CN108570500A - The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method - Google Patents
The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method Download PDFInfo
- Publication number
- CN108570500A CN108570500A CN201810392823.1A CN201810392823A CN108570500A CN 108570500 A CN108570500 A CN 108570500A CN 201810392823 A CN201810392823 A CN 201810392823A CN 108570500 A CN108570500 A CN 108570500A
- Authority
- CN
- China
- Prior art keywords
- mir
- cephalantha
- marker
- primer
- quantitative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
-
- 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 the primer combination for detecting G. cephalantha marker, include the forward primer quantitatively detected for miR 10b, the forward primer that quantitatively detects for miR 31, at least one of forward primers quantitatively detected of miR 24.Include the quantitative detecting reagent for quantitatively detecting at least one of miR 10b, 31 miR, miR 24 the invention also discloses G. cephalantha marker detection kit.The invention also discloses a kind of quantitative detecting methods of G. cephalantha marker.
Description
Technical field
The present invention relates to biotechnologies, are tried more particularly to a kind of combination of G. cephalantha marker primer, detection
Agent box and its quantitative detecting method.
Background technology
G. cephalantha (Squamous Cell Carcinoma of Head and Neck, SCCHN) is world wide
Interior 6th big kinds of tumor.G. cephalantha includes laryngocarcinoma, carcinoma of mouth, nasopharyngeal carcinoma, hypopharyngeal cancer etc..2010, U.S.'s disease control
The statistical result at center shows that global annual about 500,000 people suffer from head and neck scale carcinoma, and death toll is about 12000 people.In recent years, although
Huge progress is achieved including the various therapies such as operation, chemicotherapy, but due to many considerable factors such as technical merit
Limitation, many tumours have been developed to middle and advanced stage when finding, treatment means limitation, therapeutic effect and prognosis are poor, survival rate one
Straight relatively low, 5 years survival rates of G. cephalantha are not significantly improved, still 60% or so.And infantile tumour is small, it is less
Transfer, tumor development can effectively be controlled by such as carrying out treatment in due course, and cure rate actively develops the early stage of tumour up to 83%
It was found that, diagnosis and early treatment research, have great significance for the prevention and control of cancer.
Clinically, the detection of peripheral blood and other body fluid is small, repeatable with wound, can be examined in same time point
The advantages that surveying many index is the promising approach of disease early diagnosis.It is answered in the early prevention of the cancers such as lung cancer, liver cancer and diagnosis
In, the detection of peripheral blood tumor markers has become important clinical foundation, as alpha-fetoprotein, cancer antigen 125, people's cancer embryo are anti-
Original etc..With the relevant serum of oral squamous cell carcinoma (OSCC) and saliva tumor markers such as Cyfra21-1, TPS, CEA, SCC,
Also there are many researchs by CA125 and CA19-9, however it is in G. cephalantha disease diagnoses, due to lacking sensitivity and specificity,
It is not used widely.
Therefore, neck can be opened up as the tumor markers and its detection method that G. cephalantha diagnoses by finding
The new method of portion's squamous carcinoma early diagnosis, has urgent clinical demand and wide application prospect.
Invention content
Based on this, it is necessary to provide a kind of combination of the G. cephalantha marker primer that simplicity uses, detection kit and
Its quantitative detecting method.
A kind of primer combination for detecting G. cephalantha marker includes the forward direction quantitatively detected for miR-10b
Primer, the forward primer quantitatively detected for miR-31, at least one of miR-24 forward primers quantitatively detected.
In one of the embodiments, primer combination include the forward primer quantitatively detected for miR-10b, it is described
Two or three in the forward primer that is quantitatively detected for miR-31, the forward primer quantitatively detected for miR-24
Combination.
The sequence such as SEQ ID of the forward primer quantitatively detected for miR-10b in one of the embodiments,
Shown in NO.1.
The sequence such as SEQ ID of the forward primer quantitatively detected for miR-31 in one of the embodiments,
Shown in NO.2.
The sequence such as SEQ ID of the forward primer quantitatively detected for miR-24 in one of the embodiments,
Shown in NO.3.
A kind of G. cephalantha marker detection kit, including be used to quantitatively detect miR-10b, miR-31, miR-24
At least one of quantitative detecting reagent.
The quantitative detecting reagent includes described for detecting G. cephalantha marker in one of the embodiments,
Primer combination.
The quantitative detecting reagent further includes reverse primer in one of the embodiments, the sequence of the reverse primer
As shown in SEQ ID NO.4.
The quantitative detecting reagent further includes PCR reaction reagents in one of the embodiments, the PCR reaction reagents
Including reverse transcription PCR reaction reagent and real-time quantitative PCR reaction reagent.
A kind of quantitative detecting method of G. cephalantha marker, includes the following steps：
The detected sample of predetermined amount is taken, total serum IgE is extracted；
The total serum IgE is mixed with reverse transcription PCR reaction reagent, it is anti-to carry out reverse transcription PCR using the total serum IgE as template
It answers, obtains cDNA；
By the primer combination for being used to detect G. cephalantha marker and reverse primer, the cDNA and reality
When quantitative PCR reaction reagent mix, be template progress quantitative PCR reaction using the cDNA, miR-10b described in quantitative amplification,
MiR-31 or miR-24；And
The result of quantitative PCR reaction is analyzed.
The detected sample is serum or blood plasma in one of the embodiments,.
Using miR-10b, miR-31 or miR-24 as G. cephalantha marker miR- is utilized using total serum IgE as template
The primer that the primer and miR-24 that primer that 10b is quantitatively detected, miR-31 are quantitatively detected quantitatively detect, carries out PCR reactions, passes through
The expression quantity of miR-10b, miR-31 or miR-24 are analyzed, can as G. cephalantha early diagnose judgement according to
According to winning valuable rescue time for clinic diagnosis.Using the G. cephalantha marker detection kit as pre- gauge head
The external detection method of neck squamous cell carcinoma morbidity property, has the advantages that of low cost, easy to operate, high specificity, or even can be real
Existing Non-invaive examination.
Description of the drawings
Fig. 1 be one embodiment of the invention serum in miR-10b relative expression quantity schematic diagram；
Fig. 2 be one embodiment of the invention serum in miR-31 relative expression quantity schematic diagram；
Fig. 3 be one embodiment of the invention serum in miR-24 relative expression quantity schematic diagram.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, by the following examples, it and combines attached
Figure carries out the combination of G. cephalantha marker primer, detection kit and its quantitative detecting method of the present invention further detailed
It describes in detail bright.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not intended to limit the present invention.
It is beyond imagination important due to being played in expression regulation of the miRNA (microRNA) after genetic transcription
Effect, thus it there are relevances below with disease：First, the variation of miRNA may be the cause of disease, when small core
Disorderly expression first has occurred in ribosomal ribonucleic acid itself, for example the original miRNA expression quantity for inhibiting disease promotive factor reduces
, or the miRNA expression quantity of disease inhibiting factor is inhibited to increase, final result can all lead to one system of downstream
The variation of row gene expression and the whole disorder of certain accesses, and then the generation that induces an illness.Secondly, the change of miRNA
Change is also likely to be disease as a result, this is because when disease (such as cancer) occurs, and can lead to loss, the gene of chromosome segment
Mutation or chromosome segment violent amplification, if miRNA is placed exactly in this varied sections, then its table
Highly significant variation will occur up to amount.Therefore, theoretically miRNA molecule can be used as a new class of disease completely
Marker, its specific variations are inevitable associated with disease generation development.
To find G. cephalantha marker and accurately being detected to it, inventor is attached to Shandong University first
The 364 G. cephalantha samples and 66 normal structures for belonging to Shandong Prov. Hospital carry out differential genes expression analysis, obtain difference
The microRNA of different expression is miR-10b, miR-31, miR-24.Whether can make for verification miR-10b, miR-31, miR-24
For the marker of G. cephalantha, to the patient of 78 customary G. cephalantha resections of the attached Shandong Prov. Hospital of Shandong University
Tumor tissues carry out pathological analysis, evaluate the complete implementations of histological grade, transfer and tumor capsule of tumour and right
Organize corresponding serum using the expression of real-time quantitative PCR detection miR-10b, miR-31, miR-24.With 80 normal human serums
In microRNA levels be control, show miR-10b, miR-31, miR- in G. cephalantha patients serum by verification
24 expression quantity is higher than normal person, and miR-10b, miR-31, miR-24 can be as the markers of G. cephalantha.
By the above-mentioned research to serum or blood plasma miRNA and the correlation of G. cephalantha, inventor proposes
The specific miRNA being stabilized using in serum or blood plasma detects marker as G. cephalantha, establishes a kind of body
The method for the specific miRNA being stabilized in outer detection serum or blood plasma, by detecting specific miRNA
Specific variations occur to carry out early diagnosis, disease identification and course of disease monitoring, recurrence and prognosis, the complication of G. cephalantha
Prediction, while can further carry out drug effect judgement, medicine guide, individualized treatment, effective components of Chinese medicinal screening and population
The researchs such as classification.
The embodiment of the present invention proposes a kind of G. cephalantha marker first, is included in human serum or blood plasma and stablizes
In the presence of and at least one of detectable miRNA maturation body miR-10, miR-31 and miR-24.The incidence
Squamous carcinoma marker expression quantity in serum or blood plasma of the G. cephalantha Patient Sample A with normal human's sample is different, by right
The expression quantity of the G. cephalantha marker of tested sample is detected, and judges whether differential expression, to by
Examination person is estimated with the presence or absence of the illness probability of G. cephalantha.
It, may be such as tested by accidental sexual factor if be only detected to a kind of G. cephalantha marker
The interference of the personal physical condition of person, ambient enviroment, other factors such as with the G. cephalantha marker expression correlative factor,
Keep the confidence level of testing result not high.Preferably, the G. cephalantha marker includes in miR-10, miR-31 and miR-24
Two or three of combination.To arbitrary two kinds of combinations of three kinds of G. cephalantha markers or the expression of three kinds of combinations
Amount carries out comprehensive analysis, and whether there is the risk with G. cephalantha to subject assesses, and improves forecasting reliability.
The marker for improving single is difficult to caused by the individual difference (i.e. age, sex, race, diet and environment etc.) overcome
Low specificity and muting sensitivity significantly improve the clinical recall rate of disease and realize the early stage diagnosis and treatment of disease.
The embodiment of the present invention provides a kind of quantitative detecting method of G. cephalantha marker, includes the following steps：
S100 takes the detected sample of predetermined amount, extracts total serum IgE；
S200 mixes the total serum IgE with reverse transcription PCR reaction reagent, and reverse transcription is carried out by template of the total serum IgE
PCR reacts, and obtains cDNA；
S300, by the forward primer quantitatively detected for miR-10b, for the miR-31 forward primers quantitatively detected and use
One kind and reverse primer, the cDNA in the forward primer that miR-24 is quantitatively detected and real-time quantitative PCR reaction reagent
Mixing, using the cDNA as template progress quantitative PCR reaction, miR-10b, miR-31 or miR-24 described in quantitative amplification；And
S400 analyzes the result of quantitative PCR reaction.
In the step s 100, the total serum IgE of extraction can be the RNA of all categories or be total in detected sample
MicroRNA can increase the accuracy of test result using total microRNA as the template of subsequent experimental, avoid removing
The interference of other kinds of RNA outside microRNA.
In step S400, the interpretation of result method of the PCR amplification may include electrophoresis and real time fluorescent quantitative point
Analysis method.The electrophoresis is by carrying out electrophoresis to the reaction solution obtained after S300, passing through the molecular weight or electricity of electrophoretic band
The depth of band of swimming analyzes the expression quantity of miR-10b, miR-31 or miR-24 to be amplified.The Real time PCR
To analyze miR-10b, miR-31 or miR-24 to be amplified by analyzing the fluorescence curve of real-time quantitative fluorescence PCR reaction
Expression quantity.
Detected sample used in the detection method can be serum or blood plasma, and the serum or blood plasma can come
Derived from the attached Shandong Prov. Hospital subject live body of Shandong University, tissue, organ and/or corpse.
In one embodiment, the method for detecting miRNA is reverse transcriptase polymerase chain reaction method (RT-
PCR).In the RT-PCR method, the S400 steps are the PCR product that obtains to S300 into row agarose gel electrophoresis,
Gel after electrophoresis observes result and takes pictures come the molecular weight and the depth of analytical electrophoresis band in the UV lamp after being dyed with EB.
In another embodiment, the method for detecting miRNA is real-time quantitative PCR reaction method (Real-time
PCR).In the Real-time PCR methods, the step S300 further includes：Fluorescence probe such as EVAGREEN dyes are added
Material carries out PCR reactions.The step S400 is to carry out analyzing and processing data to the fluorescence data of the obtained PCR products of S300 and compare
Compared with as a result, analyzing amount of the patients serum/plasma sample relative to miRNA in normal serum/blood plasma by fluorescence data
Variation.
The embodiment of the present invention also provides a kind of primer combination for detecting the G. cephalantha marker, including is used for
Forward primer that miR-10b is quantitatively detected, the forward primer quantitatively detected for miR-31 quantitatively detect just for miR-24
To at least one of primer.The primer combination can be applied to the quantitative detecting method of the G. cephalantha marker
In, the PCR primer in quantitatively being detected as the G. cephalantha marker, the specific expressed G. cephalantha mark
Object.It is analyzed by the amplification amount to the G. cephalantha marker after PCR, to judge that the G. cephalantha of subject is suffered from
Sick correlation.
Preferably, primer combination includes the forward primer quantitatively detected for miR-10b, is quantitatively examined for miR-31
Arbitrary two kinds of combination in the forward primer of survey, the forward primer quantitatively detected for miR-24 or three kinds of group
It closes.Comprehensive point is carried out by the expression quantity of arbitrary two kinds of combinations or three kinds of combinations to three kinds of G. cephalantha markers
Analysis whether there is the risk with G. cephalantha to subject and assess, improves forecasting reliability.Preferably, described
Primer combination includes the forward primer quantitatively detected for miR-10b, for the miR-31 forward primers quantitatively detected and is used for
Three kinds of combination of the forward primer that miR-24 is quantitatively detected.
In one embodiment, the sequence such as SEQ ID NO.1 institutes of the forward primer quantitatively detected for miR-10b
Show, the sequence of the forward primer quantitatively detected for miR-31 is described fixed for miR-24 as shown in SEQ ID NO.2
The sequence of the forward primer of detection is measured as shown in SEQ ID NO.3.In practical applications, the primer sequence may exist 1-3
The deviation of a base does not interfere with the result of amplification.
The embodiment of the present invention additionally provides a kind of kit for detecting G. cephalantha marker, namely predicts, examines
It is disconnected, differentiate and/or the kit of evaluation G. cephalantha, the kit include for quantitatively detect miR-10b, miR-31,
The quantitative detecting reagent of at least one of miR-24.Preferably, the quantitative detecting reagent includes above-mentioned for detecting neck
The primer of portion's squamous carcinoma marker combines, and the primer combination can carry out miR-10b, miR-31 or miR-24 quantitative special
Property detection.Due to the statistics of the expression quantity progress to G. cephalantha patient and miR-10b, miR-31 or miR-24 of normal person
Credit is analysed, and judges whether that there are a critical value (cut-off values) with G. cephalantha disease, by the kit
Testing result is compared with the critical value and can be estimated to risk.Preferably, the kit further includes anti-
To primer and PCR reaction reagents.The reverse primer is universal primer, for quantitatively being detected just with the miR-10b of specificity
The forward primer that the forward primer or miR-24 quantitatively detected to primer, miR-31 quantitatively detects is coordinated, anti-by PCR
The expression quantity of detection miR-10b, miR-31 or miR-24 should be quantified.In one embodiment, the sequence of the reverse primer such as SEQ
Shown in ID NO.4.In practical applications, the primer sequence may exist the deviation of 1-3 base, not interfere with the knot of amplification
Fruit.
Specifically, the forward primer quantitatively detected for miR-10b, the forward direction quantitatively detected for miR-31
Primer, the forward primer quantitatively detected for miR-24, the sequence of the reverse primer are as shown in table 1.
1 primer sequence of table
| miRNA | Corresponding primer sequence (5 ' -3 ') | Sequence number |
| miR-10b | ACACTCCAGCTGGGTACCCTGTAGAACCGAA | SEQ ID NO.1 |
| miR-31 | ACACTCCAGCTGGGATGGCAATATGTTGGC | SEQ ID NO.2 |
| miR-24 | ACACTCCAGCTGGGTGGCTCAGTTCAGCAG | SEQ ID NO.3 |
| Reverse primer | TGGTGTCGTGGAGTCG | SEQ ID NO.4 |
Preferably, may include reverse transcription PCR reaction step and quantitative PCR reaction step to the quantitative detection of microRNA
Suddenly, cDNA is obtained by reverse transcription PCR reaction step, cDNA is carried out by primer combination and reverse primer cooperation special
Property quantitative PCR reaction come specific expressed microRNA.The PCR reaction reagents include reverse transcription PCR reaction reagent and in real time
Quantitative PCR reaction reagent, the reverse transcription PCR reaction reagent and real-time quantitative PCR reaction reagent include polymerase, dNTP, ATP
Deng.It is furthermore preferred that by screen combined with the primer of the relevant specific variations of G. cephalantha, reverse primer and
PCR reaction reagents combine, and collectively constitute the kit of detection G. cephalantha marker.
The G. cephalantha marker detection kit is using miR-10b, miR-31 or miR-24 as G. cephalantha
Marker, using total serum IgE as template, the primer and miR-24 that the primer, the miR-31 that are quantitatively detected using miR-10b are quantitatively detected are fixed
The primer of detection is measured, specific PCR reaction is carried out, is analyzed by the expression quantity to miR-10b, miR-31 or miR-24,
The basis for estimation that can be early diagnosed as G. cephalantha, only clinic diagnosis does not win valuable rescue time, and has
There is the advantages of of low cost, easy to operate, high specificity.
The detection sample of the G. cephalantha marker detection kit can be easier to obtain, it is preferred that the detection
Sample is the blood that can be collected into routine physical examination.Since miRNA molecule is by 19-23 nucleotide units group
At, in structure particularity and opposite stability, be present in serum or blood plasma, the multi-signal as body tissue's organ
Molecule.Blood can be recycled to whole body institute in a organized way, and to cell delivery nutrition and remove waste, thus blood can reflect it is whole
The physiological and pathological situation of a body, testing result have directive significance to health.
Embodiment 1
Step S100, the extraction of total microRNA and concentration mensuration in blood
Total microRNA in tissue specimen is extracted using microRNA extracts kits (precious bioengineering Co., Ltd).
Extraction process is：With all plastic products used of DEPC (pyrocarbonic acid diethyl ester) aqueous solution soaking of 0.1% mass concentration and
Glassware is stayed overnight, and following experimental implementation is then carried out：(1) 78 G. cephalantha patients and 80 normal persons are collected respectively
Blood sample is put into liquid nitrogen transport, is preserved in -80 degrees Celsius of refrigerators, using effective within 6 months in cryopreservation tube；(2) to
2mL microRNA extracts reagents (microRNAiso Plus) are added in the blood sample of 5mL steps (1), aspirate repeatedly, mix
Even, absorption is transferred in the 1.5mL centrifuge tubes of RNA enzyme, is stored at room temperature 15min；(3) add in the liquid obtained to step (2)
Enter 0.2mL chloroforms, violent mixing 45s or instrument shake 30s, stand 30min at room temperature；(4) 4 degrees Celsius, 12000rpm centrifugations
15min carefully takes supernatant totally to be gone in the 1.5mL centrifuge tubes of RNA enzyme to another；(5) it is added into the centrifuge tube equipped with supernatant
0.5mL isopropanols, the mixing that turns upside down slowly (avoiding acutely shaking makes RNA be broken), stand 15~30min at room temperature；(6)
4 degrees Celsius, 12000rpm centrifugations 10min abandons supernatant；(7) 75% alcohol of l mL is added, in shaking mixing on swirl mixing device；
(8) 4 degrees Celsius, 12000rpm is centrifuged 5 minutes；(9) supernatant is removed, the supernatant on residual precipitate object and centrifuge tube is absorbed with filter paper
Centrifuge tube is placed 5~10min in superclean bench by liquid；(10) 0.1% mass concentration is added in the sediment of centrifuge tube
20~50 μ L of DEPC aqueous solutions, obtain total microRNA solution；(11) take total microRNA solution that 2 μ L steps (10) obtain molten
In 0.1mL water, survey respectively under wavelength 230nm, 260nm and 280nm the absorbance OD230, OD260 of total microRNA solution and
OD280；(12) the total microRNA solution examples extracted are in 80 degrees Celsius of preservations.
Step S200, Poly (A) tailing method modifies microRNA and reverse transcription PCR
(1) the total microRNA samples for taking 300-500ugS100 step extractions, utilize Reverse Transcriptase kit (precious bioengineering
Co., Ltd, SYBR PrimeScriptTM miRNART-PCR kits), PCR instrument (Thermo Scientific companies,
Model Aapplied Biosystems 7900HT Fast Real Time PCR System) on carry out reverse transcription PCR reaction,
Obtain cDNA；
(2) water of RNA enzyme is removed in addition in the cDNA reaction solutions handled to the first step, complements to 100 μ L of total amount of liquid,
Form cDNA dilutions.
Step S300, real-time fluorescence quantitative PCR
(1) real-time quantitative PCR is grasped according to the specification of SYBR PrimeScriptTM miRNART-PCR kits
Make, the miR-10b forward primer, the miR-31 that quantitatively detect quantitatively are detected described in 1 μ L forward primer and described is added
One kind in the forward primer that miR-24 is quantitatively detected, and it is dilute that the cDNA that reverse primer, 2 μ L second steps obtain described in 1 μ L is added
Liquid is released, reaction liquid is configured on ice by component；
(2) in PCR instrument (Thermo Scientific companies, model Aapplied Biosystems 7900HT Fast
Real Time PCR System) on, carry out real-time fluorescence quantitative PCR reaction；
Step S400, analysis of experimental results
Acquire S300 one-step fluorescence quantitatives PCR fluorescence datas obtained by the reaction, through PCR control analysis software (Thermo companies,
Model Aapplied Biosystems 7900HT Fast Real Time PCR System) cycle threshold (Ct values) is obtained,
The quantitative standard curves of PCR are made, the expression quantity of microRNA is calculated.
- 3 and table 2 are please referred to Fig.1, respectively using the microRNA of 78 G. cephalantha patients and 80 normal persons as mould
Plate as a contrast (U6 is the snRNA that molecular weight is 100bp, the internal reference molecule as miRNA experiment) with U6 obtains
To the relative expression quantity result of specific miR-10b, miR-31 or miR-24.
The average expression amount result of table 2microRNA
Determine the cut-off values (critical value) of each miRNA
According to sensibility and maximum specificity principle, the data of above-mentioned PCR reaction results are analyzed, compare neck
The experimental data of portion's squamous cell carcinoma patients and normal person makes ROC curve (the IBM SPSS Statistic 16.0 under miRNA expression
Software), the cut-off values that miRNA is expressed in serum are determined according to sensitivity and specificity.
Cut-off values in serum：
miR-10b 3.0×10-3
miR-31 2.7×10-4
miR-24 9.2×10-3
Subject's detection is carried out using G. cephalantha marker detection kit
The blood sample for taking 5mL subject, the forward primer quantitatively detected using miR-10b, the miR-31 are quantitatively examined
It is anti-to carry out quantitative PCR according to the experimental procedure of embodiment 1 for the forward primer that the forward primer of survey and the miR-24 are quantitatively detected
It answers.Quantitative PCR result obtained by the reaction and the cut-off values of miR-10b, miR-31 and miR-24 are compared, if tested
The expression value of miR-10b, miR-31 or miR-24 of person are more than cut-off values, then judge that the subject exists and suffer from incidence
The risk of squamous carcinoma disease.Further, if the expression value of miR-10b, miR-31 and miR-24 of subject is all higher than cut-off
Value then judges that probability of the subject with G. cephalantha disease is very big.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Shandong Prov. Hospital
<120>The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
acactccagc tgggtaccct gtagaaccga a 31
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acactccagc tgggatggca atatgttggc 30
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
acactccagc tgggtggctc agttcagcag 30
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tggtgtcgtg gagtcg 16
Claims (11)
1. a kind of primer combination for detecting G. cephalantha marker, which is characterized in that quantitatively examined including being used for miR-10b
In the forward primer of survey, the forward primer quantitatively detected for miR-31, the forward primer quantitatively detected for miR-24 extremely
Few one kind.
2. the primer combination according to claim 1 for detecting G. cephalantha marker, which is characterized in that described to draw
Object combination includes the forward primer quantitatively detected for miR-10b, the forward primer quantitatively detected for miR-31, described
Two or three of combination in the forward primer quantitatively detected for miR-24.
3. the primer combination according to claim 1 for detecting G. cephalantha marker, which is characterized in that the use
In the forward primer that miR-10b is quantitatively detected sequence as shown in SEQ ID NO.1.
4. the primer combination according to claim 1 for detecting G. cephalantha marker, which is characterized in that the use
In the forward primer that miR-31 is quantitatively detected sequence as shown in SEQ ID NO.2.
5. the primer combination according to claim 1 for detecting G. cephalantha marker, which is characterized in that the use
In the forward primer that miR-24 is quantitatively detected sequence as shown in SEQ ID NO.3.
6. a kind of G. cephalantha marker detection kit, which is characterized in that including being used to quantitatively detect miR-10b, miR-
31, the quantitative detecting reagent of at least one of miR-24.
7. G. cephalantha marker detection kit according to claim 6, which is characterized in that the quantitative detection examination
Agent includes the primer combination for detecting G. cephalantha marker as described in any one of claim 1 to 5.
8. G. cephalantha marker detection kit according to claim 7, which is characterized in that the quantitative detection examination
Agent further includes reverse primer, and the sequence of the reverse primer is as shown in SEQ ID NO.4.
9. G. cephalantha marker detection kit according to claim 7, which is characterized in that the quantitative detection examination
Agent further includes PCR reaction reagents, and the PCR reaction reagents include reverse transcription PCR reaction reagent and real-time quantitative PCR reaction examination
Agent.
10. a kind of quantitative detecting method of G. cephalantha marker, includes the following steps：
The detected sample of predetermined amount is taken, total serum IgE is extracted；
The total serum IgE is mixed with reverse transcription PCR reaction reagent, reverse transcription PCR reaction is carried out by template of the total serum IgE, obtains
To cDNA；
By the primer combination according to any one of claims 1 to 5 for detecting G. cephalantha marker, and reversely
Primer, the cDNA and the mixing of real-time quantitative PCR reaction reagent carry out quantitative PCR reaction by template of the cDNA, quantitative to expand
Increase described miR-10b, miR-31 or miR-24；And
The result of quantitative PCR reaction is analyzed.
11. the quantitative detecting method of G. cephalantha marker according to claim 10, which is characterized in that described to be checked
Sample is serum or blood plasma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810392823.1A CN108570500A (en) | 2018-04-27 | 2018-04-27 | The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810392823.1A CN108570500A (en) | 2018-04-27 | 2018-04-27 | The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108570500A true CN108570500A (en) | 2018-09-25 |
Family
ID=63575417
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810392823.1A Pending CN108570500A (en) | 2018-04-27 | 2018-04-27 | The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108570500A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110143950A1 (en) * | 2008-06-12 | 2011-06-16 | Keio University | Diagnosis/Treatment Option for Head-and-Neck Tumor Using Micro-RNA as Biomarker |
| CN103805603A (en) * | 2014-01-20 | 2014-05-21 | 南京医科大学附属南京儿童医院 | Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof |
| CN105219867A (en) * | 2015-11-02 | 2016-01-06 | 杨廷稳 | For miRNA biomarker and the detection kit of diagnosing gastric cancer |
-
2018
- 2018-04-27 CN CN201810392823.1A patent/CN108570500A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110143950A1 (en) * | 2008-06-12 | 2011-06-16 | Keio University | Diagnosis/Treatment Option for Head-and-Neck Tumor Using Micro-RNA as Biomarker |
| CN103805603A (en) * | 2014-01-20 | 2014-05-21 | 南京医科大学附属南京儿童医院 | Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof |
| CN105219867A (en) * | 2015-11-02 | 2016-01-06 | 杨廷稳 | For miRNA biomarker and the detection kit of diagnosing gastric cancer |
Non-Patent Citations (6)
| Title |
|---|
| C-J LIU等: "Increase of microRNA miR-31 level in plasma could be a potential marker of oral cancer", 《ORAL DISEASES》 * |
| JINGZHU ZHAO等: "miR-24 promotes the proliferation, migration and invasion in human tongue squamous cell carcinoma by targeting FBXW7", 《ONCOLOGY REPORTS》 * |
| PENNHMP116: "【求助】microRNA引物设计（帮朋友代发）", 《丁香园论坛》 * |
| YA-CHING LU等: "Oncogenic Function and Early Detection Potential of miRNA-10b in Oral Cancer as Identified by microRNA Profiling", 《CANCER PREVENTION RESEARCH》 * |
| 李燕主编: "《精编分子生物学实验技术》", 30 September 2017, 世界图书出版公司 * |
| 胡安等: "MicroRNA与头颈部肿瘤", 《中国耳鼻咽喉颅底外科杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112522A (en) | Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification | |
| CN105624303B (en) | Cattle and sheep pig Brucellacanis parting fluorescence PCR detection kit and its preparation and application | |
| Gaba et al. | Salivary biomarkers and their efficacies as diagnostic tools for oral squamous cell carcinoma: systematic review and meta‐analysis | |
| CN103667444B (en) | A kind of tumor marker relevant to carcinoma of the pancreas and application thereof | |
| CN109055557A (en) | One kind serum miRNA marker relevant to cancer of pancreas auxiliary diagnosis and its application | |
| CN107267659A (en) | Detect the purposes of the product of TRIM genes and/or protein level | |
| CN106755309A (en) | Application of the molecular marked compound in cancer of pancreas prognosis evaluation product is prepared | |
| CN108570500A (en) | The combination of G. cephalantha marker primer, detection kit and its quantitative detecting method | |
| CN110331208A (en) | Application of the molecular target in adenocarcinoma of lung Combining diagnosis | |
| CN113151462B (en) | Application of lung cancer prognosis diagnosis marker and detection kit | |
| CN109852698A (en) | Detect application and the kit of the reagent of 32 expression of ring finger protein | |
| CN101760518A (en) | Method for extracting live bacteria RNA in Mycobacterium tuberculosis and detection kit thereof | |
| CN109825585A (en) | A kind of biomarker of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation | |
| CN109136373A (en) | It is a kind of for early diagnosing the lncRNA detection kit and its application of lung cancer metastasis | |
| CN108998528B (en) | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof | |
| CN113470754A (en) | Gene marker for tumor prognosis evaluation, evaluation product and application | |
| CN113470813A (en) | Survival rate prognosis model for liver cancer patient | |
| CN113450917A (en) | Application of biomarker in prediction of liver cancer prognosis | |
| CN113241181A (en) | Prognosis risk assessment model and assessment device for liver cancer patient | |
| Mengual et al. | Quantitative RNA analysis from urine using real time PCR | |
| CA2818890C (en) | Method and system for prognosis and treatment of diseases using portfolio of genes | |
| CN111172285A (en) | miRNA group for early diagnosis and/or prognosis monitoring of pancreatic cancer and application thereof | |
| CN110273000A (en) | Detect application and the kit of the reagent of 8 expression of zinc finger protein 46 | |
| CN109609614A (en) | Detect the purposes of TRIM2, TRIM4, TRIM32 and/or TRIM46 gene or protein product | |
| EP2596131A2 (en) | Gene expression profiling for the identification of lung cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180925 |