CN105176983A - Kit for detecting esophageal squamous carcinoma associated serum miRNAs genes - Google Patents
Kit for detecting esophageal squamous carcinoma associated serum miRNAs genes Download PDFInfo
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Abstract
The invention discloses a kit for detecting esophageal squamous carcinoma associated serum miRNAs genes, and belongs to the technical field of molecular biology. According to the technical scheme, the kit for detecting the esophageal squamous carcinoma associated serum miRNAs genes comprises a detection reagent which is prepared from stem loop primers and RNase-free water and used for the diagnosing esophageal squamous carcinoma associated serum miRNAs genes. The invention further discloses a use method of the kit for detecting the esophageal squamous carcinoma associated serum miRNAs genes. A real-time fluorescent quantitative polymerase chain reaction (qPCR) technology is directly adopted to conduct detection on multiple esophageal squamous carcinoma associated serum miRNAs genes in serum, and by means of the method, the minimally invasive diagnosis of early esophageal squamous carcinoma, large-scale screening and prognosis evaluation are achieved.
Description
Technical field
The invention belongs to technical field of molecular biology, being specifically related to a kind of test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene.
Background technology
MicroRNAs(miRNAs) be the endogenous non-coding strand tiny RNA of class high conservative in sequence, be about 19-25nt(FinneganEF etc., MicroRNAbiogenesis:regulatingtheregulators.
critRevBiochemMolBiol, 2013,48 (1): 51-68).MiRNAs is being permitted to play an important role in polygenic post-transcriptional control.Each miRNA can identify the mRNAs of hundreds of target genes, and the imbalance of single miRNA can affect whole idiotype network.MiRNAs gene number, far fewer than mRNAs, is the biomarker (LuJ etc., the MicroRNAexpressionprofilesclassifyhumancancers. that are easy to screen and verify
nature, 2005,435 (7043): 834-838).Different cancer types has special miRNAs express spectra.In addition, in the generation of cancer, development, transfer and treatment, miRNAs ANOMALOUS VARIATIONS can illustrate the Evolution Mechanism of cancer.Therefore, research emphasis is turning to the pass between miRNA and tumour to fasten by increasing researchist.
Worldwide, the mortality ratio of the esophageal carcinoma is in the 6th of cancer mortality, and the sickness rate of the male sex is 3-4 doubly (JemalA etc., the Globalcancerstatistics. of women
caCancerJClin, 2011,61 (2): 69-90).Esophageal squamous cell carcinoma (Esophagealsquamouscellcarcinoma, ESCC) in the middle part of oesophagus or top 1/3 is occurred in, it is the main subtypes of the Asia esophageal carcinoma, account for more than 90% (SongYM etc., Identificationofgenomicalterationsinoesophagealsquamousc ellcancer. of the esophageal carcinoma
nature, 2014,509 (7498): 91-95).Because China is located in " esophageal carcinoma band ", the M & M of esophageal squamous cell carcinoma all occupies first place in the world.Clinically, the early stage diagnosis and treatment of esophageal squamous cell carcinoma can improve the prognosis of patient significantly and reduce mortality ratio, but still lack the specific biomarker of esophageal squamous cell carcinoma and effective Wicresoft method of early diagnosis at present.Therefore patients with esophageal squamous cell carcinoma has more than 50% for middle and advanced stage (ZhangC etc., CytoplasmicexpressionoftheELAV-likeproteinHuRasapotentia lprognosticmarkerinesophagealsquamouscellcarcinoma. when making a definite diagnosis
tumorBiol, 2014,35 (1): 73-80), its 5 years survival rates are at lymphoglandula without being 20%-30% during transfer, and to have during nodus lymphoideus transferring rate be 13%.Although traditional blood serum tumor markers, be used to early diagnosis as carcinomebryonic antigen, squamous cell carcinoma antigen and detected the dynamic change of some tumour, between these blood serum tumor markers and esophageal squamous cell carcinomas, having lacked enough sensitivity and specificity.Therefore, the biomarker finding responsive, special esophageal squamous cell carcinoma early detection is the active demand reducing M & M, also becomes an active area of current research.
Because serum miRNA easily obtains, it is the sample mode of a relative noninvasive, simultaneously because serum miRNA is highly stable, favorable reproducibility, digestion and harsh environment (as multigelation, long-time room temperature placement, peracid cross alkali, extreme pH etc.) (ChenX etc., the CharacterizationofmicroRNAsinserum:anovelclassofbiomarke rsfordiagnosisofcancerandotherdiseases. of RNaseA can be resisted
cellRes2008,18 (10): 997-1006), so after reported first circulation miRNA in 2008 can be used as potential diagnosing tumor instrument, exponential growth (JarryJ etc., ThevalidityofcirculatingmicroRNAsinoncology:fiveyearsofc hallengesandcontradictions. is presented as the scientific literature of diagnosis, prognosis and early prediction human diseases about serum miRNAs
molOncol, 2014,8 (4): 819-829).Some of them research finds, serum miRNAs content has tomour specific and sexually revises, illustrate that serum miRNAs has potential (KomatsuS etc., the CirculatingmiR-18a:asensitivecancerscreeningbiomarkerinh umancancer. as biomarker for cancer
inVivo, 2014,28 (3): 293-297).Serum miRNA both can from cancerous tissue, also can from healthy tissues.In serum, miRNA and protein, lipid, external secretion body etc. form mixture, thus keep higher stability.Some serum miRNA from healthy tissues can act on tumour cell, supplements the miRNA declined in cancer cells.Once this effect can not play, tumour cell will enter transition phase.Therefore, the height of serum miRNA can reflect the dynamic variation of tumor development, provides biomarker to the diagnoses and treatment of tumour and prognosis.
Along with serum miRNA study go deep into, its as a kind of disease particularly tumor micro-wound method of early diagnosis be also day by day subject to the favor of people.There are some researches show, the generation development relationship of serum miRNA and esophageal squamous cell carcinoma is very close, may become the biomarker of oesophagus squama cancer diagnosis and prognosis.Nearest research shows, serum miRNAs is relevant to oesophagus squama cancer diagnosis and prognosis, and this shows that they also can be used as the potential mark of diagnosis and prognosis classification, contributes to going out suitable methods for the treatment of at esophageal squamous cell carcinoma early screening.Zhang Chunni seminar is one group with miR-25, miR-100, miR-193-3p, miR-194, miR-223, miR-337-5p, miR-483-5p, their content (ZhangCN etc., ExpressionprofileofmicroRNAsinserum:afingerprintforesoph agealsquamouscellcarcinoma. is detected in 111 routine patients ESCC and 111 routine normal peoples
clinChem2010,56 (12): 1871-1879) and be another group with miR-22, miR-100, miR-10a, miR-148b, miR-223, miR-133a, miR-127-3p, their content (WuCY etc., DiagnosticandprognosticimplicationsofaserummiRNApanelino esophagealsquamouscellcarcinoma. is detected in 290 routine patients ESCC and 140 routine normal peoples
plosOne, 2014,9 (3): e92292), found that these serum miRNAs content in patient ESCC is all significantly higher than normal people, and without this change in other tumours, thus the diagnosis marker that these two kinds of combinations can be used as esophageal squamous cell carcinoma is proposed.In addition, much research is to serum miRNAs Receiver operating curve (Receiveroperatingcharacteris-ticcurve, ROC) analyze, and the serum/plasma miRNA serum s calculating ROC area under curve (Areaundercurve, AUC) to assess candidate is to the predictive ability of esophageal squamous cell carcinoma.AUC is more close to 1, and accuracy is higher, and in pertinent literature, AUC great majority all show higher accuracy (ZouKH etc., Receiver-operatingcharacteristicanalysisforevaluatingdia gnostictestsandpredictivemodels.
circulation, 2007,115 (5): 654-657).
Often specificity is lower as diagnosing tumor mark for a kind of serum miRNA, to combine and miRNA express spectra can improve the accuracy of diagnosis, but the latter measures time-consuming and costly, affects its widespread use if measure multiple miRNAs.Therefore, select suitable miRNA to measure its ratio vary and then can obtain higher accuracy by less mensuration.The result of study displays such as Komatsu, make observation index by the ratio vary of miR-21 and miR-375 content in blood plasma, and the miRNA that can improve blood plasma is used as sensitivity and the specificity of the biomarker of diagnosis.The AUC of relatively single miRNA, the AUC value of miR-21 and miR-375 content ratio is 0.816.In blood plasma, the ratio of miR-21 and miR-375 content can distinguish Healthy People and patient with esophageal carcinoma, there is the susceptibility of 88% and specificity (KomatsuS etc., the CirculatingmicroRNAsinplasmaofpatientswithoesophagealsqu amouscellcarcinoma. of 70%
brJCancer, 2011,105 (1): 104-111).Wang etc. think after analyzing by Meta-analysis method: esophageal squamous cell carcinoma serum miRNAs can as a kind of new Wicresoft, the biomarker with superior diagnostic feature.Compared to saliva miRNAs, plasma/serum miRNAs has better diagnostic feature (WangYY etc., IdentificationofmicroRNAsasnovelbiomarkersfordetectinges ophagealsquamouscellcarcinomainAsians:ameta-analysis.
tumorBiol, 2014,35 (11): 11596-11604).
The main detection method of serum miRNAs has: chip technology, qPCR, Northern hybridization, in situ hybridization, high-flux sequence etc.Fly when holding high with low density, Solexa, the U.S., the serum/plasma miRNA serum express spectra of Agilent, Toray3D-GenemicroRNA high throughput testing esophageal squamous cell carcinoma time, from 11-219 not etc., and repetition rate is extremely low for the discrepant quantity of detected miRNAs.When different experiments room is detected with identical chip technology or the different chip technology of same use for laboratory, miRNAs differential expression quantity is little, but Species differences is larger.And the most frequently used detection method is qPCR detection.Although most of seminars choice for use TaqMan probe detects the expression level of serum miRNA, but it is expensive that the incontrovertible fact is probe, the probe price of single miRNA is that 1000 yuan (150uL) can only detect more than 40 sample, therefore, need to find economic, efficient with special method and detect the relevant serum miRNAs of esophageal squamous cell carcinoma, for the detection of expression of corresponding miRNAs relevant to esophageal squamous cell carcinoma and the detection of esophageal squamous cell carcinoma provide effective way, early screening and the diagnosis of esophageal squamous cell carcinoma will be contributed to.
In sum, this area adopts the gene marker miRNAs gene of esophageal squamous cell carcinoma in stem ring primer qPCR technology for detection serum little, therefore can develop this technology and be applied in the early diagnosis of esophageal squamous cell carcinoma.
Summary of the invention
The technical problem that the present invention solves there is provided a kind of test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene.
The present invention adopts following technical scheme for solving the problems of the technologies described above, a kind of test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene, it is characterized in that including the detection reagent that stem ring primer and RNase-free water for diagnosing esophageal squamous cell carcinoma associated serum miRNAs gene are prepared, wherein esophageal squamous cell carcinoma associated serum miRNAs gene is hsa-miR-10a-5p gene, hsa-miR-16-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-107 gene, hsa-miR-127-3p gene, hsa-miR-129-5p gene, hsa-miR-148b-3p gene, hsa-miR-155-5p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-375 gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene or hsa-miR-1322 gene are at least one in following 22 kinds of stem ring primers for diagnosing the stem ring primer of esophageal squamous cell carcinoma associated serum miRNAs gene:
(1) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.1 of hsa-miR-10a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAA-3’；
(2) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.2 of hsa-miR-16-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCGCCAAT-3’；
(3) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.3 of hsa-miR-18a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTATCT-3’；
(4) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.4 of hsa-miR-21-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAACA-3’；
(5) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.5 of hsa-miR-22-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAGTT-3’；
(6) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.6 of hsa-miR-25-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAGAC-3’；
(7) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.7 of hsa-miR-31-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCTAT-3’；
(8) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.8 of hsa-miR-100-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAG-3’；
(9) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.9 of hsa-miR-107 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGATAG-3’；
(10) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.10 of hsa-miR-127-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCCAA-3’；
(11) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.11 of hsa-miR-129-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACGCAAGC-3’；
(12) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.12 of hsa-miR-148b-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAAAG-3’；
(13) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.13 of hsa-miR-155-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACCCCT-3’；
(14) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.14 of hsa-miR-194-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCACA-3’；
(15) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.15 of hsa-miR-200c-3p gene;
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCATC-3’；
(16) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.16 of hsa-miR-223-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGGGGT-3’；
(17) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.17 of hsa-miR-337-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAACTCC-3’；
(18) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.18 of hsa-miR-375 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCACGC-3’；
(19) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.19 of hsa-miR-483-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTCCCT-3’；
(20) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.20 of hsa-miR-548k gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCAAA-3’；
(21) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.21 of hsa-miR-1246 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCCTGCT-3’；
(22) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.22 of hsa-miR-1322 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCAGCAT-3’。
Further restriction, described hsa-miR-10a-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-127-3p gene, hsa-miR-148b-3p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene and hsa-miR-1322 gene are up-regulated gene, hsa-miR-107 gene, hsa-miR-129-5p gene, hsa-miR-155-5p gene and hsa-miR-375 gene are down-regulated gene, has-miR-16-5p gene is reference gene.
The using method of the test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene of the present invention, is characterized in that comprising the steps:
(1) extract the total serum IgE of serum sample to be detected, use micro-spectrophotometer to measure concentration and the purity of RNA;
(2) carry out reverse transcription with stem ring primed reverse transcription testing sample to be measured, reverse transcription system is 20 μ L, and by above-mentioned mixed solution in 42 DEG C of reaction 50min, 70 DEG C of heating 15min reverse transcription RNA are cDNA;
(3) adding qPCR system with forward primer and universal primer is 15 μ L;
(4) qPCR reaction conditions is: the denaturation stage, and temperature is 95 DEG C, and the time is 5min; In the circulating reaction stage, denaturation temperature is 95 DEG C, and the time is 10s, and annealing temperature is 60 DEG C, and the time is 34s, 40 circulations; The solubility curve stage, denaturation temperature 95 DEG C, time 15s, annealing temperature 60 DEG C, time 60s, denaturation temperature 95 DEG C, time 15s.
Further restriction, the detailed process of step (1) is: by the fresh blood collected temperature bath 20min in 37 DEG C of thermostat water baths; The centrifugal 5min of room temperature 3000rpm, gets supernatant, goes precipitation; At 4 DEG C, the centrifugal 10min of 12000rpm, remove precipitation, packing supernatant, is stored in-80 DEG C; Take out sample, after sample dissolution, in 4 DEG C by centrifugal for 12000g 10min, stay supernatant; Get 100 μ L serum, add 300 μ LRNase-free water, vortex mixes; Add 400 μ LTRIzol, concussion mixing, room temperature places 5min, in 4 DEG C by centrifugal for 12000g 10min, removes precipitation; The TRIzol reagent of every 1mL adds 0.2mL chloroform, vortex 15s, and room temperature places 2-3min, in 4 DEG C by centrifugal for 12000g 15min, gets supernatant; Every 1mLTRIzol reagent adds 0.5mL Virahol, fully mixes, and room temperature places 10min, in 4 DEG C by centrifugal for 12000g 15min, removes supernatant; Add the ethanol that 1mL volume fraction is 75%, vortex mixes, and in 4 DEG C by centrifugal for 7500g 5min, stay precipitation, and dry RNA precipitates; Add 20 μ LRNase-free water dissolution RNA, and at 55-60 DEG C of temperature bath 15min.
Compared to existing detection method, the present invention has following advantage: in prior art, the miRNAs of TaqMan probe qPCR method to esophageal squamous cell carcinoma tissue is the most often adopted to detect, the undeniable fact is that TaqMan probe is expensive, increase the burden of patient, simultaneously collecting sample can cause traumatic to patient, and can not early diagnosis, brings misery to patient; This test kit directly adopts stem ring primer qPCR method to detect miRNAs gene relevant to esophageal squamous cell carcinoma multiple in serum, gets final product the detection sample of direct economy, can accomplish again Wicresoft's esophageal squamous cell carcinoma early diagnosis, extensive examination and prognosis evaluation.Because miRNAs multiple in patients with esophageal squamous cell carcinoma serum exists high expression level, can be used as its diagnosis esophageal squamous cell carcinoma and gene marker of prognosis evaluation, therefore adopt technical scheme of the present invention can reach the advantages such as easy collection, Micro trauma and fast, economical detection by serum for the detection of esophageal squamous cell carcinoma associated serum miRNAs genetic expression.In addition, adopt the solution of the present invention to detect the multiple expression with esophageal squamous cell carcinoma associated serum miRNAs gene simultaneously, in engineering practice, how making the composition of multiple stem ring primer in qPCR reaction, show good validity and specificity is a very large screening engineering, and comprise multiple stem ring primer in stem ring Primer composition of the present invention can be efficient, specificity significantly with respective for goal gene carry out specific binding, and it is efficient, fast, economically by demonstrating correct detected result, greatly improve detection efficiency, reduce testing cost, for the detection of expression of corresponding and esophageal squamous cell carcinoma associated serum miRNAs gene and the detection of esophageal squamous cell carcinoma provide a kind of effective way.
Accompanying drawing explanation
Fig. 1,2,3 is experimental results that in the embodiment of the present invention, stem ring primer detects miRNAs genetic expression relevant to esophageal squamous cell carcinoma in 19 routine patients with esophageal squamous cell carcinoma perioperatively serum samples, Fig. 1 is miR-129-5p relative expression in patients with esophageal squamous cell carcinoma perioperatively serum, Fig. 2 is miR-223-3p relative expression in patients with esophageal squamous cell carcinoma perioperatively serum, and Fig. 3 is miR-483-5p relative expression in patients with esophageal squamous cell carcinoma perioperatively serum.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.
Embodiment 1
The Design and synthesis of stem ring primer
By online database miBase(http: //www.mirbase.org/) 22 kinds and esophageal squamous cell carcinoma associated serum miRNAs gene are analyzed, the design stem ring primer of about 50nt and the quantitative primer gene to be detected of 16-25nt, its stem ring primer sequence is as follows:
(1) for the stem ring primer of hsa-miR-10a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAA-3’（SEQIDNO.1）；
(2) for the stem ring primer of hsa-miR-16-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCGCCAAT-3’（SEQIDNO.2）；
(3) for the stem ring primer of hsa-miR-18a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTATCT-3’（SEQIDNO.3）；
(4) for the stem ring primer of hsa-miR-21-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAACA-3’（SEQIDNO.4）；
(5) for the stem ring primer of hsa-miR-22-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAGTT-3’（SEQIDNO.5）；
(6) for the stem ring primer of hsa-miR-25-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAGAC-3’（SEQIDNO.6）；
(7) for the stem ring primer of hsa-miR-31-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCTAT-3’（SEQIDNO.7）；
(8) for the stem ring primer of hsa-miR-100-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAG-3’（SEQIDNO.8）；
(9) for the stem ring primer of hsa-miR-107 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGATAG-3’（SEQIDNO.9）；
(10) for the stem ring primer of hsa-miR-127-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCCAA-3’（SEQIDNO.10）；
(11) for the stem ring primer of hsa-miR-129-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACGCAAGC-3’（SEQIDNO.11）；
(12) for the stem ring primer of hsa-miR-148b-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAAAG-3’（SEQIDNO.12）；
(13) for the stem ring primer of hsa-miR-155-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACCCCT-3’（SEQIDNO.13）；
(14) for the stem ring primer of hsa-miR-194-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCACA-3’（SEQIDNO.14）；
(15) for the stem ring primer of hsa-miR-200c-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCATC-3’（SEQIDNO.15）；
(16) for the stem ring primer of hsa-miR-223-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGGGGT-3’（SEQIDNO.16）；
(17) for the stem ring primer of hsa-miR-337-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAACTCC-3’（SEQIDNO.17）；
(18) for the stem ring primer of hsa-miR-375 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCACGC-3’（SEQIDNO.18）；
(19) for the stem ring primer of hsa-miR-483-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTCCCT-3’（SEQIDNO.19）；
(20) for the stem ring primer of hsa-miR-548k gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCAAA-3’（SEQIDNO.20）；
(21) for the stem ring primer of hsa-miR-1246 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCCTGCT-3’（SEQIDNO.21）；
(22) for the stem ring primer of hsa-miR-1322 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCAGCAT-3’（SEQIDNO.22）。
The esophageal squamous cell carcinoma associated serum miRNAs gene illustrated is needed to be hsa-miR-10a-5p gene, hsa-miR-16-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-107 gene, hsa-miR-127-3p gene, hsa-miR-129-5p gene, hsa-miR-148b-3p gene, hsa-miR-155-5p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-375 gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene or hsa-miR-1322 gene.Wherein hsa-miR-107 gene, hsa-miR-129-5p gene, hsa-miR-155-5p gene and hsa-miR-375 gene are down-regulated gene, hsa-miR-10a-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-127-3p gene, hsa-miR-148b-3p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene and hsa-miR-1322 gene are up-regulated gene, has-miR-16-5p gene is reference gene.
Synthesize the above-mentioned nucleotide sequence designed, stem ring primer entrusts the synthesis of Invitrogen biotech firm.
Embodiment 2
The preparation of detection reagent
Stem ring primer used in test kit is dissolved in RNase-free water formulated.
Embodiment 3
Stem ring primer qPCR method detects the expression experiment of miRNAs gene relevant to esophageal squamous cell carcinoma in patients with esophageal squamous cell carcinoma serum.
Test in accordance with the following steps:
1, serum sample pre-treatment to be detected is specially: by the fresh blood collected temperature bath 20min in 37 DEG C of thermostat water baths; The centrifugal 5min of room temperature 3000rpm, gets supernatant, removes precipitation; In 4 DEG C, the centrifugal 10min of 12000rpm, remove precipitation, packing supernatant, is stored in-80 DEG C; Take out sample, after sample dissolution, in 4 DEG C by centrifugal for 12000g 10min, stay supernatant; Get 100 μ L serum, add 300 μ LRNase-free water, vortex mixes; Add 400 μ LTRIzol, concussion mixing, room temperature places 5min, in 4 DEG C by centrifugal for 12000g 10min, goes precipitation; The TRIzol reagent of every 1mL adds 0.2mL chloroform, vortex 15s, and room temperature places 2-3min, in 4 DEG C by centrifugal for 12000g 15min, gets supernatant; Every 1mLTRIzol reagent adds 0.5mL Virahol, fully mixes, and room temperature places 10min, in 4 DEG C by centrifugal for 12000g 15min, removes supernatant; Add the ethanol that 1mL volume fraction is 75%, vortex mixes, and in 4 DEG C by centrifugal for 7500g 5min, stay precipitation, and dry RNA precipitates; Add 20 μ LRNase-free water dissolution RNA, and at 55-60 DEG C of temperature bath 15min.
2, carry out reverse transcription with stem ring primer pair testing sample to be measured, reaction system is 20 μ L, mixing; Above-mentioned mixed solution is reacted 50min at 42 DEG C, 70 DEG C of heating 15min.
3, qPCR process is specially:
3.1, adding qPCR system with forward primer and universal primer is 15 μ L;
3.2, qPCR reaction conditions is: the denaturation stage, and temperature is 95 DEG C, and the time is 5min; In the circulating reaction stage, denaturation temperature is 95 DEG C, and the time is 10s, and annealing temperature is 60 DEG C, and the time is 34s, 40 circulations; The solubility curve stage, denaturation temperature 95 DEG C, time 15s, annealing temperature 60 DEG C, time 60s, denaturation temperature 95 DEG C, time 15s.
3.3, qPCR result as Figure 1-3.
Interpretation of result: for experimental group, there is signal in the serum miRNAs gene relevant to esophageal squamous cell carcinoma, and negative control group fails to detect signal in qPCR detects.Illustrate that the inventive method can be used for detecting the expression with esophageal squamous cell carcinoma associated serum miRNAs gene in serum.QPCR experimental result is as shown in Fig. 1 (miR-129-5p), Fig. 2 (miR-223-3p) and Fig. 3 (miR-483-5p), the expression amount of major part (16/19) patients with esophageal squamous cell carcinoma operation consent serum miR-129-5p is significantly lower than postoperative expression amount, and in serum the result of miR-223-3p and miR-483-5p is in contrast, relative to preoperative, most of patients with esophageal squamous cell carcinoma Post operation serum miR-223-3p(18/19) and expression amount miR-483-5p(14/19) significantly reduce.Describing miR-129-5p is down-regulated gene, miR-223-3p and miR-483-5p is up-regulated gene, above result further illustrates, the method adopting stem ring primer qPCR to detect the serum miRNAs relevant to esophageal squamous cell carcinoma is feasible, and more direct economy, can realize the diagnosis of the early stage esophageal squamous cell carcinoma of Wicresoft, extensive examination and prognosis evaluation simultaneously.In addition, found for the method validation detecting the serum miRNAs gene relevant to esophageal squamous cell carcinoma by order-checking, above-mentioned experimental result is accurately, it serves to show test kit of the present invention and the accuracy of method in the screening detecting the serum miRNAs gene relevant to esophageal squamous cell carcinoma and esophageal squamous cell carcinoma.
Embodiment above describes ultimate principle of the present invention, principal character and advantage; the technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; under the scope not departing from the principle of the invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the scope of protection of the invention.
<110> He'nan Normal University
<120> mono-kind is for detecting the test kit of esophageal squamous cell carcinoma associated serum miRNAs gene
<160>22
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>1
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAA
<210>1
<211>51
<212>DNA
<213> artificial sequence
<400>2
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCGCCAAT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>3
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTATCT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>4
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAACA
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>5
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAGTT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>6
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAGAC
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>7
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCTAT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>8
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAG
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>9
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGATAG
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>10
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCCAA
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>11
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACGCAAGC
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>12
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAAAG
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>13
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACCCCT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>14
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCACA
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>15
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCATC
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>16
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGGGGT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>17
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAACTCC
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>18
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCACGC
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>19
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTCCCT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>20
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCAAA
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>21
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCCTGCT
<210>1
<211>50
<212>DNA
<213> artificial sequence
<400>22
GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCAGCAT
Claims (4)
1. one kind for detecting the test kit of esophageal squamous cell carcinoma associated serum miRNAs gene, it is characterized in that including the detection reagent that stem ring primer and RNase-free water for diagnosing esophageal squamous cell carcinoma associated serum miRNAs gene are prepared, wherein esophageal squamous cell carcinoma associated serum miRNAs gene is hsa-miR-10a-5p gene, hsa-miR-16-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-107 gene, hsa-miR-127-3p gene, hsa-miR-129-5p gene, hsa-miR-148b-3p gene, hsa-miR-155-5p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-375 gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene or hsa-miR-1322 gene are at least one in following 22 kinds of stem ring primers for diagnosing the stem ring primer of esophageal squamous cell carcinoma associated serum miRNAs gene:
(1) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.1 of hsa-miR-10a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAA-3’；
(2) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.2 of hsa-miR-16-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCGCCAAT-3’；
(3) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.3 of hsa-miR-18a-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTATCT-3’；
(4) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.4 of hsa-miR-21-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAACA-3’；
(5) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.5 of hsa-miR-22-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAGTT-3’；
(6) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.6 of hsa-miR-25-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCAGAC-3’；
(7) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.7 of hsa-miR-31-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCTAT-3’；
(8) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.8 of hsa-miR-100-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCACAAG-3’；
(9) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.9 of hsa-miR-107 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGATAG-3’；
(10) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.10 of hsa-miR-127-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCCAA-3’；
(11) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.11 of hsa-miR-129-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACGCAAGC-3’；
(12) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.12 of hsa-miR-148b-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACAAAG-3’；
(13) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.13 of hsa-miR-155-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACACCCCT-3’；
(14) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.14 of hsa-miR-194-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCACA-3’；
(15) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.15 of hsa-miR-200c-3p gene;
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCCATC-3’；
(16) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.16 of hsa-miR-223-3p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTGGGGT-3’；
(17) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.17 of hsa-miR-337-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAACTCC-3’；
(18) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.18 of hsa-miR-375 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACTCACGC-3’；
(19) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.19 of hsa-miR-483-5p gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCTCCCT-3’；
(20) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.20 of hsa-miR-548k gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACAGCAAA-3’；
(21) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.21 of hsa-miR-1246 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCCTGCT-3’；
(22) for the stem ring primer of nucleotide sequence as shown in SEQIDNO.22 of hsa-miR-1322 gene:
5’-GTCGTATCCATGGCAGGGTCCGAGGTATTCGCCATGGATACGACCAGCAT-3’。
2. the test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene according to claim 1, it is characterized in that: described hsa-miR-10a-5p gene, hsa-miR-18a-5p gene, hsa-miR-21-5p gene, hsa-miR-22-3p gene, hsa-miR-25-3p gene, hsa-miR-31-5p gene, hsa-miR-100-5p gene, hsa-miR-127-3p gene, hsa-miR-148b-3p gene, hsa-miR-194-5p gene, hsa-miR-200c-3p gene, hsa-miR-223-3p gene, hsa-miR-337-5p gene, hsa-miR-483-5p gene, hsa-miR-548k gene, hsa-miR-1246 gene and hsa-miR-1322 gene are up-regulated gene, hsa-miR-107 gene, hsa-miR-129-5p gene, hsa-miR-155-5p gene and hsa-miR-375 gene are down-regulated gene, has-miR-16-5p gene is reference gene.
3. a using method for the test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene according to claim 1, is characterized in that comprising the steps:
(1) extract the total serum IgE of serum sample to be detected, use micro-spectrophotometer to measure concentration and the purity of RNA;
(2) carry out reverse transcription with stem ring primer pair testing sample to be measured, reverse transcription system is 20 μ L, and by above-mentioned mixed solution in 42 DEG C of reaction 50min, 70 DEG C of heating 15min reverse transcription RNA are cDNA;
(3) adding qPCR system with forward primer and universal primer is 15 μ L;
(4) qPCR reaction conditions is: the denaturation stage, and temperature is 95 DEG C, and the time is 5min; In the circulating reaction stage, denaturation temperature is 95 DEG C, and the time is 10s, and annealing temperature is 60 DEG C, and the time is 34s, 40 circulations; The solubility curve stage, denaturation temperature 95 DEG C, time 15s, annealing temperature 60 DEG C, time 60s, denaturation temperature 95 DEG C, time 15s.
4. the using method of the test kit for detecting esophageal squamous cell carcinoma associated serum miRNAs gene according to claim 3, is characterized in that the detailed process of step (1) is: by the fresh blood collected temperature bath 20min in 37 DEG C of thermostat water baths; The centrifugal 5min of room temperature 3000rpm, gets supernatant, goes precipitation; At 4 DEG C, the centrifugal 10min of 12000rpm, remove precipitation, packing supernatant, is stored in-80 DEG C; Take out sample, after sample dissolution, in 4 DEG C by centrifugal for 12000g 10min, stay supernatant; Get 100 μ L serum, add 300 μ LRNase-free water, vortex mixes; Add 400 μ LTRIzol, concussion mixing, room temperature places 5min, in 4 DEG C by centrifugal for 12000g 10min, removes precipitation; The TRIzol reagent of every 1mL adds 0.2mL chloroform, vortex 15s, and room temperature places 2-3min, in 4 DEG C by centrifugal for 12000g 15min, gets supernatant; Every 1mLTRIzol reagent adds 0.5mL Virahol, fully mixes, and room temperature places 10min, in 4 DEG C by centrifugal for 12000g 15min, removes supernatant; Add the ethanol that 1mL volume fraction is 75%, vortex mixes, and in 4 DEG C by centrifugal for 7500g 5min, stay precipitation, and dry RNA precipitates; Add 20 μ LRNase-free water dissolution RNA, and at 55-60 DEG C of temperature bath 15min.
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| CN105950708A (en) * | 2016-03-31 | 2016-09-21 | 浙江大学 | Fluorogenic quantitative PCR detection primer and method for miR-100 content |
| EP3494230A4 (en) * | 2016-08-04 | 2020-07-29 | Baylor Research Institute | Methods for diagnosing and treating esophageal cancer |
| US11603566B2 (en) | 2016-08-04 | 2023-03-14 | Cancer Diagnostics Research Innovation | Methods for diagnosing and treating esophageal cancer |
| CN106119393A (en) * | 2016-08-25 | 2016-11-16 | 朱伟 | A kind of blood plasma miRNA mark relevant to esophageal squamous cell carcinoma auxiliary diagnosis and application thereof |
| CN106119393B (en) * | 2016-08-25 | 2020-04-07 | 朱伟 | Plasma miRNA marker related to esophageal squamous carcinoma auxiliary diagnosis and application thereof |
| CN109852686A (en) * | 2019-02-26 | 2019-06-07 | 浙江大学医学院附属妇产科医院 | The internal reference collection of excretion body miRNA a kind of and more internal reference combination with standard sizing techniques |
| WO2021162065A1 (en) * | 2020-02-10 | 2021-08-19 | 国立大学法人広島大学 | Method for aiding in detection of esophageal squamous cell carcinoma |
| CN115595369A (en) * | 2022-10-31 | 2023-01-13 | 中国医学科学院肿瘤医院（Cn） | Liquid biopsy prediction model based on esophageal precancerous lesion or esophageal cancer miRNAs, diagnosis kit and application |
| CN115595369B (en) * | 2022-10-31 | 2023-06-30 | 中国医学科学院肿瘤医院 | Liquid biopsy prediction model based on esophageal precancerous lesions or esophageal cancer miRNAs, diagnosis kit and application |
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Application publication date: 20151223 |