CN109055555A - A kind of lung cancer transfer diagnosis marker and its kit and application in early days - Google Patents
A kind of lung cancer transfer diagnosis marker and its kit and application in early days Download PDFInfo
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- CN109055555A CN109055555A CN201810983160.0A CN201810983160A CN109055555A CN 109055555 A CN109055555 A CN 109055555A CN 201810983160 A CN201810983160 A CN 201810983160A CN 109055555 A CN109055555 A CN 109055555A
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to medical diagnosis on disease prevention technique fields, specifically disclose a kind of for the lung cancer LncRNA ENSG00000270607 marker that transfer diagnoses in early days and its kit and application.The sequence of the LncRNA ENSG00000270607 is as shown in SEQ ID NO:1, it especially can be used as and distinguish the molecular marker whether patients with lung cancer shifts, kit provided by the invention detects quick and convenient, accuracy rate and high sensitivity, high specificity, the early stage transfer diagnosis and prediction that lung cancer may be implemented, can be widely used in the early stage transfer diagnosing and treating of medical industry lung cancer disease.
Description
Technical field
The present invention relates to medical diagnosis on disease prevention technique fields, more particularly, to a kind of lung cancer early stage transfer diagnostic markers
Object LncRNA ENSG00000270607 and its kit and application.
Background technique
Lung cancer is the highest malignant tumour of morbidity and mortality in worldwide.According to the World Health Organization (WHO)
" report of world's cancer " display issued recently, the newly-increased cases of lung cancer 1,800,000 in the whole world in 2012, accounts for the 13% of cancer morbidity,
The relevant death toll of lung cancer accounts for the nearly one third of the relevant death toll of all cancers.Between Past 30 Years, the lung in China
Mortality of carcinoma rises 46.5%, and disease incidence increases by 26.9% every year on average, every year on average nearly 400,000 deaths and lung
Cancer is related.Non-small cell lung cancer clinical therapeutic efficacy is still unsatisfactory at present, and prognosis is poor, and five year survival rate is only 10%
~15%.Even clinical data shows that the patient of IA phase also dies of recurrence and transfer after tumour is cut off completely mostly.Advanced stage
Having the patients with lung cancer of distant metastasis of human, then for five year survival rate less than 1%, median survival time is about only 13 months.Due to lung
The mechanism shortage of carcinogenesis is fully understood, and the diagnosis of lung cancer lacks always important effective early warning molecule.Therefore, lung cancer has become
China's increasing degree is maximum, endangers malignant tumour the most serious, and the recurrence and transfer of non-small cell lung cancer are its lethal masters
Want reason.Currently, spiral CT is the most common means for clinically detecting lung cancer, but there are the false positives of height ratio；And it is super
The patients with lung cancer for crossing 50% has been in advanced stage in diagnosis incipience, and the median survival time of Patients with Advanced Lung Cancer is less than 18
Month.Due to various reasons, existing molecular marker is difficult to enter clinical application for lung cancer early diagnosis.
Past is concentrated mainly on the gene of coding protein in genome about the research of cancer, and for non-coding region
To the regulating and controlling effect of cancer, there are also many still open questions.Long non-coding RNA (long non-coding RNA, referred to as
LncRNA it is) a kind of transcript for being defined as greater than 200 nucleotide, lacks the RNA molecule of protein coding potential.It has been reported that
LncRNA and the process for arriving kinds of tumors occurrence and development, and the processes such as the apoptosis of lncRNAs and tumour cell, transfer and drug resistance
It is closely related.In addition, can be sent out in the detection such as the Precancerous Lesion and lesion tissue of tumor patient, blood, urine and saliva
The lncRNA of existing tumour-specific prompts it can be as a kind of potential diagnosing tumor and prognostic factor, however lncRNA exists
Specific clinical meaning, function and mechanism in tumor development do not illustrate yet.
It is nearest the study found that circulating tumor cell and tumour relevant DNA and RNA are relatively stable in human peripheral,
It is used as the tool of in-vitro diagnosis.And blood sample is clinically easy to collect, and most to the extent of injury of patient
It is small.Therefore, the inner link between lncRNA and lung cancer is inquired into, to excavate novel lung cancer metastasis specificity marker in blood
Object, the method developed new early diagnosis and/or treat lung cancer is task very urgent in current lung cancer research.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the above-mentioned deficiency of the prior art, a kind of lung cancer early stage transfer is provided and is examined
Disconnected marker LncRNA ENSG00000270607, the marker LncRNAENSG00000270607 is in cancerous lung tissue
Expression is apparently higher than cancer beside organism, and whether the differentiation that the LncRNA ENSG00000270607 marker can be specific occurs
The lung cancer patient of transfer and normal person.
It is early in preparation lung cancer that another object of the present invention is to provide the LncRNA ENSG00000270607 markers
Phase shifts the application in diagnostic kit.
Another object of the present invention is to provide the primer pairs and probe of one group of detection LncRNA ENSG00000270607.
Another object of the present invention is to provide the primer pairs and probe to prepare the reagent for detecting other metastases
And/or the application in kit.
It is early in preparation lung cancer that another object of the present invention is to provide the LncRNA ENSG00000270607 markers
Application in phase diversion medicaments.
Another object of the present invention is to provide a kind of lung cancer early stage transfer diagnostic kits.
To achieve the goals above, the present invention is achieved by following scheme:
The present invention separates from human blood and detects lung cancer early stage transfer specificity LncRNA ENSG00000270607
In significantly high expression.In addition, LncRNA ENSG00000270607 does not shift patient's to differentiation lung cancer metastasis patient and lung cancer
For high sensitivity up to 90.53%, specificity is up to 88.13%.Therefore, LncRNA ENSG00000270607 is a kind of novel lung
Metastasis of cancer blood molecules diagnostic marker can significantly improve accuracy, the sensibility and specificity of diagnosis.
Therefore, nucleotide sequence LncRNA as shown in SEQ ID NO:1 is claimed in the present invention
Application of the ENSG00000270607 as marker in lung cancer early stage transfer diagnosis.
Nucleotide sequence LncRNA ENSG00000270607 as shown in SEQ ID NO:1 mark is also claimed in the present invention
Application of the will object in preparation lung cancer early stage transfer diagnostic kit.
The present invention be also claimed the specificity amplification primer of one group of detection LncRNA ENSG00000270607 to and visit
Needle, nucleotide sequence is successively as shown in NO:2~4 SEQ ID；5 ' ends of the Taqman probe are combined with fluorescent material
FAM, 3 ' ends, which are combined with, is quenched substance BHQ1.
LncRNA ENSG00000270607-F (SEQ ID NO:2):
5'-TGGGCAAGTTATTTAAGC-3'；
LncRNA ENSG00000270607-R (SEQ ID NO:3):
5'-CAACCATCTCATTTAATCCA-3'；
LncRNA ENSG00000270607 fluorescence probe (SEQ ID NO:4):
5’-FAM-AATGTCGTCAAGCCTCCACCT-BHQ1-3’。
It preferably, further include primer pair and probe for expanding reference gene ACTB, nucleotide sequence is successively such as SEQ
Shown in NO:5~7 ID, 5 ' ends of the Taqman probe are combined with fluorescent material CY5, and 3 ' ends, which are combined with, is quenched substance BHQ2.
ACTB-F (SEQ ID NO:5): 5 '-TCACCCACACTGTGCCCATCTACGA-3 '；
ACTB-R (SEQ ID NO:6): 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 '；
ACTB fluorescence probe (SEQ ID NO:7):
5’-CY5-ATGCCCTCCCCCATGCCATCCTGCGT-BHQ2-3’。
Meanwhile the application of above-mentioned primer pair and probe in preparation lung cancer early stage transfer diagnostic kit is also protected in the present invention
It protects in range.
A kind of lung cancer early stage transfer diagnostic kit, the kit include qualitative or quantitative detection
The reagent of LncRNAENSG00000270607 expression quantity.
Preferably, the reagent is the primer pair and probe of specific amplification LncRNA ENSG00000270607, including
Upstream primer F and downstream primer R and probe, successively as shown in NO:2~4 SEQ ID, the Taqman is visited nucleotide sequence
5 ' ends of needle are combined with fluorescent material FAM, and 3 ' ends, which are combined with, is quenched substance BHQ1.
LncRNA ENSG00000270607-F (SEQ ID NO:2):
5'-TGGGCAAGTTATTTAAGC-3'；
LncRNA ENSG00000270607-R (SEQ ID NO:3):
5'-CAACCATCTCATTTAATCCA-3'；
LncRNA ENSG00000270607 fluorescence probe (SEQ ID NO:4):
5’-FAM-AATGTCGTCAAGCCTCCACCT-BHQ1-3’。
Preferably, the kit include further include primer pair and probe for expanding reference gene ACTB, including it is upper
Swim primers F and downstream primer R and probe, nucleotide sequence is successively as shown in NO:5~7 SEQ ID, the Taqman probe
5 ' ends be combined with fluorescent material CY5,3 ' ends, which are combined with, is quenched substance BHQ2.
ACTB-F (SEQ ID NO:5): 5 '-TCACCCACACTGTGCCCATCTACGA-3 '；
ACTB-R (SEQ ID NO:6): 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 '；
ACTB fluorescence probe (SEQ ID NO:7):
5’-CY5-ATGCCCTCCCCCATGCCATCCTGCGT-BHQ2-3’。
Preferably, the kit also contains positive control solution and negative controls；The positive control solution is LncRNA
ENSG00000270607 standard items；The negative controls are no template blank control.
It is further preferred that the kit also contain RNA reverse transcription needed for reagent.
Preferably, the quantitative fluorescent PCR reaction system is grouped as by each group of following volumes: 5 μ of TaqmanPremix
2 μ L, cDNA template of L, Taqman probe, 3 μ L, total system are 10 μ L.
Preferably, the quantitative fluorescent PCR reaction condition are as follows: response procedures be 95 DEG C of 10min, 95 DEG C of 15s, 55 DEG C
1min, 40 circulations.
The application method of mentioned reagent box includes the following steps:
S1. blood is acquired, the total serum IgE in blood plasma is provided；
S2. by total serum IgE reverse transcription at cDNA；
S3. the expression quantity of real-time quantitative PCR detection LncRNA ENSG00000270607；
Measure area AUC under the ROC curve of sample；Complete unworthy diagnostic test AUC is 0.5, ideal diagnosis examination
Testing AUC is 1；It is generally believed that diagnostic value is lower when AUC is between 0.5~0.7, with certain when between 0.7~0.9
Diagnostic value, at 0.9 or more, diagnostic value is higher.
Compared with prior art, the invention has the following advantages:
High expression is presented in marker LncRNA ENSG00000270607 of the present invention in blood samples of patients, being capable of conduct
The molecular marker of lung cancer early stage transfer diagnosis, especially can be used as and distinguishes the molecular marked compound whether patients with lung cancer shifts, spirit
Sensitivity is 90.53%, specificity 88.13%, reliability with higher.Kit detection provided by the present invention is quickly square
Just, accuracy rate and high sensitivity, high specificity may be implemented the early stage transfer diagnosis and prediction of lung cancer, can be widely used in curing
The early stage for treating industry lung cancer disease shifts diagnosing and treating.
Detailed description of the invention
Fig. 1 is to detect LncRNA in the Serum of Patients with Lung Cancer of non-diverting patient and transfer by qPCR
The expression quantity of ENSG00000270607.
Fig. 2 is to indicate LncRNA ENSG00000270607 to the diagnostic value of lung cancer metastasis patient.
Fig. 3 is the high expression by cell vitro invasion testing inspection LncRNA ENSG00000270607 to non-small cell
The ability of lung carcinoma cell vitro invasion.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The verifying of 1 lung cancer metastasis characteristic LncRNA ENSG00000270607 of embodiment
Numerous studies discovery of the present inventor team by early period, LncRNA shown in SEQ ID NO:1
ENSG00000270607 is related to lung cancer metastasis diagnosis, can be used as lung cancer early stage transfer blood molecules diagnosis marker.It is sharp below
With to 2017 tumour hospital of Nian9Yue Jian Zhongshan University outpatient services or in hospital 154 Patients with Non-small-cell Lung from June, 2017
Serum specimen carries out sonde method PCR detection to LncRNA ENSG00000270607, thus to lung cancer metastasis characteristic LncRNA
ENSG00000270607 is verified.Specifically comprise the following steps:
1, the measurement of plasma sample total serum IgE (TRIZOL LS method) and RNA concentration is extracted
(1) sample homogenizes: taking 0.25mL sample that 0.75ml TRIzol LS reagent is added, is blown up and down with pipettor even several
It is secondary, keep sample uniform；The biofluid high for the polluters content such as whole blood is first diluted with no RNase water by 1:1；Work as sample
When less than 0.25mL, for convenience of RNA is extracted, volume can be supplemented with RNase water to 0.25mL.
(2) it mutually separates: the sample after homogenizing being stored at room temperature 5 minutes, so that ribosome divides completely in homogenised sample
From.Chloroform is then added for the ratio of 0.75:0.2 with TRIzol LS reagent and chloroform volume ratio, covers tightly the lid of centrifuge tube,
It is placed in concussion instrument and shakes 30 seconds, stand 2~15 minutes at room temperature.Then at 4 DEG C, 12000rpm is centrifuged 15 minutes.Centrifugation
Afterwards, mixture is separated into 3 layers, and upper layer is clear water phase, and middle layer, lower layer is red phenol-chloroform phase, and RNA is present in water
Xiangli, the TRIzol LS amount of reagent that water phase volume uses when being about 70% homogeneous.
(3) RNA precipitate: the water phase after separation is transferred to a new pipe, is added by 0.75mL TRIzol LS reagent
The ratio of 100% isopropanol of 0.5mL is added in water phase, and the centrifuge tube mixing isopropanol that repeatedly turns upside down from water phase to precipitate
Then RNA stands sample 30 minutes in -20 DEG C of environment to precipitate RNA, is finally centrifuged 15 in 4 DEG C, 12000rpm centrifuge
Minute.
(4) RNA is washed: the supernatant in centrifuge tube being outwelled, 1mL is added with every 0.75mL TRIzol LS reagent
The ratio of 75% ethyl alcohol washs RNA precipitate, and be then vortexed concussion, mixing sample, with 7500rpm revolving speed in last 4 DEG C of centrifuges
Supernatant is abandoned in centrifugation 5 minutes.
(5) RNA is resuspended: by the sample vacuum after washing or being air-dried 5~10min of RNA precipitate, then uses nothing
RNA precipitate is resuspended in RNase water (20~50 μ L), and pipettor gently blows solution several times up and down, then in 55~60 DEG C of water bath
10~15min of middle incubation is kept in -40 or -80 DEG C of refrigerators.
(6) RNA is quantitative: first after the Nucleic Acid module of starting microplate reader Nanodrop 1000, mentioning according to screen
Show and cleans pedestal without RNA enzyme water with 2 μ L；Then RNA40 mode is selected, compares blank solution Blank with 2 μ L；2 μ L are then added
RNA solution Measure.It is finally detected with microwell plate quantitative system: sharing 24 holes, three holes that the rightmost side one arranges are sky
White control, remaining 21 holes can be added RNA sample and be detected；Slide is covered after sample-adding, is put into support plate, and DNA_ is used
RNA quantification program readings.
(7) quantitative result interpretation: the A260/280=2 of pure rna, when A260/280 be between 1.7~2 it is normal, when
Show there is protein or phenol pollution when A260/280 is less than 1.7；Show there may be isothiocyanic acid residual when A260/280 is greater than 2.0
It deposits.A230 indicates that there are some pollutants, such as carbohydrate, salt (guanidine salt), purer nucleic acid A260/A230 in sample
Ratio be greater than 2.0.
2, the reverse transcription of total serum IgE
(1) MMLVReverse Transcriptase (Promega) reverse transcription is used, reaction system described in allocation list 1:
1 reverse transcription reaction system of table
| Component | Volume |
| Random Primer(10μM) | 1μL |
| RNA template | 1μg |
| RNase-free H2O | Surplus |
| Total volume | 13μL |
It is placed in PCR instrument, 70 DEG C, 5 minutes, cools down 5min on ice immediately after.
(2) reaction system described in table 2 in above-mentioned reaction system carries out reverse transcription reaction:
2 reverse transcription reaction system of table
It after mixing gently, is placed in PCR instrument, 40 DEG C, 60 minutes；70 DEG C, 5 minutes；Product is reversed to be placed in 4 DEG C of of short duration guarantors
It deposits, or carries out subsequent operation.
3, the expression quantity of real-time quantitative PCR detection LncRNA ENSG00000270607
(1) real-time quantitative PCR detection primer and probe
LncRNA ENSG00000270607-F (SEQ ID NO:2):
5'-TGGGCAAGTTATTTAAGC-3'；
LncRNA ENSG00000270607-R (SEQ ID NO:3):
5'-CAACCATCTCATTTAATCCA-3'；
LncRNA ENSG00000270607 fluorescence probe (SEQ ID NO:4):
5’-FAM-AATGTCGTCAAGCCTCCACCT-BHQ1-3’。
Internal reference ACTB:
ACTB-F (SEQ ID NO:5): 5 '-TCACCCACACTGTGCCCATCTACGA-3 '；
ACTB-R (SEQ ID NO:6): 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 '；
ACTB fluorescence probe (SEQ ID NO:7):
5’-CY5-ATGCCCTCCCCCATGCCATCCTGCGT-BHQ2-3’。
(2) real-time quantitative PCR
Real-time quantitative PCR reaction uses Roche480Probe Master real-time fluorescence quantitative PCR system,
Utilize CFX96TMReal-Time PCRDetection Systems (C1000Touch PCR instrument, Bio-rad) is operated.
The amplified production length of quantitative PCR is the most suitable (can extend to 300bp) with 80bp~150bp.Reaction system such as 3 institute of table
Show:
3 quantitative fluorescent PCR reaction system of table
Reaction condition: 95 DEG C of 15min；95 DEG C of 15s, 60 DEG C of 45s (acquisition signal), repeat 40 circulations；4℃∞.
(3) data are analyzed: this experiment uses relative quantitative analysis method.Each sample does three multiple holes, as a result takes three
The mean CT-number of multiple holes.LncRNA mean CT-number-reference gene mean CT-number of △ Ct=mesh；△ △ Ct=△ Ct transfer-△
Ct is not shifted；Relative expression quantity=log2^ (- △ △ Ct).This kit uses ATCB for internal reference, calculates LncRNA
The relative expression quantity of ENSG00000270607.Data are analyzed with SPSS20.0 software, with target lncRNA relative expression quantity
For independent variable, group is dependent variable, establishes the Logistic regression model of lung cancer early stage transfer diagnosis, the degree of fitting of regression model
Using likelihood ratio test, regression parameter estimated value uses non-parametric test method, and according to ROC curve and area under the curve (AUC) is commented
Estimate the sensitive experiment result of target lncRNA diagnosis.
4, the verifying of lung cancer metastasis characteristic LncRNA ENSG00000270607
(1) expression of the target lncRNA in test set sample
The quantitative expression result of LncRNA ENSG00000270607 is as shown in the figure: with non-diverting group of ratio of lung cancer, lung cancer turns
Blood plasma LncRNA ENSG00000270607 significantly raises (P < 0.05) with respect to internal reference expression quantity in shifting group.
(2) ROC curve of target lncRNA is analyzed in test set
With the phase of LncRNA ENSG00000270607 in non-diverting group of all samples of test set lung cancer metastasis group and lung cancer
To internal reference expression quantity as independent variable (setting X=LncRNENSG00000270607 relative expression quantity), using group as dependent variable
(lung cancer metastasis group is set as 1,0) non-diverting group of lung cancer sets, and draws ROC curve accordingly.
Target lncRNA concentrates the effect of diagnosing transfer as shown in table 4 in verifying, and ROC curve is as shown in Fig. 2, ROC
Area under the curve is 0.818, and best cutoff value is 2.16 (diagnosing wealthy value), at best cut off value sensitivity be
90.53%, specificity is 88.13%.Area AUC refers to as the intrinsic accuracy of diagnostic test Authentic Assessment under ROC curve
It marks oneself to be commonly recognized, complete unworthy diagnostic test AUC is 0.5, and ideal diagnostic test AUC is 1.It is generally believed that AUC
Diagnostic value is lower when between 0.5~0.7, with certain diagnostic value when between 0.7~0.9, examines at 0.9 or more
Disconnected value is higher.Therefore, LncRNA ENSG00000270607 diagnosing transfer has certain diagnostic value.
4 target lncRNA of table concentrates the effect of diagnosing transfer in verifying
2 cell vitro invasion testing inspection LncRNA ENSG00000270607 of embodiment is to non-small cell lung cancer cell body
The influence of outer invasive ability
(1) pre-treatment of sample
The Matrigel (BD company) for being stored in -80 DEG C is taken out first, is put in ice chest and is embedded with ice, then by ice chest
Be put in 4 DEG C of chromatography cabinets makes the Matrigel of frost thaw overnight；Take the Matrigel to have thawed and basal medium (i.e. not
Culture medium containing fetal calf serum) with VMatrigel/VBasal mediumMixed liquor is packed into volume after sufficiently diluting by=1/9 ratio
In the EP pipe of 1.5mL and it is inserted on ice.
(2) cell vitro invasion testing inspection
Cell (24Well Transwell Permeable Supports) is placed in 24 orifice plates, takes 50 μ L above-mentioned dilute
The mixed liquor released uniformly is laid on cell upper layer bottom and pays attention to being sure not to generate bubble；37 DEG C of incubators stand 0.5~2 hour, pass through
Shaking observation, whether it solidifies；Will be ready cells trypsinised and count, culture medium is removed by centrifugation, takes it
In 2 × 104A cell is resuspended to 200 μ L with basal medium and is added to ready cell upper layer in 24 orifice plates, to 24 orifice plates
The basal medium for containing 10% fetal calf serum is added in interior cell lower layer；37 DEG C after incubator culture 24 hours, utilize liposome to turn
It is transferred to pSIN-Vector control plasmid and pSIN-LncRNA ENSG00000270607 plasmid respectively in dye normal direction cell, 36 is small
When after take out cell and absorb base foundation culture medium with big pipette tips, be slowly added into phosphate buffer washing 2 times, and with small cotton swab
The heart wipes the cell Matrigel of cell upper layer bottom, acts soft in order to avoid destroying cell film；With methanol/glacial acetic acid (VMethanol/
VGlacial acetic acid=3/1) mixed liquor fixes the cell on 10~15 minutes cell films, is then slowly added into phosphate buffer and washs 1 time,
Crystal violet is redyed 10~15 minutes, uses tap water rinse, then to remove extra blue；Finally it is placed in microscope (brightfield mode
That is BF mode) under observe, take pictures and take figure；When counting statistics result, count on as far as possible all cells across cell film or
Randomly select that 10 visuals field are for statistical analysis, with guarantee result it is objective effectively.
(3) result
As a result as shown in figure 3, high expression LncRNA ENSG00000270607 group passes through the non-small of cell film than control group
The quantity of cell lung cancer cell increased significantly, and show that the high expression of LncRNA ENSG00000270607 promotes non-small cell lung cancer
Cells in vitro invasive ability.
Diagnostic value of the embodiment 3LncRNA ENSG00000270607 to lung cancer metastasis patient
A kind of lung cancer early stage transfer diagnostic kit, including for detecting LncRNA ENSG00000270607 marker
Specificity amplification primer to and probe, and primer pair and probe for expanding reference gene, including upstream primer F and downstream
Primer R and probe.The amplimer of the LncRNA ENSG00000270607 to and probe and reference gene primer pair
Distinguish with probe as follows:
LNCRNA ENSG00000270607:
LNCRNA ENSG00000270607-F (SEQ ID NO:2):
5'-TGGGCAAGTTATTTAAGC-3'；
LNCRNA ENSG00000270607-R (SEQ ID NO:3):
5'-CAACCATCTCATTTAATCCA-3'；
LNCRNA ENSG00000270607 fluorescence probe (SEQ ID NO:4):
5’-FAM-AATGTCGTCAAGCCTCCACCT-BHQ1-3’。
Internal reference ACTB:
ACTB-F (SEQ ID NO:5): 5 '-TCACCCACACTGTGCCCATCTACGA-3 '；
ACTB-R (SEQ ID NO:6): 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 '；
ACTB fluorescence probe (SEQ ID NO:7):
5’-CY5-ATGCCCTCCCCCATGCCATCCTGCGT-BHQ2-3’。
The Taqman mixed liquor includes following components: primer pair, probe, dNTP, magnesium ion and other salt ions etc..
The quantitative fluorescent PCR reaction condition are as follows: response procedures are 95 DEG C of 15min, 95 DEG C of 15s, 60 DEG C of 45s (acquisition letters
Number), repeat 40 circulations.
The kit also contains positive control solution (i.e. LncRNA ENSG00000270607 standard items), negative controls
(i.e. blank control).
The kit also includes MMLV Reverse Transcriptase (Promega) reverse transcription system；The RNA
Reverse transcription reaction system is made of following each component: 1 μ L Random Primer, 1 μ g RNA template and surplus RNase-
free H2O complements to 13 μ L.
It also needs that following reagent is added in the reverse transcription reaction system:
5μL M-MLV 5X Reaction Buffer、5μL dNTP Mix(2.5mM)、1μL M-MLV RT、0.5μ
LRNase Inhibitor(40U/μL)、0.5μLRNase-free H2O complements to 25 μ L.
The reverse transcription reaction condition is 40 DEG C, 60 minutes；70 DEG C, 5 minutes.
Mentioned reagent box can be used for the expression quantity of real-time quantitative PCR detection LncRNA ENSG00000270607.Above-mentioned examination
The application method of agent box includes the following steps:
S1. blood is acquired, the total serum IgE in blood plasma is provided；
S2. by total serum IgE reverse transcription at cDNA；
S3. the expression quantity of real-time quantitative PCR detection LncRNA ENSG00000270607；
Measure area AUC under the ROC curve of sample；Complete unworthy diagnostic test AUC is 0.5, ideal diagnosis examination
Testing AUC is 1；It is generally believed that diagnostic value is lower when AUC is between 0.5~0.7, with certain when between 0.7~0.9
Diagnostic value, at 0.9 or more, diagnostic value is higher.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Zhongshan University
<120>a kind of lung cancer early stage transfer diagnosis marker and its kit and application
<141> 2018-08-27
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1686
<212> DNA
<213>people (Homo sapiens)
<400> 1
ggggggccca cacccctgcg ttcctgaaac ccttcttttg aaaaatgccc tttcgaacgc 60
aaccgtccgc cccagagcca cgcgggcacg gctcgaggga agtttgtttc ctttttcaaa 120
ttgctccggt caccttaacg gtttgagtct tccggagtca cgcccgcagc ctcgctgccc 180
ccatctggct tgggcagggc cgaagctcgt ctcccgcttt aagagcgcgc cagccatttc 240
tctgcctgct cttccaggcc ggctgggcgg ggggcctccc aacggcttcc tctccagttt 300
tcaaaggctg cacatttcac ttaactattt gaagtattta taggcaaata caatacgcag 360
ggaagcttcc cgccccgaga cagctcctcc acgcaaccta ccacagactc tgtaattaga 420
gtggcctgaa gaagccggga gaggctccat gaccccggca ggctctggcc aaaccttgct 480
gcgtttcatg gagcccctcc ctgtccgcgc ctgggacgca ctggccccaa caggttcctc 540
gactcgaatt cgctgtttgg gttgctctgt ggtttcctcg ctgactgaaa gtggatgaga 600
ataagaagga aactgagaga tgatcaggca ggccagcctt cgcgatttat gaatgaggag 660
cctcccaccc tgtagaactg cttggattag aagtgggcaa gttatttaag cttacatgca 720
tgcttataaa gcttctgttt tcacatgtag taaggtggag gcttgacgac atttacctaa 780
tgagctgttg tttggattaa atgagatggt tggtgcatgg cagtcattta gcacaagtac 840
ctggcaatgt aagagaccaa taaaagctgg gctttatttt ccctgttgtt aaattactgc 900
tgctttcccc caaagtcatc tctgtttata aactatttgt ctttcttctt tgtttccagg 960
acactttgtg cccaccctat agcctgatct tcagtgctga tagcattgat ttagaataac 1020
tcagatgcta ttaactctgt ccccctggaa ttcatgttaa acccgtaatc cccagtgtga 1080
tggtattagg aggtggggaa ctttaggagg taataaggtc aagacagtgg agccctcatg 1140
ataggattag cactcttatt agaagagaca ggagagagat gctttcttac tgccttgtga 1200
ggacacagaa ggcagtcttc tgtggaccag gaagagggcc cttaccaaga atctgatacc 1260
gtgttggcat cctagtctca gacttccagc ctctagtact gtaagaaata aatgtttgtt 1320
gtttaagcca tcctgtttat ggtatttttg ttatagtggc ccaaagtgac aaagatctca 1380
gaccagagga aacatcaaag tcttaacctc tgaagcatta agaaatgaga aggcaccttc 1440
ttagccattc taggtagcaa ctttctactc tagttaggct gaaatgcacg gccctgtcta 1500
tgtctgtttc agttatcctc cctctctctg aaatcaagtc acacatcatt catgctactt 1560
acttaacact ttgccctttg ccatctgatg ccacctcccg tactattgtc tcccattatt 1620
taacctgtta tttattgttt catgcattca ttttcatctt agagaataaa catcttggaa 1680
ttagga 1686
<210> 2
<211> 18
<212> DNA
<213>people (Homo sapiens)
<400> 2
tgggcaagtt atttaagc 18
<210> 3
<211> 20
<212> DNA
<213>people (Homo sapiens)
<400> 3
caaccatctc atttaatcca 20
<210> 4
<211> 21
<212> DNA
<213>people (Homo sapiens)
<400> 4
aatgtcgtca agcctccacc t 21
<210> 5
<211> 25
<212> DNA
<213>people (Homo sapiens)
<400> 5
tcacccacac tgtgcccatc tacga 25
<210> 6
<211> 25
<212> DNA
<213>people (Homo sapiens)
<400> 6
cagcggaacc gctcattgcc aatgg 25
<210> 7
<211> 26
<212> DNA
<213>people (Homo sapiens)
<400> 7
atgccctccc ccatgccatc ctgcgt 26
Claims (9)
1. nucleotide sequence LncRNA ENSG00000270607 as shown in SEQ ID NO:1 is as marker in lung cancer early stage
Application in transfer diagnosis.
2. nucleotide sequence LncRNA ENSG00000270607 marker as shown in SEQ ID NO:1 is in preparation lung cancer early stage
Shift the application in diagnostic kit.
3. one group of primer pair and probe for specific amplification detection LncRNA ENSG00000270607 marker, feature
It is, including upstream primer F, downstream primer R and probe, nucleotide sequence is described successively as shown in NO:2~4 SEQ ID
5 ' ends of probe are combined with fluorescent material FAM, and 3 ' ends, which are combined with, is quenched substance BHQ1.
4. application of the primer pair and probe described in claim 3 in preparation lung cancer early stage transfer diagnostic kit.
5. a kind of lung cancer early stage transfer diagnostic kit, which is characterized in that include qualitative or quantitative detection LncRNA
The reagent of ENSG00000270607 expression quantity.
6. kit according to claim 5, which is characterized in that the reagent is specific amplification LncRNA
The primer pair and probe of ENSG00000270607, including upstream primer F, downstream primer R and probe, nucleotide sequence is successively
As shown in NO:2~4 SEQ ID, 5 ' ends of the probe are combined with fluorescent material FAM, and 3 ' ends, which are combined with, is quenched substance BHQ1.
7. kit according to claim 5, which is characterized in that also containing the primer pair and spy for expanding reference gene
Needle, including upstream primer F, downstream primer R and probe, nucleotide sequence is successively as shown in NO:5~7 SEQ ID, the spy
5 ' ends of needle are combined with fluorescent material CY5, and 3 ' ends, which are combined with, is quenched substance BHQ2.
8. kit according to claim 5, which is characterized in that also contain positive control solution and negative controls.
9. kit according to claim 5, which is characterized in that also containing reagent needed for RNA reverse transcription.
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Cited By (4)
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| CN112813167A (en) * | 2021-02-08 | 2021-05-18 | 江苏省肿瘤医院 | Marker LINC01977 and application thereof |
| CN113151469A (en) * | 2021-04-19 | 2021-07-23 | 温州医科大学 | Tumor classification marker combination and application thereof |
| CN113355413A (en) * | 2020-03-02 | 2021-09-07 | 南京腾辰生物科技有限公司 | Application of molecular marker and kit in auxiliary diagnosis of cancer |
| CN114150066A (en) * | 2020-03-30 | 2022-03-08 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113355413A (en) * | 2020-03-02 | 2021-09-07 | 南京腾辰生物科技有限公司 | Application of molecular marker and kit in auxiliary diagnosis of cancer |
| CN113355413B (en) * | 2020-03-02 | 2024-04-30 | 腾辰生物科技(上海)有限公司 | Application of molecular marker and kit in auxiliary diagnosis of cancer |
| CN114150066A (en) * | 2020-03-30 | 2022-03-08 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN114277138A (en) * | 2020-03-30 | 2022-04-05 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN114317745A (en) * | 2020-03-30 | 2022-04-12 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN114317745B (en) * | 2020-03-30 | 2022-06-07 | 中国医学科学院肿瘤医院 | Application of exosomes ARPC5, ERI3 and the like in lung cancer diagnosis |
| CN114277138B (en) * | 2020-03-30 | 2022-06-24 | 中国医学科学院肿瘤医院 | Application of exosomes ARPC5, MBOAT2 and the like in lung cancer diagnosis |
| CN112813167A (en) * | 2021-02-08 | 2021-05-18 | 江苏省肿瘤医院 | Marker LINC01977 and application thereof |
| CN113151469A (en) * | 2021-04-19 | 2021-07-23 | 温州医科大学 | Tumor classification marker combination and application thereof |
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