CN101942502A - Pancreatic cancer marker, and detection method, kit and biochip thereof - Google Patents
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Abstract
The invention provides a pancreatic cancer marker, and a detection method, a kit and a biochip thereof. The pancreatic cancer marker provided by the invention contains 36 kinds of micro ribonucleic acids which stably exist in the serum/plasma of a person receiving the test and can be detected. The invention also provides a kit and a biochip of tools or elements for detecting the pancreatic cancer marker. The combination, the method, the kit and the biochip provided by the invention can be used for auxiliary diagnosis and differential diagnosis of pancreatic cancers, predication of occurrence and recrudescence of disease complications, evaluation of the curative effect, screening of active ingredients of medicaments, evaluation of pesticide effectiveness and the like, and has the advantages of wide detection range, high sensitivity, low detection cost, readily available raw materials, easy storage of samples and the like; and the method is widely applied to work related to the general survey of pancreatic cancers, improves the specificity and sensitivity which are low in the single marker due to individual difference, obviously increases clinical detection rate of pancreatic cancers and becomes an effective method for early diagnosis of pancreatic cancers.
Description
Technical field
The invention belongs to biological technical field, relate to separation, the qualitative and quantitative analysis of miRNA molecule in the human serum, also relate to the various clinical indication of carcinoma of the pancreas simultaneously.Specifically, the present invention is a kind of method that detects miRNA in carcinoma of the pancreas patients serum/blood plasma, variation by miRNA in carcinoma of the pancreas patients serum/blood plasma, in in-vitro diagnosis carcinoma of the pancreas and chronic pancreatitis, judge the carcinoma of the pancreas pathogenic process, the generation of prediction carcinoma of the pancreas complication and the probability of carcinoma of the pancreas recurrence and the prognosis of carcinoma of the pancreas, and analyze drug effect and curative effect.
Background technology
Carcinoma of the pancreas is the tumour of a kind of mortality ratio high (～99.9%, make a definite diagnosis the back).U.S.'s sickness rate: estimated new cases 32,180 examples, and accounted for 2% of all New Development cancers in 2005; U.S.'s mortality ratio: estimated death 31,800 examples in 2005 1 year, and accounted for the 4th and the 5th of all cancer associated death reasons of men and women respectively, account for the 5%-6% of all cancer mortality reasons.European Union's sickness rate: estimated new cases 55100 examples, European Union's mortality ratio in 2002: estimated death 59300 examples in 2002 1 year, difference is not obvious in different sexes and race, and as a rule patient's prognosis is relatively poor.Global carcinoma of the pancreas morbidity in 2002 and dead statistics are as shown in table 1, and wherein number of the infected is meant 2002 and finds to suffer from that the number of carcinoma of the pancreas, death toll are meant was diagnosed as carcinoma of the pancreas and dead number in 2002 till 2002.
Table 12002 year global carcinoma of the pancreas morbidity and dead statistics
Therefore, seek carcinoma of the pancreas marker and it is accurately detected the extremely urgent and important precondition of the early diagnosis and therapy that has become carcinoma of the pancreas.Although increasing disease markers has been found and has been applied to the monitoring of generaI investigation, diagnosis and the curative effect of clinical disease, their clinical application effect also exists obvious deficiency.For example, the tumor marker alpha-fetoprotein, serum lactic dehydrogenase, carcinomebryonic antigen etc. have been widely used in clinical, but these disease markers also can not satisfy the needs to early diagnosis of cancer far away, its major cause has two aspects: the sensitivity and the specificity of (1) above-mentioned disease markers are relatively low, the index that their detected result can't be made a definite diagnosis as disease; (2) early diagnostic rate of disease should present positive correlation with the effect of treatment, and above-mentioned any disease markers also is difficult to satisfy this requirement of disease early diagnosis.With the cancer is example, tumour differentiation classification specificity is strong excessively, the whole susceptibility of tumour is lower, the censorship sample is difficult to take repeatedly, sample is preserved defectives such as requirement condition height owing to exist, cost an arm and a leg simultaneously, therefore under existence conditions, be difficult to the existing tumor marker of wide popularization and application.And some traditional medicine means detect as histocyte and to exist its inherent defective, and the position of drawing materials is improper, histocyte sample material deficiency or people will cause mistaken diagnosis for lacking experience etc.Though other technology for example iconography has been widely used in the inspection and the diagnosis of disease, it still exists significant limitation on disease degree qualitative.Therefore at present be necessary very much to seek the novel, sensitive of the above-mentioned defective that can remedy existing marker and use disease detection marker easily.
MiRNA, English microRNA by name is the non-coding strand micro ribonucleic acid molecule that a class is about 19 to 23 Nucleotide.They are high conservative on evolving, and with many normal physiological activity of animal, closely related as biont growth, tissue differentiation, natural death of cerebral cells and energy metabolism etc., also exist closely simultaneously and get in touch with the generation of numerous disease and development.The nearest expression level of discovering several miRNAs in lymphocytic leukemia and the Burkitt lymphoma all has downward modulation (Lawrie CH in various degree, Gal S, Dunlop HM et al.Detection of elevatedlevels of tumor-associated microRNAs in serum of patients with diffuse largeB-cell lymphoma.BrJ Haematol 2008; 141:672-675); When analyzing the miRNA expression of comparing in people's lung cancer, the breast cancer tissue, discovery has the expression level of some tissue specificity miRNAs with respect to healthy tissues variation (Garofalo M to take place, Quintavalle C, Di Leva G etal.MicroRNA signatures of TRAIL resistance in human non-small cell lungcancer.Oncogene 2008).There are some researches prove that also miRNA has influenced the generation and the development of cardiovascular disordeies such as myocardial hypertrophy, heart failure, atherosclerosis, and close association (Tryndyak VP is arranged with metabolic diseases such as type ii diabetes, Ross SA, Beland FA, Pogribny IP.Down-regulationof the microRNAs miR-34a, miR-127, and miR-200b in rat liver duringhepatocarcinogenesis induced by a methyl-deficient diet.Mol Carcinog.2008Oct21).Exist positive connection between these experimental result prompting miRNA expression and specific variations and disease generation and the development.
Owing to play vital role beyond imagination in the expression regulation of miRNA after genetic transcription, therefore the cognation below it exists with disease: at first, the variation of miRNA may be the cause of disease, this is because the supressor of disease and the promotion factor all may be the target sites of miRNA, when disorderly expression has taken place earlier miRNA itself, promote the miRNA expression amount of the factor to reduce such as the original disease that suppresses, the miRNA expression amount that perhaps suppresses the disease supressor has raise, its net result all can cause the whole disorderly of downstream series of genes change of Expression and some path, and then the generation that induces an illness; Secondly, the variation of miRNA also may be the result of disease, this is because when disease (as cancer) takes place, can cause the losing of chromosome segment, the sudden change of gene or the violent amplification of chromosome segment, if miRNA just in time is positioned at this varied sections, its expression amount will take place to change extremely significantly so.Therefore, the miRNA molecule can be used as the new disease markers of a class fully in theory, and its specific variations is inevitable to be associated with disease generation development.MiRNA can also by miRNA that suppresses to raise in the lysis or the miRNA of crossing down-regulated expression, might greatly be alleviated the generation and the development of disease as the potential drug target simultaneously.
Domestic existing at present with the correlative study of miRNA as disease markers, as Chinese patent application CN100999765A and CN101298630A, they all choose account for the 4th of malignant tumour sickness rate colorectal carcinoma as research object, find after deliberation, during the colon benign polypus develops into malignant tumour, some miRNA molecules all exist specific variations, and have set up a kind of more responsive, more accurate method of making a definite diagnosis colorectal carcinoma in early days by the specific variations of measuring miRNA in view of the above.Yet because drawing materials of tissue sample is not easy to make the widespread use clinically of this method to be restricted.
Summary of the invention
For overcoming this defective, the inventor will study sight and invest more easily acquisition, even the blood that just can collect in the routine physical examination.Because blood can be circulated to whole body institute in a organized way, and to cell delivery nutrition and remove refuse, so blood can reflect the physiological and pathological situation of whole machine body, and its detected result has directive significance to HUMAN HEALTH.Exist multiple protein in the known blood serum, as total protein, albumin, sphaeroprotein etc., multiple lipid is as HDL cholesterol, triglyceride etc., multiple saccharic, pigment, ionogen and inorganic salt, plurality of enzymes, as amylase, alkaline phosphatase, acid phosphatase, the plain lipase of courage, zymohexase etc., also compiled multiple signaling molecule simultaneously from body tissue's organ, as cytokine, hormone etc.At present, the diagnosis of disease only is confined to above-mentioned biochemical indicator in the blood serum, the still report of serum-free/blood plasma miRNA.Thinking in people's traditional concept does not have ribonucleic acid molecule in the blood serum, though have also can be degraded to small molecule segment very soon by rnase and detect less than.But, because being 19 to 23 nucleotide units, the miRNA molecule forms, have structural singularity and relative stability, and they very likely are present in the blood serum.The inventor's early-stage Study is verified, stably have miRNA in the blood serum, and each disease there is its specific variation collection of illustrative plates (Chen et al:Characterization of microRNAs in serum:a novel classof biomarkers for diagnosis of cancer and other diseases.Cell Res.2008Oct; 18 (10): 997).
For seeking carcinoma of the pancreas certification mark thing and it accurately being detected, the inventor has carried out the research of the following aspects based on available research achievements:
(1) specific variations of blood serum miRNA in the research carcinoma of the pancreas pathogenic process;
(2) by being used to detect the biochip of blood serum miRNA and the variation that sequencing technologies is measured pancreatopathy cancer serum/blood plasma miRNA;
What (3) will screen expresses the big class blood serum miRNA molecular application of difference degree in blood serum miRNA detection technique under carcinoma of the pancreas, chronic pancreatitis and normal physiological state, preparation is applied to the biochip and the diagnostic kit in fields such as diagnosis of pancreatic cancer.
Research by above-mentioned dependency to blood serum miRNA and carcinoma of the pancreas, the applicant has proposed specific miRNA with stable existence in the blood serum as carcinoma of the pancreas certification mark thing, set up the method for the specific miRNA of stable existence in a kind of vitro detection blood serum, carry out the early diagnosis of carcinoma of the pancreas by the specific variations that detects specific miRNA, the differential diagnosis of chronic pancreatitis, disease is identified and course of disease monitoring, recurrence and prognosis, the prediction that complication takes place, can further carry out simultaneously drug effect judges, medicine guide, individualized treatment, researchs such as heap sort are planted in the effective components of Chinese medicinal screening.
Therefore, the purpose of this invention is to provide a kind of carcinoma of the pancreas marker.
Another object of the present invention provides a kind of probe combinations that is used to detect carcinoma of the pancreas cancer marker.
Another purpose of the present invention provides the purposes of above-mentioned carcinoma of the pancreas marker, comprises preparation reagent corresponding box and biochip.
Another object of the present invention provides the method that detects above-mentioned carcinoma of the pancreas cancer marker.
The objective of the invention is to realize by the following technical solutions.
On the one hand, the present invention at first provides a kind of carcinoma of the pancreas certification mark thing, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA (MaturemicroRNA), for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.Wherein, miR-320 comprises for example miR-320a, miR-320b.
Preferably, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA, for example 2,3,4,5,6 or 7 kind: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
The present invention also provides a kind of carcinoma of the pancreas marker, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA, for example 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.
Preferably, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA, for example 3,4,5,6 or 7 kind: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
Above-mentioned blood serum can derive from human body live body, tissue, organ and/or corpse.
On the other hand; the invention provides a kind of detection method of above-mentioned marker, described detection method is selected from one or more in inverse transcription polymerase chain reaction method (RT-PCR), real time fluorescent quantitative poly chain reaction method (Real-time PCR), Northern blot hybridization method (Northern blotting), rnase protection analysis method (RNase protection assay), Solexa sequencing technologies (Solexa sequencingtechnology) and the biochip method.
Preferably, described detection method is the RT-PCR method, the RT-PCR method that for example may further comprise the steps: 1) extract experimenter's the total RNA of blood serum, obtain the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
2) carry out the PCR reaction with miRNA design primer;
3) carry out the agarose gel electrophoresis of PCR product;
4) EB dyeing back observations under ultraviolet lamp;
Perhaps preferably, described detection method is the Real-time PCR method, the Real-time PCR method that for example may further comprise the steps:
1) extraction experimenter's the total RNA of blood serum obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
2) design primer with miRNA;
3) add fluorescent probe and carry out the PCR reaction;
4) detection and comparison blood serum sample are with respect to the variation of the amount of miRNA in normal serum/blood plasma.
Specifically, the method for above-mentioned 36 kinds of miRNAs in the detection provided by the invention experimenter blood serum can further be estimated the state of human body carcinoma of the pancreas.The method of stable existence and detectable 36 kinds of miRNAs comprises in the described human body blood serum: inverse transcription polymerase chain reaction method (RT-PCR); real time fluorescent quantitative poly chain reaction method (Real-time PCR); Northern blot hybridization method (Northern blotting); rnase protection analysis method (RNaseprotection assay); in Solexa sequencing technologies (Solexa sequencing technology) and the biochip method one or more.
Described RT-PCR method may further comprise the steps: (1) collects the blood serum sample, particularly, uses for example total RNA of blood serum of Trizol reagent extraction human body, obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample; (2) carry out the PCR reaction with miRNA design primer; (3) carry out the agarose gel electrophoresis of PCR product; (4) EB dyeing back observations and taking pictures under ultraviolet lamp.
Described Real-time PCR method may further comprise the steps: (1) collects the blood serum sample, particularly, uses for example Trizol reagent extraction experimenter's the total RNA of blood serum, obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample; (2) design primer with miRNA; (3) add fluorescent probe for example EVA GREEN carry out the PCR reaction; (4) analyzing and processing data and comparative result, particularly, detection is also compared the variation of blood serum sample with respect to the amount of miRNA in normal serum/blood plasma.
Described Northern blotting method may further comprise the steps: (1) collects the blood serum sample; (2) extract the total RNA of blood serum by Trizol reagent; (3) carry out sex change PAGE electrophoresis and film shift experiment; (4) preparation isotopic labeling miRNA probe; (5) carry out the film hybridization; (6) isotropic substance signal detection is as phosphorus screen scanning detecting result.
Described RNase protection assay method comprises the steps: that (1) carry out the synthetic of antisense RNA probes, isotopic labeling and purifying; (2) collect blood serum sample and extract RNA; (3) RNA after will extracting is dissolved in the hybridization buffer and adds antisense RNA probes and carries out hybridization; (4) adding the RNase Digestive system reacts; (5) carry out electrophoresis and radioautograph; (6) analytical results.
Described Solexa sequencing technology method comprises the steps: (1) collection blood serum sample; (2) extract the total RNA of blood serum by Trizol reagent; (3) carry out the PAGE electrophoresis and reclaim 17-27nt RNA molecule; (4) adaptor prime enzyme is associated in 3 of small RNA molecular ' with 5 ' end; (5) carry out RT-PCR reaction back and checking order; (6) data analysis and processing.
Described biochip method comprises the steps: that (1) is with the ripe body storehouse dot matrix of whole more than 500 miRNAs and prepare biochip; (2) collect the blood serum sample; (3) extract the total RNA of blood serum; (4) separate miRNA by post; (5) utilize the T4RNA ligase enzyme to carry out the miRNA fluorescent mark; (6) carry out hybridization with biochip; (7) Data Detection and analysis.
The present invention is by above-mentioned RT-PCR, Real-time PCR, Northern blotting, RNaseprotection assay, the variation tendency and the variable quantity of methods analysts such as Solexa sequencing technology and biochip blood serum miRNA in carcinoma of the pancreas takes place, and the dependency of they and carcinoma of the pancreas.Wherein, check and analysis miR-27a at first, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185, the variation of miR-191 in carcinoma of the pancreas, preparation blood serum miRNA biochip is measured the variation of blood serum miRNA in the various disease, simultaneously miRNA in the various disease blood serum is carried out the Solexa sequencing analysis.
Employed blood serum derives from experimenter's live body, tissue, organ and/or corpse in the aforesaid method.
The present invention also provides a kind of prediction, diagnosis, discriminating and/or estimates the method for carcinoma of the pancreas, and this method comprises the above-mentioned marker of detection, and preferably, this method comprises that the above-mentioned detection method of employing detects above-mentioned marker.
The invention provides above-mentioned nonsmall-cell lung cancer marker in preparation prediction, diagnosis, discriminating and/or the reagent of evaluation carcinoma of the pancreas or the purposes in the instrument.
The present invention also provides a kind of miRNA probe combinations that is used to detect the carcinoma of the pancreas marker, also promptly predict, diagnose and/or estimate the micro ribonucleic acid probe combinations of carcinoma of the pancreas, described probe combinations comprises one or more in the probe shown in the following nucleotide sequence, for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind; Preferably, described probe combinations comprises two or more in the probe shown in the following nucleotide sequence, for example 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind:
The present invention also provides a kind of miRNA probe combinations that is used to detect the carcinoma of the pancreas marker, also promptly predict, diagnose and/or estimate the micro ribonucleic acid probe combinations of carcinoma of the pancreas, described probe combinations comprises one or more in the probe shown in the following nucleotide sequence, for example 2,3,4,5,6 or 7 kind:
| miRNA | Corresponding probe sequence | Sequence numbering |
| miR-20a | CTACCTGCACTATAAGCACTTTA | SEQ?ID?NO.28 |
| miR-21 | TCAACATCAGTCTGATAAGCTA | SEQ?ID?NO.29 |
| miR-24 | CTGTTCCTGCTGAACTGAGCCA | SEQ?ID?NO.30 |
| miR-25 | TCAGACCGAGACAAGTGCAATG | SEQ?ID?NO.31 |
| miR-99 | CACAAGATCGGATCTACGGGTT | SEQ?ID?NO.33 |
| miR-185 | GAACTGCCTTTCTCTCCA | SEQ?ID?NO.35 |
| miR-191 | AGCTGCTTTTGGGATTCCGTTG | SEQ?ID?NO.36 |
The invention provides a kind of test kit that is used to detect the carcinoma of the pancreas marker, also promptly predict, diagnose, differentiate and/or estimate the test kit of carcinoma of the pancreas, this test kit comprises the instrument that detects above-mentioned marker.Preferably, wherein said instrument comprises the above-mentioned miRNA probe combinations that is used to detect the carcinoma of the pancreas marker; More preferably, described instrument also comprises polysaccharase, deoxyribonucleotide.The miRNA primer of the specific variations relevant with carcinoma of the pancreas that screen or its corresponding probe sequence collected in the PCR test kit (RT-PCR or Real-time PCR) can prepare the diagnosis of pancreatic cancer test kit.
The present invention also provides a kind of biochip that is used to detect the carcinoma of the pancreas marker, also promptly predicts, diagnoses, differentiates and/or estimate the biochip of carcinoma of the pancreas, and this biochip comprises the element that detects above-mentioned marker.Preferably, wherein said element comprises the above-mentioned miRNA probe combinations that is used to detect the carcinoma of the pancreas marker.With the reverse complementary sequence of the miRNA of the specific variations relevant that screen with carcinoma of the pancreas as probe points at chip, just made specially blood serum miRNA detection of biological chip at carcinoma of the pancreas.
Particularly, in above-mentioned any containing in above a kind of combination, method, test kit or the biochip to 36 kinds of miRNA markers, described evaluation experimenter's carcinoma of the pancreas state is for measuring the carcinoma of the pancreas state after the experimenter gives determinand, the activity that prevents and/or treats carcinoma of the pancreas that specifically is used to screen determinand (medicine that is used for the treatment of carcinoma of the pancreas); Described evaluation experimenter's carcinoma of the pancreas state is diagnosis and/or differential diagnosis experimenter's disease; Described evaluation experimenter's carcinoma of the pancreas state is for estimating the validity that experimenter's disease is treated; Described evaluation experimenter's carcinoma of the pancreas state predicts that for carcinoma of the pancreas is taken place the experimenter described generation carcinoma of the pancreas is specially the generation of carcinoma of the pancreas complication and/or the recurrence of carcinoma of the pancreas.Risk factor for pancreatic cancer comprises chronic pancreatitis etc., and the pathology evidence has also been found gradually the process advanced of normal pancreatic tissue-hyperplasia-carcinoma of the pancreas.Pancreatitis patient such as hereditary pancreatitis or tropical pancreatitis more early takes place, and the danger of canceration is also high more, and the time length that also promptly is exposed under the chronic inflammatory state is topmost Hazard Factor.There are the carcinoma of the pancreas and the chronic pancreatitis of chronic pancreatitis background that certain False Rate is arranged, therefore, seem particularly important in external differential diagnosis carcinoma of the pancreas and chronic pancreatitis.
It is also more loaded down with trivial details and coarse at present disease to be carried out the traditional biological chemistry and the Protocols in Molecular Biology of clinical diagnosis.The new technique that might be used for medical diagnosis on disease that development in recent years is got up has gene chip and protein (antibody) chip technology etc.The measured mRNA level of gene chip changes the change that can not reflect real protein level fully.Because proteinic biological activity and post transcriptional modificaiton such as glycosylation, phosphorylation etc. are closely related.And for numerous disease detected, biochip technology can't detect marker molecules in body fluid and the blood.Protein (antibody) chip technology and proteomic techniques also have its limitation.Particularly contain ten hundreds of albumen and polypeptide fragments in the blood serum in the human body, their concentration distribution are wide, clearly Bao Dao albumen seldom, quantification just still less.In the huge protein group of this quantity, look for the protein that close association is arranged with specified disease, and understand its effect in lesion tissue and remain an extremely large order, and lacking perfect antibody resource will be a bottleneck problem of restriction antibody chip technical development.Blood serum miRNA detection technique, biochip and diagnostic kit based on the blood serum miRNA are combined as a whole the peculiar property and the conventional molecular Biological Detection technology of blood serum miRNA dexterously, they analyze the composition of miRNA in pancreatopathy cancer serum/blood plasma in high-throughput ground apace, and clinical applicability is extremely strong.Because the physiological status variation of organ-tissue can cause the change that the blood serum miRNA is formed, so the blood serum miRNA can be used as " disease fingerprint ", the early diagnosis of realization carcinoma of the pancreas.
In sum, the present invention has following advantage:
The specific blood serum miRNA that (1) will filter out is as novel carcinoma of the pancreas marker, have the pedigree of detecting wide, highly sensitive, detect cost low, draw materials conveniently, sample advantages such as (blood serum-20 ℃ deposit get final product) easy to store, this method can be widely used in related works such as general investigation of desease, becomes the effective means of early diagnosis disease.
(2) the blood serum miRNA with low specificity and the muting sensitivity that the improvement individual difference that single marker was difficult to overcome is brought, significantly improves the early stage diagnosis and treatment of the clinical recall rate and the realization disease of disease as new disease markers.
(3) advantage of blood serum miRNA detection technique is, its detection be a series of disease-related marker, thereby can overcome difference (being age, sex, race, diet and environment etc.) between the individual patient, and this single just disease markers a subject matter can't going beyond.
In a word, the present invention can further be applied to make a definite diagnosis in early days carcinoma of the pancreas, this new blood serum carcinoma of the pancreas marker not only provides basic substance for people fully understand the mechanism of carcinoma of the pancreas on molecular level, has also quickened clinical disease diagnosis and has learned and therapeutic progress.Superiority based on the blood serum miRNA, believe in the near future, will become the part of routine physical examination to the blood serum miRNA diagnostic techniques of seriously disease such as cancer, and the relevant gene therapy of miRNA also can use widely, conquers these diseases and is no longer a dream.
Description of drawings
Below, describe embodiments of the invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1 shows the RT-PCR result of direct detected part miRNA in the normal human serum;
Fig. 2 shows and extracts in the normal human serum RNA and detect the wherein RT-PCR result of miRNA;
In Fig. 1 and Fig. 2, U6 is that molecular weight is the snRNA of 100bp, internal reference molecule as the miRNA experiment, the specific miRNA miR-181a of hemocyte (181a) represented respectively in remaining 12 code name, miR-181b (181b), miR-223 (223), miR-142-3p (142-3p), miR-142-5p (142-5p), miR-150 (150), miRNA miR-1 (1) from cardiac muscle and skeletal muscle, miR-133a (133a), miR-206 (206), miRNA miR-9 (9) from cerebral tissue, miR-124a (124a), and from the miRNA miR-122a (122a) of liver.
Fig. 3 shows the miRNA RT-PCR result of direct detected partially stabilized expression in mouse, rat, tire ox, calf and the horse serum respectively;
Fig. 4 A to 4D shows the result schematic diagram of 7 specific specificity blood serum miRNAs in normal population, chronic pancreatitis and Pancreas cancer patients cluster analysis;
Fig. 5 A to 5C shows that 7 kinds of miRNA detect the susceptibility and the specificity synoptic diagram of carcinoma of the pancreas;
Fig. 6 shows that 7 kinds of miRNA detect the figure as a result of the accuracy rate of carcinoma of the pancreas.
Embodiment
Followingly the present invention is described with reference to specific embodiment.It will be appreciated by those skilled in the art that these embodiment only are used to illustrate the present invention, the scope that it does not limit the present invention in any way.
Embodiment 1The RT-PCR of miRNA experiment in the blood serum
Use the various miRNAs of stable existence in RT-PCR scientific discovery and reference and the animal serum/blood plasma, and its expression amount is quite abundant.Concrete steps are:
(1) collection mouse, rat, normal people and some patient's blood serum;
(2) preparation cDNA sample.This operation has two kinds of schemes, a kind of scheme is for directly to carry out reverse transcription reaction with 10 μ l blood serum, another kind of is to use Trizol reagent (Invitrogen company) to extract the total RNA of blood serum (the 10ml blood serum is the RNA about the about 10 μ g of energy enrichment usually) earlier, obtains cDNA by the RNA reverse transcription reaction then.The reaction system of reverse transcription comprises 4 μ l, 5 * AMVbuffer, 2 μ l10mM each dNTP mixture (Takara company), 0.5 μ l RNase Inhibitor (Takara company), 2 μ l AMV (Takara company) and 1.5 μ l gene specific reverse primer miscellanys.Reactions steps is 16 ℃ hatched 15 minutes, and 42 ℃ were reacted 1 hour, and hatched 5 minutes for 85 ℃;
(3) PCR and electrophoresis observation.CDNA is diluted by 1/50, get the cDNA after 1 μ l dilutes, add 0.3 μ l Taq enzyme (Takara company), 0.2 μ l 10 μ M forward primers, the general reverse primer of 0.2 μ l, 10 μ M, 1.2 μ l 25mM MgCl2,1.6 μ l 2.5mM each dNTP mixture (Takara company), 2 μ, 10 * PCR buffer, 13.5 μ lH2O, 20 μ l systems are carried out PCR.The reaction conditions of PCR is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 40 circulations in 60 ℃, 1 minute.The PCR product is got 10 μ l and is carried out 3% agarose gel electrophoresis, and EB dyeing back is observed under ultraviolet lamp.
Concrete experimental result is seen Fig. 1.Fig. 1 is to be research object with the serum of taking from the normal people, serum is directly carried out the experimental result of RT-PCR.Select for use the ripe body of whole more than 500 miRNAs of people to carry out the PCR reaction, Fig. 1 is 12 kinds of miRNAs wherein.They are respectively the specific miRNA miR-181a of hemocyte, miR-181b, miR-223, miR-142-3p, miR-142-5p, miR-150, miRNA miR-1, miR-133a, miR-206 from cardiac muscle and skeletal muscle, from miRNA miR-9, the miR-124a of cerebral tissue, and from the miRNA miR-122a of liver.Above-mentioned as can be seen from the results four kinds of tissue-derived miRNAs can both detect in blood, be not that the ripe body of whole more than 500 miRNAs all has high abundance to express in blood serum, some miRNA is very micro-, even can not normally detect.
In order further to verify these miRNAs of stable existence in the blood serum, extract the RNA in the normal human serum earlier, select for use the ripe body of whole more than 500 miRNAs of people to carry out the PCR experiment then, the result is as shown in Figure 2.The result of Fig. 2 and the result of Fig. 1 are very identical, and the PCR product is single, show that these two kinds of experimental techniques can both detect the expression and the abundance of human serum miRNA, prove stably to have multiple tissue-derived miRNA in human serum.In addition, use the same method and detected the expression and the abundance of more than 500 miRNA in mouse, rat, tire ox, calf and the horse serum, the same miRNA of different tissue sources of finding has stably express in mouse, rat, tire ox, calf and horse serum, the result as shown in Figure 3.
Embodiment 2The real-time PCR of miRNA experiment in the blood serum
In order to study the special variation of blood serum miRNA in the carcinoma of the pancreas lysis, carried out the quantitative PCR experiment of blood serum miRNA.Quantitative PCR experiment principle and experimental procedure are the same with RT-PCR, and unique not being both added fluorescence dye EVA GREEN in PCR.What instrument used is ABI Prism 7300 quantitative real time PCR Instruments, and reaction conditions is to carry out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carries out 40 circulations in 60 ℃, 1 minute.Data processing method is a Δ Δ CT method, and CT is made as the cycle number of reacting when reaching thresholding, and then each miRNA can be represented with equation 2-Δ CT with respect to the expression amount of standard confidential reference items, wherein Δ CT=CT sample-CT confidential reference items.Patients serum/plasma sample and normal human serum/plasma sample are directly carried out reverse transcription reaction, react the wherein amount of contained miRNA by quantitative PCR.
Choose aplastic anemia, mammary cancer, osteosarcoma, central nervous system lymphoma, diabetic serum sample, the ripe body of whole more than 500 miRNAs of choosing simultaneously carries out the PCR experiment.Above-mentioned hemocyte specificity miR-181a, miR-181b, miR-223, miR-142-3p, miR-142-5p, the miR-150 that mentions, miRNA miR-1, the miR-133a of cardiac muscle and skeletal muscle, miR-206, from miRNA miR-9, the miR-124a of cerebral tissue, and the experimental result of in normal people and patients serum, carrying out quantitative PCR from the miRNA miR-122a of liver.The amount of miRNA has the mediation of going up downward modulation respectively with respect to the ratio of normal people's amount in aplastic anemia, mammary cancer, osteosarcoma, central nervous system lymphoma, the diabetic serum, and same tissue-derived miRNA intensity of variation difference in various disease, show that the blood serum miRNA has specific variations in various disease, they can be used as the marker of the new medical diagnosis on disease of a class.
Embodiment 3Be used to diagnose the blood serum miRNA chip of carcinoma of the pancreas
The chip operation flow process is:
(1) extract total RNA in the blood serum, the denaturing formaldehyde gel electrophoresis detects the quality of total RNA;
(2) separation of miRNA: get the total RNA of 50-100 μ g and separate miRNA with Ambion ' s miRNAIsolation Kit (Cat#.1560);
(3) fluorescent mark of miRNA sample: utilize T4RNA ligase enzyme marking method to carry out fluorescent mark, and then, be used for chip hybridization after drying up with the dehydrated alcohol precipitation;
(4) hybridization and cleaning: RNA is dissolved in (15% methane amide in the 16 μ L hybridization solutions; 0.2%SDS; 3 * SSC; 50 * Denhardt ' s solution), spend the night in 42 ℃ of hybridization.After hybridization finishes, contain 0.2%SDS earlier about 42 ℃, washed in the liquid of 2 * SSC 4 minutes, then room temperature was washed 4 minutes in 0.2 * SSC liquid, promptly can be used for scanning after slide dries;
(5) chip scanning: chip scans with LuxScan 10K/A two channels laser scanner;
(6) data extract and analysis: adopt the LuxScan3.0 image analysis software that chip image is analyzed, picture signal is converted into numerary signal, analyze with SAM at last and select difference expression gene.
The class blood serum miRNA probe that the differential expression degree under carcinoma of the pancreas and normal physiological state of quantitative PCR technique and biochip technology double verification is big is used to prepare biochip, and method is the same.This chip is compared with traditional die, and manufacture craft and operating process do not have significant improvement, but this chip has been simplified probe library, will significantly reduce the cost of manufacture and the production time of chip thus, is easy to preparation.The specific aim and the practicality of chip have also been increased simultaneously.With the practice of this chip input, only need patient's blood serum and just can find disease in early days without any need for other tissue, help to instruct and diagnose and treat.
Embodiment 4The minuteness ribonucleic acid reagent kit that is used for diagnosis of pancreatic cancer and prediction
Be used for the diagnosis of carcinoma of the pancreas, the generation of disease complication and the prediction of recurrence, therapeutic evaluation, and the manufacture craft of the minuteness ribonucleic acid reagent kit of the screening of active constituents of medicine, evaluating drug effect and operating process are based on quantitatively and sxemiquantitative round pcr and biochip technology.
At first determine to have in normal serum/blood plasma the miRNA that copies more than by the method or the PCR method of order-checking.Screen expression amount and the big class blood serum miRNA of difference degree under nonsmall-cell lung cancer and normal physiological state by quantitative PCR technique and biochip technology then, whether the index of nonsmall-cell lung cancer and diagnosis lesion degree takes place as prediction.The quantity of the blood serum miRNA of the corresponding every kind of disease that filters out at last is 36 kinds, and this is that make on the basis in chip probe storehouse optimized simplified.This test kit comprises reagent such as a collection of blood serum miRNA primer, Taq enzyme, dNTP.
All detect the comfortable hospital of sample standard deviation and are diagnosed as Pancreas cancer patients, chronic pancreatitis patient and equity age and other normal people of homogeny (contrast) in the present embodiment.
At first, adopt the method for Solexa order-checking to determine to have in normal serum/blood plasma the miRNA that copies more than, by detecting the variation of miRNA in the blood serum, filter out with normal people's (control group) and compare, 63 kinds of miRNA that change in the Pancreas cancer patients serum sample, wherein 44 miRNA raise, 19 miRNA downward modulations, and concrete outcome is as shown in table 2.
The differential expression sequencing result of miRNA in table 2 Pancreas cancer patients serum sample and the control group serum sample
By quantitative PCR technique and biochip technology, filter out expression amount and the big class blood serum miRNA of difference degree under disease and normal physiological state, reference table 2, choose wherein the mean change multiple surpass 5 and copy number greater than 10, and in conjunction with laboratory early-stage Study result, selected 36 miRNA and detected index, as the index whether prediction carcinoma of the pancreas takes place and diagnose lesion degree, concrete outcome sees Table 3.
36 kinds of miRNA that table 3 is selected
From table 3 among 36 of up-regulated kinds of miRNA, choice criteria according to mean change multiple＞2 and p value＜0.01, further optimize the molecular marked compound that 7 kinds of miRNA detect as carcinoma of the pancreas, be specially miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191, concrete outcome sees Table 4.
7 kinds of miRNA that table 4 is selected
By above-mentioned 7 kinds of miRNA are carried out cluster analysis, show that once more their expression between carcinoma of the pancreas, chronic pancreatitis and normal control serum sample there are differences.Above-mentioned 7 kinds in blood serum miRNA change specific analytical results as the specificity fingerprint of carcinoma of the pancreas in normal population and Pancreas cancer patients and see Fig. 4 A-D.As known in the figure, can make up clear and definite differentiating pancreatic cancer sample and normal sample according to these 7 kinds of miRNA, and can differentiating pancreatic cancer sample and chronic pancreatitis sample.Promptly 7 kinds of miRNA make up clear and definite differentiating pancreatic cancer sample and contrast (comprising normal people and chronic pancreatitis) sample.
The concrete data processing of cluster analysis is as follows: for training set (Fig. 4 A is 25 routine Pancreas cancer patients and 25 contrasts), checking collection (Fig. 4 B is 95 routine Pancreas cancer patients and 81 contrasts), (Fig. 4 C is 95 routine Pancreas cancer patients and 82 routine chronic pancreatitis patients to the high risk factor group; Fig. 4 D be 95 routine Pancreas cancer patients, 81 the contrast and 82 routine chronic pancreatitis patients), absolute expression values with serum miRNA in the carcinoma of the pancreas sample is converted to the multiple ratio of contrasting with normal sample respectively, and with its normalization method, cluster and drafting pattern 4A-D (adopt cluster 3.0 softwares mapping form), i.e. the analytical results that miRNA changes as the specificity fingerprint of carcinoma of the pancreas in these 7 kinds of blood serum.Fig. 4 A-D is described in detail as follows.
In Fig. 4 A, right-hand mark literal is 7 miRNA that detected, and top mark literal is respectively and detects the sample individuality, and normal represents normal people (n=25), concentrates on the figure right side; T represents carcinoma of the pancreas patient (n=25), concentrates on the figure left side.This figure has confirmed normal people and Pancreas cancer patients to be distinguished by the detection of 7 miRNA expression levels.
In Fig. 4 B, right-hand mark literal is 7 miRNA that detected, and top mark literal is respectively and detects the sample individuality, and normal (represents normal people (n=81): concentrate on the figure right side; T represents Pancreas cancer patients (n=95), concentrates on the figure left side.The further enlarged sample of this figure detects, and has verified by the detection of 7 miRNA expression levels and normal people and Pancreas cancer patients can have been distinguished.
The right-hand mark literal of Fig. 4 C is 7 miRNA that detected, and top mark literal is respectively and detects the sample individuality, and ch pan (represents chronic pancreatitis patient (n=82): concentrate on the figure right side; T represents Pancreas cancer patients (n=120), concentrates on the figure left side.The further enlarged sample of this figure detects, and has verified by the detection of 7 miRNA expression levels and chronic pancreatitis patient and Pancreas cancer patients can have been distinguished.
Fig. 4 D is depicted as take a sample this set of Fig. 4 B and Fig. 4 C, this figure right side mark literal is 7 miRNA that detected, upside mark literal is respectively and detects the sample individuality, nor represents normal people (n=81) and ch pan, and (represent chronic pancreatitis patient (n=82), normal people and chronic pancreatitis sample concentrate on the figure left field; T represents carcinoma of the pancreas patient (n=120), and the carcinoma of the pancreas sample concentrates on the figure right side area.As can be seen, 7 miRNA can separate carcinoma of the pancreas sample and contrast (comprising normal people and chronic pancreatitis sample) sample area.
Fig. 4 A, 4B and 4D are carried out the analysis that risk is given a mark, and concrete outcome sees Table 5.In table 5, first line display of form be the risk score mark of the sample of assessing; Second to eight row is illustrated respectively in the Pancreas cancer patients number that training set, checking collection and high risk factor under certain risk score mark are concentrated, chronic pancreatitis patient's number or normal people's number; Adopt statistical analysis software (SAS) to carry out statistical study, setting risk score numerical value is 6, if sample risk score 〉=6 then are divided into Pancreas cancer patients, if sample risk score＜6 are divided into the normal people.
Concrete statistical method is as follows: except controlling each the step variable in the whole process, further all data standards are turned to zero-mean and a standard deviation before data clusters.For influence and assisted layered cluster and the risk score that minimizes missing values, adopt K nearest-neighbor method K-Nearest Neighbors (KNN, a kind of method of laying the blame on (a method-based missing dataimputation)) to estimate the missing values in 19 to 20 intervals based on missing data.For example,, can be in sample A find same other K miRNA that expresses if the miRNA among the sample A has a missing values, find then comprise to case A in the most similar sample of other miRNA expression.Can estimate missing values from K the weighted mean of immediate miRNA among sample A.In weighted mean, the weighted value of each miRNA is calculated with the similarity of expressing among itself and the miRNA.Set K here and equal 9, promptly use 9 neighbours' miRNA to estimate.In addition, the estimation result who is drawn by K nearest-neighbor method KNN is very little for the influence of present result of study.All markers call rate all greater than 97.6%, and do not have the sample disappearance more than two and plural marker.
Used the hierarchical cluster that has complete association mode among the cluster 3.0 at this.In order to carry out risk score, 95% of the interval upper limit of each miRNA numeric reference in the control group is made as t, as the threshold value that the miRNA expression level of each sample correspondence is encoded.The risk score of each miRNA is designated as S, is with the calculating equation expression:
Wherein, i represents i sample,
Represent j miRNA.Consider the weight difference of each miRNA assessment nonsmall-cell lung cancer risk, set up the function of a risk score for each patient according to linear combination to the expression level of miRNA.According to the related data of K miRNA, the risk score function of i sample is:
In the equation above, sij is for miRNA among the sample i
Risk score.Ws is the weight of the risk score of miRNAj..In order to determine sign and Ws, the match of 10 single argument logistic regression models is applied to indicate the various diseases situation of risk score.As representing the weight of each miRNA in the risk score function, the sign in the regression coefficient has then determined the sign in the risk assessment function with the regression coefficient in each risk score.Then, frequency of utilization table and ROC curve are estimated the diagnosis effect in the sample colony.
The risk score of table 5 patient and contrast (normal people)
In the table, * positive prediction rate, the negative prediction rate of * *
MiRNA detects the carcinoma of the pancreas susceptibility and the specificity synoptic diagram is seen Fig. 5 A-C, if the total area (promptly detecting sample totally counts) is one, can find out the training set (Fig. 5 A) of area under curve (being confidence level) corresponding to Fig. 4 A, (promptly comprise carcinoma of the pancreas corresponding to the checking collection (Fig. 5 B) of Fig. 4 B and corresponding to the high risk factor collection of Fig. 4 D, normal people and chronic pancreatitis sample, Fig. 5 C) reach 0.995,0.987 and 0.993 respectively.
Figure 6 shows that above-mentioned 7 kinds of miRNA detect the figure as a result of carcinoma of the pancreas accuracy rate, wherein X-coordinate is the miRNA kind that is detected, ordinate zou is an area under curve, and representative adopts 7 kinds of miRNA to detect the accuracy rate (establishing the total area (promptly detecting total sample number) is 1) of nonsmall-cell lung cancer.Can find out area under curve (being accuracy rate)＞0.98.
Claims (13)
1. a carcinoma of the pancreas marker is characterized in that, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.
2. marker according to claim 1, it is characterized in that, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
3. a carcinoma of the pancreas marker is characterized in that, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.
4. according to marker according to claim 3, it is characterized in that, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
5. according to each described marker in the claim 1 to 4, it is characterized in that described blood serum derives from human body live body, tissue, organ and/or corpse.
6. the detection method of each described marker in the claim 1 to 5, it is characterized in that described detection method is selected from one or more in inverse transcription polymerase chain reaction method, real time fluorescent quantitative poly chain reaction method, Northern blot hybridization method, rnase protection analysis method, Solexa sequencing technologies and the biochip method;
Preferably, described detection method is the RT-PCR method, the RT-PCR method that for example may further comprise the steps:
1) extraction experimenter's the total RNA of blood serum obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
2) carry out the PCR reaction with miRNA design primer;
3) carry out the agarose gel electrophoresis of PCR product;
4) EB dyeing back observations under ultraviolet lamp;
Perhaps preferably, described detection method is the Real-time PCR method, the Real-time PCR method that for example may further comprise the steps:
1) extraction experimenter's the total RNA of blood serum obtains the cDNA sample by the RNA reverse transcription reaction; Perhaps collect experimenter's blood serum sample, carry out reverse transcription reaction with blood serum as damping fluid and prepare the cDNA sample;
2) design primer with miRNA;
3) add fluorescent probe and carry out the PCR reaction;
4) detection and comparison blood serum sample are with respect to the variation of the amount of miRNA in normal serum/blood plasma.
7. each described marker predicts, diagnoses, differentiates and/or estimate the reagent of carcinoma of the pancreas or the purposes in the instrument in the claim 1 to 5 in preparation.
8. miRNA probe combinations that is used to detect the carcinoma of the pancreas marker is characterized in that described combination comprises one or more in the probe shown in the following nucleotide sequence:
9. probe combinations according to claim 8 is characterized in that, described combination comprises one or more in the probe shown in the following nucleotide sequence:
10. a test kit that is used to detect the carcinoma of the pancreas marker is characterized in that, described test kit comprises the instrument that test right requires each described marker in 1 to 5.
11. test kit according to claim 10 is characterized in that, described instrument comprises claim 8 or 9 described probe combinations; Preferably, described instrument also comprises polysaccharase and/or deoxyribonucleotide.
12. a biochip that is used to detect the carcinoma of the pancreas marker is characterized in that, described biochip comprises the element that test right requires each described marker in 1 to 5.
13. biochip according to claim 12 is characterized in that, the element of described biochip comprises claim 8 or 9 described probe combinations.
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| CN111575374A (en) * | 2020-04-29 | 2020-08-25 | 肖晓莺 | Molecular marker for early pancreatic tumor detection, and detection method and application thereof |
| CN116741384A (en) * | 2023-08-14 | 2023-09-12 | 惠民县人民医院 | Bedside care-based severe acute pancreatitis clinical data management method |
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