Antibodies against human pancreatic cancer risk marker proteins are useful in immunohistochemical techniques to detect human pancreatic cancer risk marker proteins in specimens, particularly tissue samples or blood samples. Since the pancreatic cancer risk marker protein exists in a blood sample or a tissue sample, the expression level thereof can be a detection target.
Detection method
Based on differential expression of pancreatic cancer risk markers in tissue samples or blood samples, the application also provides a corresponding method for judging pancreatic cancer risk.
The present application relates to diagnostic assays for the quantitative and positional detection of protein levels or mRNA levels of pancreatic cancer risk markers. Such tests are well known in the art. The level of human pancreatic cancer risk marker protein or mRNA detected in the assay can be used to determine (including aiding in the determination of) whether there is a risk of pancreatic cancer.
A preferred method is to perform a quantitative PCR/qPCR/RT-PCR assay on mRNA or cDNA.
One preferred method is to quantitatively detect mRNA or cDNA, sequencing.
Polynucleotides of pancreatic cancer risk markers are useful for diagnosis of pancreatic cancer risk. A part or all of the polynucleotides of the present application can be immobilized as probes on a microarray or DNA chip for differential expression analysis and gene diagnosis of genes in analysis.
In addition, the application can also detect at the protein level. For example, antibodies against pancreatic cancer risk markers may be immobilized on a protein chip for detecting pancreatic cancer risk proteins in a sample.
Detection kit
Because of the correlation between pancreatic cancer risk markers and pancreatic cancer risk, pancreatic cancer risk markers can be used as judgment markers for pancreatic cancer risk.
The application also provides a kit for judging pancreatic cancer risk, which comprises a detection reagent for detecting the gene, mRNA, cDNA, protein or the combination thereof of pancreatic cancer risk markers. Preferably, the kit contains an antibody or immunoconjugate of the application, or an active fragment thereof, against a pancreatic cancer risk marker; or a primer or primer pair, probe or chip containing mRNA or cDNA specifically amplified for pancreatic cancer risk markers.
In another preferred embodiment, the kit further comprises a label or instructions.
The main advantages of the application include:
(1) Compared with the existing imaging technology, the application adopts the blood sample, is more suitable for early screening diagnosis, and has the characteristics of rapidness, convenience and low cost.
(2) Compared with the existing tumor antigen detection method, the marker combination established by the application has higher specificity and more accurate detection result.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 screening and determination of pancreatic cancer diagnostic markers
Specifically, the inventor adopts mRNA expression profiles of pancreatic cancer (cancer tissues and paracancerous tissues) in a TCGA database and a GEO database, groups samples according to the pathological stage of the pancreatic cancer, and obtains mRNA differentially expressed between a group of pancreatic cancer and normal pancreatic tissue samples by comparing the expression profiles of different sample groups. The different mRNA is used as a candidate, a Support Vector Machine (SVM) model is used for calculation, and different algorithms are adopted for calculation and screening of molecular markers on the basis of the calculation, so that a multi-marker combined classifier (containing 2 or more mRNA markers) with higher classification accuracy is obtained. The classifier composed of the mRNA markers can predict whether the sample has pancreatic cancer, and the prediction accuracy can reach 93.90 percent. Based on the set of mRNA markers of the application, RT-PCR kits can be developed for early screening of pancreatic cancer.
Example 2 verification of pancreatic cancer diagnostic markers (blood samples)
We collected 229 serum samples for verification of classifier accuracy, 131 of which pancreatic cancer and 98 of healthy volunteers.
EXAMPLE 3 pancreatic cancer diagnostic markers are used singly
4 genes with significantly higher occurrence frequency of pancreatic cancer positive rate in single use are selected from pancreatic cancer samples, and the single use effect in serum is further evaluated.
The results are shown in Table 1:
table 1 Effect of markers alone in serum
| Gene | Cut-off value | AUC | Sensitivity (%) | Specificity (%) | Accuracy (%) |
| ZHX2 | ≥1.9 | 0.841 | 97.70％ | 79.90％ | 88.80％ |
| CYYR1 | ≥2.0 | 0.637 | 62.00％ | 64.00％ | 63.00％ |
| COL10A1 | ≥1.9 | 0.801 | 84.50％ | 77.00％ | 80.75％ |
| COL17A1 | ≤1.5 | 0.551 | 40.30％ | 98.00％ | 69.15％ |
ZHX2, CYYR1, COL10A1, and COL17A1 all have good sensitivity, specificity, and accuracy when used alone. Among them, the sensitivity (%), the specificity (%) and the accuracy (%) of the single use of the ZHX2 gene are particularly outstanding.
Example 4 Combined use of pancreatic cancer diagnostic markers
In this example, the effect of detecting a combination of a plurality of pancreatic cancer diagnostic markers in a serum sample was further verified.
The results are shown in FIGS. 1-3, respectively.
FIG. 1 shows the ROC effect of the 4 marker combinations of scheme 1COL10A1, COL17A1, CYYR1 and ZHX 2.
Wherein, the scoring calculation formula is adopted as follows:
score = 3.2 x col10a1+0.1 x zhx2-2 x cyyr1-2.5 x col17a1,
the cut-off value is defined as: -1.22
The verification results of 229 samples were:
| accuracy rate of | 93.87％ |
| AUC | 0.975 |
| Sensitivity of | 93.10％ |
| Specificity (specificity) | 94.90％ |
FIG. 2 shows the ROC effect of the 3 marker combinations of scheme 2COL10A1, COL17A1 and CYYR 1.
Wherein, the scoring calculation formula is adopted as follows:
score = 3.6 co l10a1-2 cy rr 1-2.8 co l17a1
The cut-off value is defined as: -1.58
The verification results of 229 samples were:
| accuracy rate of | 93.90％ |
| AUC | 0.975 |
| Sensitivity of | 93.90％ |
| Specificity (specificity) | 93.90％ |
Fig. 3 shows the ROC effect of the 2 marker combinations of schemes 3COL10A1 and hx 2.
Wherein, the scoring calculation formula is adopted as follows:
score = 4.1 x col10a1-0.2 x hx2-5.8 x col17a1
The cut-off value is defined as: -3.75
The verification results of 229 samples were:
| accuracy rate of | 93.01％ |
| AUC | 0.970 |
| Sensitivity of | 93.10％ |
| Specificity (specificity) | 92.90％ |
Example 5 detection reagents and uses
In this example, the following detection methods and detection reagents were used for detection.
The detection method comprises the following steps:
(1) The collected serum samples extract total RNA.
(2) Removing genomic DNA from the extracted RNA, specifically: preparing a reaction mixed solution on ice according to the following components, preparing a Master Mix according to the reaction number of +2 when each reaction is carried out in order to ensure the preparation accuracy of the reaction solution, then sub-packaging the reaction mixed solution into each reaction tube, and finally adding an RNA sample. The reaction steps are as follows: 2min at 42 ℃ and 5min at 4 ℃.
| Reagent(s) | Usage amount |
| 5x gDNA Eraser Buffer | 2.0ul |
| gDNA Eraser | 1.0ul |
| Total RNA | 7.0ul |
| Total | 10.0ul |
(3) The RNA of the product is subjected to reverse transcription, and the method specifically comprises the following steps: the reaction mixture was prepared on ice. In order to ensure the accuracy of the preparation of the reaction liquid, when each reaction is carried out, master Mix is prepared according to the amount of reaction number +2, and then 10ul of the Master Mix is split into each reaction tube. Immediately after instantaneous centrifugation and mixing, reverse transcription reaction is carried out. (TB green qPCR method). The reaction steps are as follows: 15min at 37 ℃, 5sec at 85 ℃ and 5min at 4 ℃.
| Reagent(s) | Usage amount |
| Reaction liquid of step 1 | 10.0ul |
| PrimeScript RT Enzyme Mix I | 1.0ul |
| RT Prime Mix | 1.0ul |
| 5x PrimeScript Buffer 2(for Real time) | 4.0ul |
| RNase Free dH 2 O | 4.0ul |
| Total | 20.0ul |
(4) The reverse transcription product is subjected to fluorescence quantitative PCR, specifically:
plate dilution, adding 180ul of RNase Free dH2O to 20ul of reverse transcription cDNA template, and diluting template 10 times.
| Reagent(s) | Usage amount |
| cDNA template diluted 10-fold | 8.0ul |
| Primer F (10 uM) | 1.0ul |
| Primer R (10 uM) | 1.0ul |
| 2xTB Green Master Mix | 5.0ul |
| Total | 20.0ul |
Fluorescent quantitative PCR instrument, LC480II, reaction steps: pre-denaturation: 95℃30sec, PCR:45cycles (95℃5sec,60℃30sec, single), melting cure: (95 ℃ C. 5sec,60 ℃ C. 1min,95 ℃ C. Continuous)
Primer F and primer R sequences for each marker are as follows:
/>
(5) Substituting the marker gene expression level obtained by the detection in the previous step into a calculation formula to obtain a detection result.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
SEQUENCE LISTING
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Claims (7)
1. Use of a detection reagent for detecting the mRNA or cDNA expression level of a marker for risk of human pancreatic cancer, for the preparation of a diagnostic kit for determining the risk of pancreatic cancer;
wherein the pancreatic cancer risk marker comprises a combination selected from the group consisting of:
(A1) ZHX2; (A2) CYYR1; (B1) COL10A1; and (B2) COL17A1; or (b)
(A2) CYYR1; (B1) COL10A1; and (B2) COL17A1.
2. A kit comprising a detection reagent for detecting mRNA or cDNA of a marker for risk of human pancreatic cancer,
wherein, pancreatic cancer risk markers are the following combinations:
(A1) ZHX2; (A2) CYYR1; (B1) COL10A1; and (B2) COL17A1; or (b)
(A2) CYYR1; (B1) COL10A1; and (B2) COL17A1;
wherein the detection reagent comprises a primer pair or a probe, and the primer pair or the probe is used for specifically amplifying mRNA or cDNA of the pancreatic cancer risk marker.
3. The kit of claim 2, wherein the primer pair is selected from the group consisting of: primer pair for amplifying ZHX 2: SEQ ID NO. 1, SEQ ID NO. 2; primer pair for amplification of CYYR 1: SEQ ID NO. 3, SEQ ID NO. 4; primer pair for amplifying COL10 A1: 15, 16; primer pair for amplifying COL17 A1: SEQ ID NO. 17, SEQ ID NO. 18; or a combination thereof.
4. The kit of claim 2, further comprising a label or instructions for use of the kit in determining the risk of pancreatic cancer.
5. A pancreatic cancer diagnostic device, the device comprising:
(a) The input module is used for inputting pancreatic cancer risk marker data of a blood sample of a certain object;
wherein said risk marker gene comprises a combination selected from the group consisting of:
(A1) ZHX2; (A2) CYYR1; (B1) COL10A1; and (B2) COL17A1; or (b)
(A2) CYYR1; (B1) COL10A1; and (B2) COL17A1;
(b) The processing module calculates the input marker genes according to a preset scoring formula to obtain a risk score; comparing the score with the cut-off value to obtain a discrimination result, wherein when the risk score is higher than the cut-off value, the subject is prompted to be a pancreatic cancer patient; when the risk score is below the cut-off value, then prompting the subject to be a non-pancreatic cancer patient;
wherein, the scoring formula is:the method comprises the steps of carrying out a first treatment on the surface of the Wherein Wi is the weight value of each gene; the Pi is the expression level of each gene; and
(c) And the output module is used for outputting the diagnosis result.
6. The pancreatic cancer diagnostic device of claim 5 wherein when the risk marker combination is (A1) hx2; (A2) CYYR1; (B1) COL10A1; and (B2) COL17A1, the scoring formula for the risk score is: s=3.2×col10a1+0.1×hx2-2×cyyr1-2.5×col17a1;
when the risk marker combination is (A2) CYYR1; (B1) COL10A1; and (B2) COL17A1, the scoring formula for the risk score is: s=3.6×col10a1-2×cyyr1-2.8×col17a1.
7. The pancreatic cancer diagnostic device of claim 5 wherein said scoring formula is automatically calculated by designing a computer-aided program.
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| EP3159695A3 (en) * | 2013-06-20 | 2017-07-05 | The Trustees of The University of Pennsylvania | Methods for diagnosing pancreatic cancer |
| EP3522924A4 (en) * | 2016-10-06 | 2020-07-08 | The Johns Hopkins University | Large-scale epigenomic reprogramming links anabolic glucose metabolism to distant metastasis during the evolution of pancreatic caner progression |
| WO2020056162A1 (en) * | 2018-09-12 | 2020-03-19 | Oregon Health & Science University | Detecting and/or subtyping circulating hybrid cells that correlate with stage and survival |
| EP3935192A1 (en) * | 2019-03-04 | 2022-01-12 | Universiteit Antwerpen | Methylation status of gasdermin e gene as cancer biomarker |
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| CN112626218A (en) * | 2021-01-07 | 2021-04-09 | 浙江科技学院 | Gene expression classifier and in-vitro diagnosis kit for predicting pancreatic cancer metastasis risk |
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