CN104357566A - Liver cancer detection kit - Google Patents
Liver cancer detection kit Download PDFInfo
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- CN104357566A CN104357566A CN201410612498.7A CN201410612498A CN104357566A CN 104357566 A CN104357566 A CN 104357566A CN 201410612498 A CN201410612498 A CN 201410612498A CN 104357566 A CN104357566 A CN 104357566A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a liver cancer detection kit which comprises a cel-miR-39 sequence and primers for performing real-time quantification PCR on cel-miR-39, hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p,hsa-miR-136-3p,hsa-let-7b-5p,hsa-let-7f-5p,hsa-miR-16-5p,hsa-miR-432-5p,hsa-miR-4446-3p and hsa-miR-486-3p. After alpha fetoprotein is used for detecting liver cancer for a patient, and the liver cancer detection kit, disclosed by the invention, is further used for auxiliary detection to effectively eliminate the false positive result of the alpha fetoprotein detection, so that huge mental stress and property loss can be avoided for the patient.
Description
Technical field
The present invention relates to a kind of liver cancer detection kit.
Background technology
MiRNA (microRNA) is that in cell, length is the non-coding tiny RNA of 18-25 Nucleotide.MiRNA combines by the 3 ' UTR (untranslated region) with target gene mRNA is not exclusively complementary, suppresses the translation of target gene.From 1993, in nematode (Caenorhabditis elegans), identify first miRNA lin-4, increasing miRNA in different biologies identified go out.So far, found in human tissue more than 1,000 kinds of miRNA.Think at present, the Human genome of 10-30% is all subject to the regulation and control of miRNA.
The early diagnosis of cancer is successfully cured it important effect.Easy acquisition and stable biological marker can provide a series of relevant valuable oncobiology information.Alpha-fetoprotein (AFP) is the Major Serological index that current liver cancer (HCC) is diagnosed, but the Sensitivity and Specificity that alpha-fetoprotein detects is still not high, false positive rate is higher, because people are to the feared state of mind of cancer, false-positive detected result easily causes huge stress and property damage to patient.
Summary of the invention
In order to overcome in prior art, the problem that alpha-fetoprotein is higher to the detected result false positive of liver cancer, the invention provides a kind of liver cancer detection kit, for the detection of auxiliary alpha-fetoprotein to liver cancer, can effectively remove false-positive detected result.
As one aspect of the present invention, relate to a kind of liver cancer detection kit, described test kit includes cel-miR-39 sequence, and for the primer of cel-miR-39, hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-432-5p, hsa-miR-4446-3p and hsa-miR-486-3p real-time quantitative PCR.
Described test kit can also include hsa-let-7a-3p, hsa-let-7f-2-3p, hsa-miR-106a-5p, hsa-miR-1180, hsa-miR-1224-5p, hsa-miR-1255b-5p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-3158-3p, hsa-miR-3163, hsa-miR-3200-5p, hsa-miR-455-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-4770, hsa-miR-5010-5p, the primer of hsa-miR-654-3p real-time quantitative PCR.
Described test kit can also include, and has added vacuum test tube and the vacuum blood collection needle assemblies of antithrombotics.Described antithrombotics is K
3eDTA.
Described test kit can also include, Reverse Transcription and real-time quantitative PCR reagent.Described Reverse Transcription refers to the miScript II RT test kit of Qiagen company.Described real-time quantitative PCR reagent refers to the miScript SYBR Green PCR Kit of Qiagen company.
After adopting such scheme, the present invention at least can realize following beneficial effect:
Using patient after alpha-fetoprotein carries out liver cancer detection, re-using detection kit provided by the invention and carrying out auxiliary detection, effectively can remove the false positive results that alpha-fetoprotein detects, thus avoid bringing huge stress and property damage to patient.
Test kit provided by the present invention, the miRNA of employing is the miRNA that existing report has been found that, its check order joint used and amplimer all have ripe commercial goods, and can obtain from the market easily, test kit is easy to preparation.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.
Experimental example 1
With the screening of the higher miRNA of the liver cancer degree of correlation
1. separating plasma operation
1) fresh blood is taked to put into K
3eDTA vacuum test tube (BD), separated plasma in two hours after collecting blood.Before separation, blood is kept on ice always.
2) utilize refrigerated centrifuge (Centrifuge 5804R, Eppendorf), 4 DEG C of centrifugal 10 minutes of centrifugal separation plasma: 500g in two steps in blood, are transferred in 15 milliliters of new centrifuge tubes by supernatant; Centrifugal 10 minutes of 1800g, the supernatant obtained is the blood plasma for building storehouse.
3) every 8 milliliters of blood obtain about 4 milliliters of blood plasma, are separated blood plasma-80 DEG C preservation obtained.
2. the extraction of total serum IgE in blood plasma
The blood plasma (from step 1) of freezen protective at thawed on ice, and puts upside down mixing gently.
In the centrifuge tube being divided by blood plasma the nuclease free to 1.5 milliliters to pollute, often pipe 600 microlitre.
Often pipe adds the TRIZOL (Invitrogen) of equal-volume (600 microlitre), fully shakes mixing.
Room temperature left standstill after 3 minutes, and often pipe adds the chloroform (Beijing Chemical Plant) of 1/5 volume (120 microlitre), fully shakes mixing, and room temperature leaves standstill 3 minutes.
Centrifugal 15 minutes of 4 DEG C of 16000g (Centrifuge 5417R, Eppendorf).
In the centrifuge tube that transfer supernatant to 1.5 milliliter nuclease free is polluted, often pipe about 700 microlitre supernatant.
Often pipe adds Virahol (Beijing Chemical Plant) and the 0.5 microlitre glycogen (Invitrogen) of equal-volume (700 microlitre), puts upside down mixing gently.
Precipitates overnight in-80 DEG C of refrigerators.
Centrifugal 30 minutes of 4 DEG C of 16000g.
Remove solution, often pipe adds 750 microlitre 75% alcohol, turns upside down, fully washes precipitation.
Centrifugal 10 minutes of 4 DEG C of 16000g.
Abundant removal solution, at room temperature drying precipitated extremely precipitation just becomes transparent.
Often pipe adds the distilled water that 10 microlitre nuclease free are polluted, and dissolves 15 minutes, and fully mix under room temperature.
The total serum IgE of all pipes is merged, and measures the amount of total serum IgE with nano-drop 1000 (Thermo Scientific).
Get 1 microgram RNA, be placed on 37 DEG C of evaporates to dryness, make RNA final volume be 5 microlitres.
Below, the reagent used in step 3-6 is all from TruSeq Small RNA Sample Prep Kits (Illumina).
The connection of 3.3 ' joint
Add 1 microlitre in 5 microlitre total serum IgE (from step 2), after dilution, concentration is the 3 ' joint (Illumina) of 0.25 nmole, mixing, 70 DEG C of heat shocks 2 minutes, and is placed on rapidly on ice.
Following reagent is added successively: 1 microlitre T4RNA Ligase 2,2 microlitre Ligation Buffer, 1 microlitre RNase Inhibitor in 200 microlitre thin-walled PCR pipe.
Solution is mixed, is placed in PCR instrument, hatches 2 hours for 28 DEG C.
Add 1 microlitre Stop Solution, after mixing, 28 DEG C 15 minutes.
The connection of 4.5 ' joint
After getting 1.1 microlitre dilutions, concentration is the 5 ' joint (Illumina) of 0.25 nmole, 70 DEG C of heat shocks 2 minutes, and is placed on rapidly on ice.
1.1 microlitre ATP and 1.1 microlitre T4 RNA Ligase 1 successively in 200 microlitre thin-walled PCR pipe of existing 1.1 microlitre 5 ' joints.
3 microlitre said mixtures are added in 11 microlitre RNA samples.
Solution is mixed, is placed in PCR instrument, hatches 1 hour for 28 DEG C.
5. reverse transcription synthesis cDNA
Add successively in 200 microlitre thin-walled PCR pipe, the RNA of 14 microlitres connections 5 ' and 3 ' joint and 1 microlitre RNA RT primer, 70 DEG C of heat shocks 2 minutes, and be placed on rapidly on ice.
Add following reagent (Illumina) successively: 4 microlitre 5X First Strand Buffer, 2 microlitre 100 mmole DTT, 1 microlitre 12.5 mmole dNTPs, 1 microlitre RNase Inhibitor, 1 microlitre SuperScript II Reverse Transcriptase.
By solution mix, be placed in PCR instrument, program is: 25 DEG C 10 minutes, 42 DEG C 65 minutes, 70 DEG C 15 minutes, 4 DEG C of maintenances.
6.PCR increases
Obtain after reverse transcription step, be equipped with in 200 microlitre thin-walled PCR pipe of 24 microlitre cDNA samples, add following reagent (Illumina) successively: 26 microlitre PCR mixtures, 1 microlitre RNA PCR primer, 1 microlitre RNA Index primer.
Above solution is mixed, is placed in PCR instrument.Before sample is placed, PCR instrument is preheated to more than 65 DEG C.
PCR is circularly set as follows:
98 DEG C 30 seconds;
15 circulations:
98 DEG C 10 seconds, 60 DEG C 30 seconds, 72 DEG C 15 seconds;
72 DEG C 5 minutes.
7. electrophoresis and glue reclaim
Pre-race 15%TBE-PAGE glue 15 minutes under voltage 100 volts.
Often add 10 microlitre 6 × Gel loading dye (Fermentas) in pipe PCR primer, mixing.
Each sample spot, in continuous print three loading wells, eachly adds 20 microliters of sample in the air.Sample and Low MW Ladder (Bioo Scientific) have the spacing of at least one loading wells.
Voltage 200 volts, run about 50 minutes, just run out of to light blue dyestuff below.
At room temperature contaminate glue 15 minutes with SYBR Gold (Invitrogen), afterwards under ultraviolet lamp, the glue of clip size at about 140bp is cut.
The embedding tube using 200 microlitre thin-walled PCR pipe and 1.5 milliliters of centrifuge tubes to do, utilizes whizzer, and blob of viscose is become little particle, is beneficial to the recovery of nucleic acid.
Add 300 microlitre Elution Buffer in the little glue particle that each sample obtains, utilize vertical mixed instrument, at room temperature dissolve and spend the night.
At room temperature centrifugal 2 minutes of 16000g.
Supernatant is transferred to (Ambion) in centrifugal column, at room temperature centrifugal 2 minutes of 16000g, with fully except the particle that removes photoresist.
Be transferred to by filtered liquid in 1.5 milliliters of centrifuge tubes that new free nucleic acid pollutes, often pipe adds 750 microliter anhydrous ethanol (Beijing Chemical Plant), 30 lli are NaOAc (Beijing Chemical Plant), the 1 microlitre Co-precipitant (Bioo Scientific) of 3 moles often liter.
-20 DEG C precipitate two hours.
Centrifugal 30 minutes of 4 DEG C of 16000g.
Remove supernatant gently, wash precipitation with 800 microlitre 75% ethanol.
Centrifugal 10 minutes of 4 DEG C of 16000g.Remove supernatant, and at room temperature drying precipitated extremely precipitation just becomes transparent.
Often pipe adds the distilled water that 10 microlitre nuclease free are polluted, and dissolves 15 minutes, and fully mix under room temperature.
8. the quality examination in storehouse
Agilent Bioanalyzer is used to analyze the clip size of storehouse amplifying nucleic acid.
Real-time quantitative PCR is used to detect the nucleic acid amount containing joint sequence in storehouse.Because the primer of real-time quantitative PCR is (this process can be undertaken by joint provider) designed according to the nucleotide sequence of joint, therefore, the nucleic acid be connected with both-end joint is only had to be detected.When detected result show nucleic acid amount be all greater than 3.5 nmoles often rise time, can be used for two generations order-checking.
Sample is carried out the order-checking of two generations.
9. sequencing data analysis
Original fastq data are carried out low-quality value, remove joint (being provided by Illumina company), retain the process that insertion sequence length is greater than 16nt, and be fasta form by data transformations, identical sequence merged simultaneously.
Utilize blast software package to be compared by the reference sequences that the data processed and miRBase are downloaded, comparison requires: e value is less than 0.05; First must mate completely; 3 ' end allows to move to left 1 or move to right 3; 1 position is allowed not mate.
Record data total amount, the sequence number of miRNA in comparison, the miRNA species number that the species number of miRNA and sequence number are greater than 10.
By at least in a sample expression amount sequence number be greater than the kind of the miRNA of 10 and expression amount gathers.
The DEseq software package in R software is utilized to find the miRNA of tumour blood plasma and normal plasma two differences between samples expression.
Experimental result
After sample blood plasma miRNA Jian Ku, the order-checking of two generations, sample master data is as shown in table 1.
The sequencing data information of table 1:14 plasma sample
By to after sequencing data carried out data filter, went joint and miRBase comparison two generations, in each sample be the data volume of miRNA at about 20%-70%, the miRNA kind that reservation queue number is greater than 10, each sample miRNA kind is about 300.
The miRNA having remarkable differential expression in tumor sample and normal sample is found by DESeq software, the miRNA that wherein P value is less than 0.05 has 29 kinds, analyzed by complete interlock cluster (complete linkage clustering), after 29 kinds of miRNA combination, effectively can distinguish tumor sample and normal sample.29 kinds of miRNA are the hsa-let-7a-3p in miRBase, hsa-let-7b-5p, hsa-let-7f-2-3p, hsa-let-7f-5p, hsa-miR-100-5p, hsa-miR-106a-5p, hsa-miR-10a-5p, hsa-miR-1180, hsa-miR-1224-5p, hsa-miR-1255b-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-miR-16-5p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-30b-5p, hsa-miR-3158-3p, hsa-miR-3163, hsa-miR-3200-5p, hsa-miR-432-5p, hsa-miR-4446-3p, hsa-miR-455-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-4770, hsa-miR-486-3p, hsa-miR-5010-5p, hsa-miR-654-3p and hsa-miR-99a-5p.Wherein, hsa-let-7a-3p, hsa-let-7f-2-3p, hsa-miR-100-5p, hsa-miR-10a-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-30b-5p, hsa-miR-455-5p, hsa-miR-4770, hsa-miR-654-3p, hsa-miR-99a-5p raise in liver cancer patient; Hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-486-3p, hsa-miR-4446-3p, hsa-miR-432-5p, hsa-miR-5010-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-3158-3p, hsa-miR-106a-5p, hsa-miR-1180, hsa-miR-3200-5p, hsa-miR-1224-5p, hsa-miR-1255b-5p, hsa-miR-3163 lower in liver cancer patient.
Clinical diagnosis often adopts real-time quantitative PCR to carry out quantitative analysis to miRNA, Given this resolving power of method, need choose the higher miRNA of expression amount further for clinical diagnosis in above-mentioned 29 kinds of miRNA.This patent is selected in 14 samples of order-checking, and in a sample in office, expression amount is higher than the miRNA of 100, obtains 12 kinds of miRNA.Analyze through complete interlock cluster (complete linkage clustering), liver cancer patient and normal individual still can distinguish by these 12 kinds of blood plasma miRNA.In these 12 kinds of miRNA, hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p and hsa-miR-136-3p of liver cancer patient raise, and hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-432-5p, hsa-miR-4446-3p and hsa-miR-486-3p lower.
Experimental example 2
The preparation of liver cancer detection kit and use
The test kit that the present embodiment provides, include cel-miR-39 sequence, and for the primer of cel-miR-39, hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-432-5p, hsa-miR-4446-3p and hsa-miR-486-3p real-time quantitative PCR.
The primer of real-time quantitative PCR is provided by Rui Bo biotech firm.
The test kit that the present embodiment provides, can also include the primer of hsa-let-7a-3p, hsa-let-7f-2-3p, hsa-miR-106a-5p, hsa-miR-1180, hsa-miR-1224-5p, hsa-miR-1255b-5p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-3158-3p, hsa-miR-3163, hsa-miR-3200-5p, hsa-miR-455-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-4770, hsa-miR-5010-5p and hsa-miR-654-3p real-time quantitative PCR.
The test kit that the present embodiment provides, can also include, and has added antithrombotics (K
3eDTA) vacuum test tube and vacuum blood collection needle assemblies.
The test kit that the present embodiment provides, can also include, reverse transcription and real-time quantitative PCR reagent, such as: miScript II RT test kit (Qiagen) and miScript SYBR Green PCR Kit (Qiagen).
The present embodiment provides test kit usage as follows:
Hospital uses alpha-fetoprotein to detect the result of liver cancer for after the positive to patient, uses this test kit to detect further.
1. blood plasma collection be separated
With having added antithrombotics (K
3eDTA) heparin tube collects Fresh blood sample about 1 milliliter, separated plasma in two hours after collecting blood.Before separation, blood is kept on ice always.
With refrigerated centrifuge (Centrifuge 5804R, Eppendorf), 4 DEG C of centrifugal 10 minutes of centrifugal separation plasma: 500g in two steps in blood, are transferred to supernatant in new centrifuge tube; Centrifugal 10 minutes of 1800g, the supernatant obtained is stand-by.
2. the miRNA reverse transcription in blood plasma is become cDNA
The cel-miR-39 (final concentration is that 2 nmoles often rise) as the contrast of miRNA quantify foreign is added in blood plasma, use 13 microlitre blood plasma and Qiagen miScript II RT test kit, for the reversion of miRNA, often the final volume of pipe reversion system is 20 microlitres.Following reagent is added successively: 13 microlitre medium, 4 microlitre 5x miScript HiSpec Buffer, 2 microlitre 10x miScript Nucleics Mix, 1 microlitre miScript Reverse Transcriptase Mix in eight connecting leg PCR pipe.
Setting program in PCR instrument:
37 DEG C 60 minutes;
95 DEG C 5 minutes;
4 DEG C of maintenances.
After centrifugal, often pipe adds 80 microlitre H
2o ,-20 DEG C of placements.
3. real-time quantitative PCR detects
Use 1 microlitre inversion product and miScript SYBR Green PCR Kit test kit, for the quantitative PCR of miRNA, often the final volume of pipe reversion system is 20 microlitres.Following reagent is added successively: 10 microlitre 2X QuantiTect SYBR Green PCR Master Mix, 1 microlitre inversion product, 2 microlitre 10X miSript Universal Primer, 0.8 microlitre Forward primer (5 micromole), 6.2 microlitres are not containing the water of RNA enzyme in 96 hole PCR plate.
Setting program on real-time PCR:
95 DEG C 15 minutes;
40 circulations:
94 DEG C 15 seconds,
55 DEG C 30 seconds,
70 DEG C 30 seconds
4. Analysis of test results
With the expression amount of cel-miR-39 for reference, when calculating miRNA expression amount, utilize Δ Ct=Ct (miRNA)-Ct (cel-miR-39), the relative expression quantity of Δ Δ Ct=Δ Ct (patient)-Δ Ct (normal individual), miRNA is 2-Δ Δ Ct.
Detect through alpha-fetoprotein, be judged to be the patient of liver cancer patient, by analyzing the expression amount of miRNA in patients blood plasma and normal plasma, if obviously do not meet hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p, hsa-miR-136-3p in patients blood plasma to raise, the rule that hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-432-5p, hsa-miR-4446-3p, hsa-miR-486-3p lower, be then judged as that alpha-fetoprotein detects false positive.
When using whole 29 kinds of miRNA as detection indicator, if obviously do not meet hsa-let-7a-3p in patients blood plasma, hsa-let-7f-2-3p, hsa-miR-100-5p, hsa-miR-10a-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-30b-5p, hsa-miR-455-5p, hsa-miR-4770, hsa-miR-654-3p and hsa-miR-99a-5p raises, hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-486-3p, hsa-miR-4446-3p, hsa-miR-432-5p, hsa-miR-5010-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-3158-3p, hsa-miR-106a-5p, hsa-miR-1180, hsa-miR-3200-5p, hsa-miR-1224-5p, the rule that hsa-miR-1255b-5p and hsa-miR-3163 lowers, then be judged as that alpha-fetoprotein detects false positive.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (7)
1. a liver cancer detection kit, it is characterized in that, described test kit includes cel-miR-39 sequence, and for the primer of cel-miR-39, hsa-miR-10a-5p, hsa-miR-100-5p, hsa-miR-30b-5p, hsa-miR-99a-5p, hsa-miR-1271-5p, hsa-miR-136-3p, hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-432-5p, hsa-miR-4446-3p and hsa-miR-486-3p real-time quantitative PCR.
2. test kit according to claim 1, it is characterized in that, described test kit also includes the primer of hsa-let-7a-3p, hsa-let-7f-2-3p, hsa-miR-106a-5p, hsa-miR-1180, hsa-miR-1224-5p, hsa-miR-1255b-5p, hsa-miR-204-5p, hsa-miR-2392, hsa-miR-3158-3p, hsa-miR-3163, hsa-miR-3200-5p, hsa-miR-455-5p, hsa-miR-4732-3p, hsa-miR-4732-5p, hsa-miR-4770, hsa-miR-5010-5p and hsa-miR-654-3p real-time quantitative PCR.
3. test kit according to claim 1, is characterized in that, described test kit also includes, and has added vacuum test tube and the vacuum blood collection needle assemblies of antithrombotics.
4. test kit according to claim 3, is characterized in that, described antithrombotics is K
3eDTA.
5. test kit according to claim 1, is characterized in that, described test kit also includes, Reverse Transcription and real-time quantitative PCR reagent.
6. test kit according to claim 5, is characterized in that, described Reverse Transcription refers to the miScript II RT test kit of Qiagen company.
7. test kit according to claim 5, is characterized in that, described real-time quantitative PCR reagent refers to the miScript SYBR Green PCR Kit of Qiagen company.
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Cited By (7)
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| CN104878012A (en) * | 2015-05-22 | 2015-09-02 | 中国人民解放军第二军医大学 | Application of Hsa-miR-3200-5P in preparation of reagents or kits for early screening or diagnosis of Brachyury positive tumours |
| CN105274227A (en) * | 2015-10-26 | 2016-01-27 | 上海交通大学 | MicroRNA SDA detecting method based on AgNCs/HpDNA probes |
| CN105274226A (en) * | 2015-10-26 | 2016-01-27 | 上海交通大学 | MicroRNA SDA (strand-displacement amplification) detection method based on AgNCs/HpDNA probes |
| CN105664163A (en) * | 2016-02-01 | 2016-06-15 | 北京泱深生物信息技术有限公司 | Application of mir-5010 and mature miRNA (micro ribonucleic acid) of mir-5010 in preparation of OSA (osteosarcoma) diagnosis and treatment preparation |
| CN105944102A (en) * | 2016-07-06 | 2016-09-21 | 北京泱深生物信息技术有限公司 | Relation between abnormal expression of genes and different differentiation degrees of liver cancer and application thereof |
| CN107964563A (en) * | 2017-07-10 | 2018-04-27 | 广西中医药大学附属瑞康医院（广西中西医结合医院） | A kind of liver cancer detection kit |
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| CN104878012B (en) * | 2015-05-22 | 2018-03-09 | 中国人民解放军第二军医大学 | Applications of the 5p of Hsa miR 3200 in preparing early screening or diagnosing Brachyury positive tumors reagent or kit |
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| CN105944102A (en) * | 2016-07-06 | 2016-09-21 | 北京泱深生物信息技术有限公司 | Relation between abnormal expression of genes and different differentiation degrees of liver cancer and application thereof |
| CN107964563A (en) * | 2017-07-10 | 2018-04-27 | 广西中医药大学附属瑞康医院（广西中西医结合医院） | A kind of liver cancer detection kit |
| CN112294960A (en) * | 2020-10-30 | 2021-02-02 | 浙江大学 | Use of micro-molecule RNA-30b-5p as target molecule |
| CN112294960B (en) * | 2020-10-30 | 2022-02-08 | 浙江大学 | Use of micro-molecule RNA-30b-5p as target molecule |
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