CN109913547A - A kind of blood plasma excretion body miRNA molecule marker and its application in reagent preparation box - Google Patents

Abstract

The invention discloses a kind of blood plasma excretion body miRNA biomarker and its applications in the micro digital pcr kit early diagnosed for cervical carcinoma, and the RNA sequence of the molecular labeling is as shown in SEQ ID NO:1~SEQ ID NO:8.It is detected based on marker of the present invention, high sensitivity, specificity is good, and AUC may be up to 0.992, and diagnosis effect is good.In test data, according to the expression quantity of 2 miRNAs therein, the clinical samples of 1 grade of Cervical intraepitheliaI neoplasia or less He 2 grades of Cervical intraepitheliaI neoplasia or more can be effectively distinguished.Novel blood plasma excretion body miRNA molecule marker disclosed by the invention has the characteristic of good diagnosis index, that is developed is used to detect the diagnostic kit and detection method of this 8 miRNA, the kit is made of specificity amplification primer and general PCR amplification reagent, it can be diagnosed effective for cervical carcinoma screening, clinical use and promotional value with higher.

Description

A kind of blood plasma excretion body miRNA molecule marker and its in reagent preparation box Using

Technical field

The invention belongs to pharmaceutical technology field, in particular to blood plasma excretion body miRNA biomarker and cervical carcinoma to be used for And the kit of precancerous lesion early screening diagnosis.

Background technique

With the increase of morbidity and mortality, cancer has become the main reason for endangering world's public health.Cervical carcinoma Number of the infected be 98.9 people in every 100,000 people, death toll is every ten Wan Renzhong, 30.5 people, this two indexs are pernicious in women It is number two respectively in tumour, three.It is shown according to newest statistical data, cervical carcinoma is that 20-39 years old female cancer is dead The second largest reason, coverage is wide, and the extent of injury is deep.Pap smear detection or new Bai Shi liquid basal cell in cervical carcinoma screening The diagnosis that detection effectively improves earlier stage cancer patients or precancerous lesion patient is learned, the hair of cervical carcinoma is considerably reduced Sick rate, and improve 5 years survival rates of cervical cancer patient.But the detection of the Pap smear of different regions and hospital and new Bai Shi Liquid based cytology test diagnosis has differences；And both screening methods of cervical cancer are in many regional especially agricultures of China Village is not widely used.Cytolgical examination is pure artificial diagosis, and low efficiency, professional is again serious deficient, high-quality The cytology screening of amount is difficult to comprehensively universal.Currently, vaginoscopy is still the goldstandard of standard diagnostics cervical carcinoma, due to This method has traumatic and limitation, is not particularly suited for the inspection of each patient.

MiRNA is a kind of endogenic, non-coding RNA that length is about 22nt, inhibits or lures after mainly passing through transcription The degradation of mRNA is led to adjust the expression and function of oncogene or tumor suppressor gene.A large number of studies show that miRNA is swollen in difference It is specific expressed in tumor, and normal and tumor tissues can be distinguished.In addition, also there is research to confirm, circulation miRNA be can be used as The diagnosis marker of various diseases, including cancer.But miRNA is recycled as diagnosis marker because it is easy to be released by damaged cell The influence for the miRNA put has certain limitation.Therefore, clinically it is badly in need of new non-invasive relatively reliable and convenient and fast Cervical carcinoma early diagnosis marker.

Excretion body (exosome) is the multivesicular body of a kind of particular size secreted by living cells, current more generally accepted theory Method is a kind of nanoscale lipid bilayer vesica of 30-100nm.It is internal containing various active molecule, such as DNA, RNA and albumen Matter etc., in cellular communication, immune response plays an important role in a variety of physiological reactions such as mass exchange.Due to stabilization Physiological property, the database of comparatively perfect is supported and reasons, the microRNA therein such as research method are very popular.By close Research over year confirms that excretion body microRNA and disease have relationship closely, such as participates in immunological diseases, virus sense Dye, neurodegenerative disease are propagated, and cancer occurs and shifts etc..

Current miRNA quantitative verification, is divided into two kinds, and one is tailing reverse transcription methods, and one is stem ring reverse transcription methods.Add It is mature miRNA plus poly (A) tail that tail reverse transcription method, which is using poly (A) polymerase, is then carried out with anchor primer Reverse transcription finally uses and carries out fluorescence quantitative PCR detection to miRNA with by the complementary reverse primer of sequence label.Stem ring is anti- Transcription method is using the reverse transcriptase primer that can be folded into loop-stem structure, by carrying out reverse transcription with the 3 ' of miRNA end base complementrities, The first chain of cDNA is generated, fluorescence quantitative PCR detection is carried out.There are three types of method, spectrophotometry bases for current nucleic acid molecules quantitative It is quantified in the absorbances of nucleic acid molecules；Real-time fluorescence quantitative PCR (Real Time PCR) is based on Ct value, and Ct value just refers to can To detect the corresponding recurring number of fluorescent value；Digital pcr is newest quantitative technique, carried out by single-molecule PCR method based on Several nucleic acid quantifications, compared to qPCR, digital pcr can directly count the number of DNA molecular, be to the absolute fixed of initial sample Amount.Digital pcr mainly uses the micro-fluidic or droplet method of the popular research field of present analysis chemistry, after Macrodilution Nucleic acid solution is dispersed in the microreactor or droplet of chip, and the nucleic acid-templated number of each reactor is less than or is equal to 1.This Sample passes through after PCR cycle, has the reactor of a nucleic acid templates that will provide fluorescence signal, not the reactor of template Just there is no fluorescence signal.According to the volume of relative scale and reactor, so that it may extrapolate the nucleic acid concentration of original solution.It can Detection accurate and that repeatability is splendid is carried out to the slight change of a small amount of miRNA.

Summary of the invention

For deficiency present in the early diagnosis of above-mentioned cervical carcinoma, the purpose of the present invention is to provide a kind of blood plasma excretion bodies MiRNA biomarker and the droplet type digital pcr kit early diagnosed for cervical carcinoma, the present invention use 8 and uterine neck Work is used in combination in cytology detection used in the closely related blood plasma excretion body miRNA or these miRNA of cancer and at present clinic It is examined for diagnosing early cervical carcinoma better than existing cytology for cervical carcinoma early diagnosis marker using the method for digital pcr The clinical means such as survey and gynecatoptron have preferable specificity and sensitivity.Meanwhile the present invention also developed for detect this 8 The diagnostic kit and detection method of a miRNA, the kit is by specificity amplification primer and general PCR amplification reagent set At the preferable clinical value with cervical carcinoma early diagnosis provides new think of to improve cervical carcinoma early diagnosis level Road and method.

The purpose of the present invention: in view of the deficiencies of the prior art, provide a kind of blood plasma excretion body miRNA molecule marker and its Application in micro digital pcr kit of the preparation for cervical carcinoma early diagnosis.

The present invention adopts the following technical scheme: a kind of novel blood plasma excretion body microRNA molecule marker, derives from Blood plasma excretion body, wherein the microRNA is selected from the group: let-7a-3p, let-7d-3p, miR-30d-5p, miR-144- 5p, miR-182-5p, miR-183-5p, miR-215-5p and miR-4443, or combinations thereof, their RNA sequence is respectively such as SEQ Shown in ID NO:1~SEQ ID NO:8.

A kind of microRNA finger-print for the diagnosis of cervical carcinoma early screening, the finger-print include being selected from the group 2 kinds and the above microRNA:let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182-5p, miR- 183-5p, miR-215-5p and miR-4443.

The invention further relates to application of the above-mentioned molecular labeling in kit of the preparation for cervical carcinoma screening diagnosis.As It is preferred that kit includes following marker: let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182- 5p, miR-183-5p, miR-215-5p and miR-4443；

Further, the kit includes to use following four internal reference: miR-128-3p, miR-129-5p, miR- 320a and let-7i-5p.

The beneficial effects of the present invention are: using heretofore described blood plasma excretion body microRNA molecule marker as palace Neck cancer early diagnosis marker, high sensitivity, specificity is good, and AUC may be up to 0.992, and diagnosis effect is good.In test data In, according to the expression quantity of 2 miRNAs therein, it can effectively distinguish 1 grade of Cervical intraepitheliaI neoplasia or less and cervical intraepithelial neoplasia Become 2 grades or more of clinical samples.Novel blood plasma excretion body miRNA molecule marker disclosed by the invention has good diagnosis The characteristic of index, that is developed is used to detect the diagnostic kit and detection method of this 8 miRNA, and the kit is by special Property amplimer and general PCR amplification reagent composition, can be diagnosed effective for cervical carcinoma screening, clinic with higher makes With and promotional value.

Detailed description of the invention

Fig. 1 is the blood plasma excretion body miRNA molecule marker that screening verification of the present invention is used for the diagnosis of cervical carcinoma early screening Specific flow chart.

Fig. 2 is 8 miRNA combinations (let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182- 5p, miR-183-5p, miR-215-5p, and miR-4443) in 121 samples classification capacity assessment.(A) principal component point Analysis, (B) clustering, (C) ROC curve.As a result, it has been found that two groups of samples of CINI- and CINII+ can be compartmentalized well.? In clustering, the sample of only 2 CINII+ groups is mistakenly allocated in CINI- group.Divided together using 8 miRNAs When class, ROC curve shows preferable classifying quality, and AUC value reaches 0.992 (95% confidence interval is 0.978-1).

Fig. 3 is the independent evaluations of the otherness and classifying quality to the single miRNA molecule of 8 miRNA.(A-B) training The expression of 8 miRNA and ROC analysis in data.(C-D) 8 in the cancer beside organism of independent cervical cancer tissues and pairing The qRT-PCR of miRNA verifies situation.(A) and (C) is 4 downward miRNA, and (B) and (D) is 4 up-regulation miRNA.Fold Change is the ratio that miRNA expression changes in the cancer beside organism of cervical cancer tissues and pairing.4 in CINII+ group on The miRNA of tune, classifying quality is successively are as follows: let-7a-3p (FDR < 0.001, AUC=0.797), let-7d-3p (FDR < 0.001, AUC=0.887), miR-30d-5p (FDR < 0.001, AUC=0.89) and miR-215-5p (FDR < 0.001, AUC= 0.804) (Fig. 3 A).Other 4 miRNA significantly lowered in CINII+ group, classifying quality be successively miR-182-5p (FDR < 0.001, AUC=0.828), miR-183-5p (FDR < 0.001, AUC=0.797), miR-144-5p (FDR < 0.001, AUC= And miR-4443 (FDR < 0.001, AUC=0.854), (Fig. 3 B) 0.888).Group by independent cervical cancer tissues and the cancer of pairing Knit qRT-PCR verifying discovery 5 miRNAs (let-7d-3p, let-7a-3p, miR-30d-5p, miR-183-5p and miR- 182-5p) consistent with the variation tendency of excretion body miNRA, miR-144-5p and miR-4443 are compared with excretion body surface reaches, in group Inconsistent variation tendency is shown in knitting.MiR-215p-5p differential expression between cancerous tissue and cancer beside organism is not significant (Fig. 3 C-3D).

Fig. 4 is ddPCR verifying of 2 differential expression miRNAs in 203 independent blood plasma excretion body samples.A)let- R_score value and ROC curve of the 7d-3p in CINI- group and CINII+；B) miR-30d-5p is in CINI- group and CINII+ group R_score value and ROC curve；C) miR-30d-5p and let-7d-3p combines the ROC curve between CINI- and CINII+ group； D) it is applied alone miRNA expression value or cytoscopy result and miRNA expression value compared with the ROC curve of cytoscopy result.It surveys Ordinal number is remarkably decreased in CINII+ group according to display miR-30d-5p and let-7d-3p, and ddPCR experiment also demonstrates that the decline becomes Gesture (Fig. 4 A-B).The independent sorting effect of this 2 miRNAs shows 0.79 and 0.822 AUC value.Join in independent test sample The expression of results for using this 2 miRNAs is closed, the AUC value of ROC curve reaches 0.828, and (95% confidence interval is 0.736- 0.902), the expression of results and cytology testing result of this 2 miRNAs of conjunctive use, the AUC value of ROC curve reach 0.887 (Fig. 4 D).

Specific embodiment

Below by specific embodiment, the present invention is further elaborated, it should explanation, following the description merely to It explains the present invention, its content is not defined.

The acquisition methods of molecular labeling of the present invention are as follows:

(1) collecting 121 parts of peripheral blood samples includes 23 healthy volunteers, I grades of patients (CINI) of 5 precancerous lesions, and 59 A precancerous lesion II is to III level patient (CINII-III), and 34 cervical cancer patients (suffer from by CC, including 21 cervical squamous cell carcinomas (SCC) Person and 13 adenocarcinoma of the uterine cervix (ACC) patients).

(2) with containing ACD anti-coagulants heparin tube collect peripheral blood take a blood sample 8 hours in 4 DEG C under the conditions of 1900 × g from The heart 10 minutes, light yellow supernatant is then carefully drawn, then 16000 × g is centrifuged 10 minutes under the conditions of 4 DEG C, by upper plasma point It is filled in the EP pipe of the free nucleic acid of 1.5ml.

(3) blood plasma is centrifuged 15 minutes removal cool insoluble proteins with 3000 × g, and 400 μ l supernatants is taken to be put into the seedless acid pollution of sterilizing EP pipe in.The sterile PBS of 10ml is added in Reconstitute Thromboplastin D*, is put into 37 DEG C of water-baths Preheating.Reagent equivalent after preheating is added in blood plasma, is blown and beaten and is mixed with pipette tips.Mixed liquor after mixing is put into 37 DEG C of water 15 minutes in bath, react it sufficiently, 10000 × g is centrifuged 5 minutes.About 750 μ l supernatants are gently pipetted to new 1.5ml In EP pipe, for the extraction of next step excretion body, tube bottom white fiber sediment is discarded.

(4) every 500 μ l supernatant is added the excretion body precipitation solution of 125 μ l SBI companies, and is blown and beaten and mixed with pipette tips.To The RNaseA that Sigma company is added in the mixed liquor of previous step makes final 10 μ g/mL of enzyme concentration.4 DEG C of 12 hours of placement.With The RNase inhibitor of source of mouse is added in the concentration of 150units/mL, is blown and beaten several times up and down with pipette tips, until being uniformly mixed.Room temperature item Under part, 1500 × g is centrifuged 30 minutes, and excretion body precipitating reagent complex will be sink to EP pipe tube bottom, and supernatant abandoning is carefully drawn with pipette tips To waste liquid cylinder.With the cleaning tube wall that the sterile PBS of 500 μ l is careful, then draw into waste liquid cylinder.EP pipe is placed into centrifuge In, 1500 × g of room temperature is centrifuged 5 minutes, is moved to abandon and is left a small amount of residual liquid.Excretion nanocrystal composition is resuspended with the sterile PBS of 25 μ l.

(5) miRNA in excretion body is extracted using the miRNeasy Micro Kit of Qiagen company.Use Qubit 2.0 fluorimeters detect the concentration of miRNA, and then the small RNA chip of sampling Agilent is in 2100 bioanalysis of Agilent Instrument goes the size and concentration of detection excretion body miRNA.

(6) the multichannel tiny RNA library reagent preparation box construction cDNA library of NEBNext series is used, 6ng or so is small RNA is as the initial amount for building library, and recurring number is using 17 circulations.CDNA library is sequenced on channel according to same cannot contain phase The same principle for building library call number, every 24 samples are one group.According to 2100 bioanalysis of Agilent measurement miRNA concentration and Mean size calculates its molal weight, and the ratio of each sample equimolar quality is uniformly mixed, upper machine sequencing.Sequencing uses The HiSeq X10 sequenator of illumina, every 24 samples generate data volume (average each sample > 500 of about 120G or so Ten thousand reads).

(7) sequencing data is compared no duplicate human genome reference sequences (American National Biotechnology Information The GRh37/hg19 that center is established), comparison process uses Burrows-Wheeler (BWA) alignment parameters.One nucleotide is not The reading matched is not allowed to comparison process.Successful data are compared, the analysis in downstream is used for, to calculate each miRNA's Expression.Hsa.gff3 file is downloaded from miRbase database, and this document includes all at present to have found and delivered in the mankind miRNAs.Sequencing data is handled with SAMtools and BEDtools, calculates the expression quantity of the miRNA in each sample.Using The method of log2 (RPM+1) carrys out standardized data, the upper machine batch effect of data is removed with R software package Combat, from original number The miRNAs that 312 log2 (RPM+1) > 1 are picked in carries out subsequent analysis.

(8) 23 healthy volunteers and 5 CINI grades of patients are merged into one group and is known as 1 grade of Cervical intraepitheliaI neoplasia or less (CINI-) group.It is examined using displacement T to compare the miRNA of CINI- group and CINII-III group expression, identifying 61 has significantly The miRNA (p < 0.01) of difference.The miRNA for comparing CINI- group and ACC group and SCC group is expressed, and is found 13 and 7 respectively and is shown The miRNAs (FDR < 0.01) for writing differential expression, wherein having 4 miRNAs is to co-own.By 59 CINII-III patients and 34 cervical cancer patients merge into 2 grades of one group of Cervical intraepitheliaI neoplasia or more (CINII+) group, identify 37 differential expressions MiRNA, miRNA raised including 9 and 28 downwards.Using the algorithms selection best features vector of random forest, screening To 8 miRNA (let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182-5p, miR-183-5p, MiR-215-5p and miR-4443) it can best distinguish CINI- and CINII+ grouping.

(9) it is tested by the qRT-PCR to this 8 miRNA in independent 25 pairs of cervical cancer tissues and its adjacent normal tissue Card.Wherein there are 5 miRNAs (let-7d-3p, let-7a-3p, miR-30d-5p, miR-183-5p and miR-182-5p) and outer The variation tendency for secreting body miNRA is consistent, in addition to this, two miR-144-5p and miR-4443 with excretion body surface reach compared with, Inconsistent variation tendency is shown in tissue.MiR-215p-5p differential expression between cancerous tissue and cancer beside organism is not shown It writes (Fig. 3).It can be seen from the figure that 4 miRNA raised in CINII+ group, classifying quality is successively are as follows: let-7a-3p (FDR < 0.001, AUC=0.797), let-7d-3p (FDR < 0.001, AUC=0.887), miR-30d-5p (FDR < 0.001, ) and miR-215-5p (FDR < 0.001, AUC=0.804) (Fig. 3 A) AUC=0.89.Under other 4 significant in CINII+ group The miRNA of tune, classifying quality be successively miR-182-5p (FDR < 0.001, AUC=0.828), miR-183-5p (FDR < 0.001, AUC=0.797), miR-144-5p (FDR < 0.001, AUC=0.888) and miR-4443 (FDR < 0.001, AUC= 0.854), (Fig. 3 B).5 miRNAs (let- of independent cervical cancer tissues and the cancer beside organism qRT-PCR of pairing verifying discovery 7d-3p, let-7a-3p, miR-30d-5p, miR-183-5p and miR-182-5p) with the variation tendency one of excretion body miNRA It causes, miR-144-5p and miR-4443 show inconsistent variation tendency compared with excretion body surface reaches in the tissue.miR- 215p-5p differential expression between cancerous tissue and cancer beside organism is not significant (Fig. 3 C-3D)

(10) in 203 independent samples, (including 50 healthy volunteers, 34 CINI patients, 56 CINII-III suffer from Person, 63 CC patients) blood plasma excretion body in for verifying consistent 5 miRNAs of trend in overlying tissue sample pick 2 A highly expressed miRNAs carries out droplet type number ddPCR verifying.Sequencing data shows that miR-30d-5p and let-7d-3p exists It is remarkably decreased in CINII+ group, ddPCR experiment also demonstrates the downward trend (Fig. 4 A-B).MiR-30d-5p and let-7d-3p Independent sorting effect show 0.79 and 0.822 AUC value (Fig. 4).The expression of results of this 2 miRNAs of conjunctive use, ROC The AUC value of curve reaches 0.828 (95% confidence interval is 0.736-0.902), the expression knot of this 2 miRNAs of conjunctive use Fruit and cytology testing result, the AUC value of ROC curve reach 0.887 (Fig. 4 D).

What the kit was detected method particularly includes:

1. the separation of blood treatment, blood plasma excretion body and the extraction of excretion body RNA

Blood is collected in BD company ACD blood sampling tube pipe, is existed with the peripheral blood that the heparin tube containing ACD anti-coagulants is collected 1900 × g is centrifuged 10 minutes under the conditions of 4 DEG C in blood sampling 8 hours, then carefully draws light yellow supernatant, then under the conditions of 4 DEG C 16000 × g is centrifuged 10 minutes, and upper plasma is dispensed into the EP pipe of the free nucleic acid of 1.5ml.

Blood plasma is centrifuged 15 minutes removal cool insoluble proteins with 3000 × g, and 400 μ l supernatants is taken to be put into the EP for the seedless acid pollution that sterilizes Guan Zhong.The sterile PBS of 10ml is added in Reconstitute Thromboplastin D*, is put into 37 DEG C of water-baths pre- Heat.Reagent equivalent after preheating is added in blood plasma, is blown and beaten and is mixed with pipette tips.Mixed liquor after mixing is put into 37 DEG C of water-baths 15 minutes in pot, react it sufficiently, 10000 × g is centrifuged 5 minutes.About 750 μ l supernatants are gently pipetted to new 1.5ml EP Guan Zhong discards tube bottom white fiber sediment for the extraction of next step excretion body.

Reagent (SBI, EXOQ20A-1) is extracted using blood plasma excretion body and extracts serum excretion body, excretion is added in 500ul supernatant Body extracts reagent 125ul., and blown and beaten and mixed with pipette tips.The RNaseA that Sigma company is added in mixed liquor one step up makes most Whole enzyme concentration is 10 μ g/mL.4 DEG C of 12 hours of placement.The RNase inhibitor of source of mouse is added with the concentration of 150units/mL, uses Pipette tips are blown and beaten several times up and down, until being uniformly mixed.Under room temperature, 1500 × g is centrifuged 30 minutes, excretion body precipitating reagent complex It will be sink to EP pipe tube bottom, supernatant is carefully drawn with pipette tips and is abandoned to waste liquid cylinder.With the cleaning tube wall that the sterile PBS of 500 μ l is careful, so After draw into waste liquid cylinder.EP pipe is placed into centrifuge, 1500 × g of room temperature is centrifuged 5 minutes, is moved to abandon and is left a small amount of Liquid Residue Body.Excretion nanocrystal composition is resuspended with the sterile PBS of 25 μ l.

The miRNA in excretion body is extracted using the miRNeasy Micro Kit of Qiagen company, concrete operations are according to examination Agent box specification carries out.Use the concentration of Qubit 2.0 or 3.0 fluorimeters detection miRNA.

2. the DDPCR of blood plasma excretion body RNA is detected

Use let-7a-3p (SEQ IN NO.1), let-7d-3p (SEQ IN NO.2), miR-30d-5p (SEQ IN NO.3),miR-144-5p(SEQ IN NO.4),miR-182-5p(SEQ IN NO.5),miR-183-5p(SEQ IN NO.6), miR-215-5p (SEQ IN NO.7), miR-4443 (SEQ IN NO.8) and 4 internal reference let-7i-5p (SEQ IN NO.9), miR-128-3p (SEQ IN NO.10), miR-129-5p (SEQ IN NO.11), miR-320a (SEQ IN NO.12) specific probe (table 1) detects the expression of each miRNA in blood plasma excretion body.It is examined according to Qubit 2.0 or 3.0 fluorimeters The miRNA concentration of survey guarantees each by each Sample Dilution to the accordingly suitable concentration groped during preliminary experiment The positive number of drops of the ddPCR of miRNA is in credible range.Utilize Biorad QX200 droplet digital pcr system (ddPCR system System) carry out digital pcr amplification.Use the matched Biorad QX200 of dye method^TMddPCR^TMEvaGreen Supermix is surveyed Determine the expression value of miRNA in blood plasma excretion body.The system of each ddPCR reaction solution is 20 μ l, specifically matches such as table 2.By 20 μ l Mixed liquor pipettes in disposable drop generator tube, and 70 μ l drop formation oil are added in the corresponding position of each sample, uses QX200 Droplet generator generates the Water-In-Oil drop of about 40 μ l.40 μ l droplets of generation are moved into 96 orifice plates, after being sealed with aluminium film, 4 DEG C of avoid light place preservations.Mixed liquor is put into thermal cycler, is reacted as follows: 95 DEG C initial denaturation 5 minutes, 95 DEG C denaturation 30 seconds, 60 DEG C of annealing, which add, to be extended 1 minute, and above two step recycles 40 times, was then pre-chilled 5 minutes for 4 DEG C, 90 DEG C make enzyme inactivate 10 points Clock is finally arranged in 4 DEG C and saves to reinforce the stability of dyestuff.The warming and cooling rate of all above steps is maintained at 2 DEG C/s, compared with Slow warming and cooling rate is the consistency in order to guarantee Water-In-Oil drop heating and cooling.

1 miRNA primer sequence table of table

MiRNA title	Primer sequence	Remarks
			let-7a-3p	cgcgcgctatacaatctactgtctttc	Verify miRNA
let-7d-3p	gctatacgacctgctgcctttct	Verify miRNA
			miR-144-5p	ccgcgcgggatatcatcatatactgtaag	Verify miRNA
miR-182-5p	cgtttggcaatggtagaactcacact	Verify miRNA
			miR-183-5p	gcgtatggcactggtagaattcact	Verify miRNA
miR-215-5p	gcgcgatgacctatgaattgacagac	Verify miRNA
			miR-30a-5p	gcgtgtaaacatcctcgactggaag	Verify miRNA
miR-30d-5p	cgtgtaaacatccccgactggaag	Verify miRNA
			miR-4443	gcttggaggcgtgggtttt	Verify miRNA
let-7i-5p	gcgtgaggtagtagtttgtgctgtt	Internal reference miRNA
			miR-128-3p	cgtcacagtgaaccggtctcttt	Internal reference miRNA
miR-129-5p	ctttttgcggtctgggcttgc	Internal reference miRNA
			miR-320a	aaaagctgggttgagagggcg	Internal reference miRNA

2 DDPCR reaction system of table

1 reverse transcription cDNA template of table	1~8 μ l
		QX200ddPCR EvaGreen Supermix(2x)	10μl
The end the F primer (1 μM) of miRNA	1μl
		The end the R primer (1 μM) of miRNA	1μl
Sterile water	It is settled to 20 μ l

After the completion of amplification, the ratio of positive drop and negative drop, to calculate the absolute of miRNA in its initial sample Expression value.The end the R primer of digital pcr, it is proposed that cooperation Reverse Transcriptase kit is used together.

The result of more internal reference combination with standard methods is set as R_score value, the expression of each miRNA is evaluated with it.R_ The specific algorithm of score is as follows:

Wherein, R_score indicates the miRNA expression value of each verifying sample and the ratio of the sum of internal reference miRNAs expression value Value；Ddexp_test indicates the absolute expression value on the ddPCR of each verifying sample；Ddexp_con indicates each verifying sample 4 internal references ddPCR on absolute expression value.

Fig. 2 is 8 miRNA combinations (let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182- 5p, miR-183-5p, miR-215-5p, and miR-4443) in 121 samples classification capacity assessment.(A) principal component point Analysis, (B) clustering, (C) ROC curve.As a result, it has been found that two groups of samples of CINI- and CINII+ can be compartmentalized well.? In clustering, the sample of only 2 CINII+ groups is mistakenly allocated in CINI- group.Divided together using 8 miRNAs When class, ROC curve shows preferable classifying quality, and AUC value reaches 0.992 (95% confidence interval is 0.978-1).

Sequence table

<110>Hospital for Gynaecology and Obstetrics Attached to Medical Hospital of Zhejiang

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Claims (5)

1. a kind of novel blood plasma excretion body microRNA molecule marker, which is characterized in that blood plasma excretion body is derived from, wherein The microRNA is selected from the group: let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182-5p, MiR-183-5p, miR-215-5p and miR-4443, or combinations thereof, their RNA sequence is respectively such as SEQ ID NO:1~SEQ Shown in ID NO:8.

2. a kind of microRNA finger-print for the diagnosis of cervical carcinoma early screening, which is characterized in that the finger-print packet It includes and is selected from the group 2 kinds and the above microRNA:let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR- 182-5p, miR-183-5p, miR-215-5p and miR-4443.

3. a kind of application of the molecular labeling described in claim 1 in kit of the preparation for cervical carcinoma screening diagnosis.

4. application according to claim 3, which is characterized in that kit includes following marker: let-7a-3p, let- 7d-3p, miR-30d-5p, miR-144-5p, miR-182-5p, miR-183-5p, miR-215-5p and miR-4443.

5. application according to claim 4, which is characterized in that the kit includes to use following four internal reference: miR- 128-3p, miR-129-5p, miR-320a and let-7i-5p.