CN105154560B - PCR kit for fluorescence quantitative and detection method and purposes for oophoroma - Google Patents
PCR kit for fluorescence quantitative and detection method and purposes for oophoroma Download PDFInfo
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- CN105154560B CN105154560B CN201510613041.2A CN201510613041A CN105154560B CN 105154560 B CN105154560 B CN 105154560B CN 201510613041 A CN201510613041 A CN 201510613041A CN 105154560 B CN105154560 B CN 105154560B
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- 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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The present invention relates to technical field of medical examination, and in particular to a kind of PCR kit for fluorescence quantitative and detection method and purposes for oophoroma.The kit includes reverse transcription PCR reaction solution, fluorescence quantitative PCR reaction solution, standard items and the primer sequence for ETV4, FOXM1 and/or LSR specific mrna reverse transcription.It is detected using detection kit of the invention for tumour-specific mRNA, it is high to the detection sensitivity of oophoroma, while sample collection is convenient, it is simple and efficient to handle.
Description
Technical field
The present invention relates to technical field of medical examination, and in particular to a kind of fluorescent quantitative poly chain type for oophoroma
React (PCR) kit and detection method and purposes.
Background technique
The oophoroma of early stage resectability with higher, but most of women have been in advanced stage when making a definite diagnosis,
Therefore, the patient that can be survived 5 years only accounts for 44%.Oophoroma early stage lacks specific clinical indication, tumor cell tissue's credit class
Complexity, there is no effective screening test that can make a definite diagnosis oophoroma at present, and early detection relies primarily on malignant tumor of ovary and boundary
The DNA quantitative analysis of property tumour is as diagnosis basis, and method has chromosome analysis, quiescence cells art and flow cytometry, method
Complicated and sensitivity is low；Existing ovary carcinoma marker HE4 and CA125 specificity is also very limited.Therefore, using tomour specific
Property mRNA carry out diagnosis of ovarian cancer with apparent advantage, a cancer cell may have hundreds of mRNA mutation to several thousand copies
Area, it is sensitiveer compared to DNA and tumor marker protein detection reliable.
ETS is in bird marrow earliest by U.S. Frederick National Cancer Institute molecular weight tumor laboratory at red thin
Born of the same parents, which increase, to be found in the gene order of syndrome virus E26.ETV4 is also once claimed as a member in ETS family PEA3 subfamily
For adenovirus E 1 A enhancer binding protein.For FOXM1 as a member in Fox transcription factor family, major function is to pass through tune
Ganglion cell ensures that cell mitogen is proliferated to the process of M phase by G1 phase to S phase, G2 phase.The FOXM1 of normal level is controlled
Cell growth, when cell periodically divides, FOXM1 has coordinated inhereditary material and has been assigned in two daughter cells.However, working as FOXM1
When overexpression, which loses the control to cell growth, allows cell overpreading.FOXM1 not only can be thin by improving
Born of the same parents' proliferative capacity promotes the formation of infantile tumour, can also enhance the transfer of tumour, invasive ability in advanced tumor.LSR (steatolysis
Activation lipoprotein receptor) content equally show the correlation with tumour.
Summary of the invention
The present invention needs the problem of the prior art that solves to be：It is existing for oophoroma detection based on DNA analysis
Kit and device, complicated for operation and sensitivity is low, existing kit and device based on the detection of oophoroma marker protein,
Specificity and sensitivity are limited.
To solve the above-mentioned problems, the present invention provides following technical solutions：
The present invention provides a kind of PCR kit for fluorescence quantitative for oophoroma, including box body 1, described in box body 1
Including first area 11, second area 12 and third region 13, wherein the first area 11 includes standard quality control 4, described
Second area 12 includes primer pipe 5, and the third region 13 includes anti-for ETV4, FOXM1 and/or LSR specific mrna
The Reverse transcription-PCR of transcription reacts the water pipe 6 of liquid pipe 2, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Preferably, the standard quality control 4 includes the plasmid pipe containing ETV4, the plasmid pipe containing FOXM1, and containing LSR's
It is more than one or more of plasmid pipe.
Preferably, the primer pipe 5 includes ETV4 specific primer pipe, and FOXM1 specific primer pipe and LSR are special
It is more than one or more of specific primer pipe.
Preferably, the described RT-PCR reaction liquid pipe 2 be include that reverse transcriptase, nucleic acid inhibitor, oligo (dT) draw
The reaction liquid pipe of object, dNTPs and buffer.
Preferably, the fluorescence quantitative PCR reaction solution pipe 3 be include fluorescent dye, archaeal dna polymerase, dNTPs gentle
The reaction liquid pipe of fliud flushing.
Specifically, the present invention provides following technical solutions：
The present invention provides a kind of PCR kit for fluorescence quantitative for oophoroma, the kit includes being used for
Reverse transcription PCR reaction solution, fluorescence quantitative PCR reaction solution, the standard items of ETV4, FOXM1 and/or LSR specific mrna reverse transcription
And primer sequence.
Preferably, the reverse transcription PCR reaction solution include reverse transcriptase, nucleic acid inhibitor, oligo (dT) primer,
DNTPs and buffer.
Preferably, the concentration of the reverse transcriptase is 30~80U/ μ l, and the concentration of nucleic acid inhibitor is 3~8U/ μ l,
The concentration of oligo (dT) primer is 50~80 μM, and the concentration of dNTPs is 1~3mM, and the buffer includes 200~300mM
Trishydroxymethylaminomethane (Tris-HCl), the potassium chloride (KCl) of 300~500mM, 10~30mM magnesium chloride (MgCl2)
With the dithiothreitol (DTT) (DTT) of 30~80mM.
Preferably, the fluorescence quantitative PCR reaction solution includes fluorescent dye, archaeal dna polymerase, dNTPs and buffer.
Preferably, the concentration of the archaeal dna polymerase is 50~80U/ml, and the concentration of dNTPs is 0.4~1mM, described slow
Fliud flushing includes the trishydroxymethylaminomethane (Tris-HCl) of 80~120mM, and the potassium chloride (KCl) of 100~200mM and 8~
Magnesium chloride (the MgCl of 15mM2)。
Preferably, the standard items include the plasmid containing ETV4, the plasmid containing FOXM1, and in the plasmid containing LSR
It is more than one or more.
Preferably, the primer sequence includes the primer sequence for ETV4 specific amplification, for FOXM1 specificity
It is more than one or more of primer sequence of amplification, and primer sequence for LSR specific amplification.
Preferably, the primer sequence for ETV4 specific amplification includes primer 1 and primer 2, wherein primer 1
Sequence is：The sequence of (5 '-ggatacttggaccagcaagt-3 '), primer 2 is：(5'-gacttgatggcgatttgt-3')；
Primer sequence for FOXM1 specific amplification includes primer 3 and primer 4, and wherein the sequence of primer 3 is：(5'-
Caaacagcggaacaaact-3 '), the sequence of primer 4 is：(5'-tctgcttgattagactcct-3')；
Primer sequence for LSR specific amplification includes primer 5 and primer 6, and the sequence of primer 5 is：(5'-
Atatctgaaagcatgccct-3 '), the sequence of primer 6 is：(5'-tggaagaggatcaccacgt-3').
Preferably, the concentration of the primer 1 for ETV4 is：0.5~1 μM, the concentration of primer 2 is 0.5~1 μM；
The concentration of the primer 3 for FOXM1 is 0.5~1 μM, and the concentration of primer 4 is 0.5~1 μM；
The concentration of the primer 5 for LSR is 0.5~1 μM, and the concentration of primer 6 is 0.5~1 μM.
Preferably, the kit further includes the water of nuclease free.
The present invention also provides a kind of detection methods of PCR kit for fluorescence quantitative for oophoroma, including walk as follows
Suddenly：
(1) reverse transcription reaction：Separately sampled mRNA, reverse transcription PCR reaction solution obtain sample by reverse transcription reaction
cDNA；
(2) quantitative fluorescent PCR reacts：Fluorescence quantitative PCR reaction solution, template cDNA, primer are separately added into reaction tube
Sequence reacts to obtain ETV4, FOXM1 and/or LSR after mixing by quantitative fluorescent PCR；
Wherein, the template cDNA includes：Step (1) preparation sample cDNA, ETV4 containing testing gene, FOXM1 and/or
The standard items of LSR.
Preferably, the reverse transcription reaction condition setting of the step (1) is 37 or 42 DEG C of 20~30min of incubation；Then
It is placed under the conditions of 94~95 DEG C, reacts 5~10min.
Preferably, the amplification condition of the quantitative fluorescent PCR of the step (2) be set as 94~95 DEG C of initial denaturations 2~
5min, 1 circulation；94~95 DEG C of 15~20s, 55~60 DEG C of 40~60s, 40~50 circulations；Last 60~95 DEG C 1~
2min, 1 circulation.
Preferably, the sample includes being taken from the cervix for being tested individual.
It is relevant to oophoroma in detection that the present invention also provides kits described above and the detection method
Application in gene specific mRNA level in-site.
ETV4 is a member of ETS family polyoma enhancer activator (PEA) 3 subfamily, have complicated structure and
Function finds it in a variety of organs of human embryos phase and manhood by the analysis to ETV4 transcription factor mRNA level in-site
There is low-level expression in normal tissue.In the comparative study of normal tissue and cancerous tissue, by the mRNA for detecting ETV4
Level discovery has notable difference between the two.ETV4 is related with normal cell division, but when with other Gene Fusions, then goes out
The abnormal activity now over-expressed, its overexpression lead to the vicious transformation of cell, enhance the invasion and transfer of tumour cell
Property.
FOXM1 is a kind of hypotype of FOX transcription factor family, and expression and transcriptional activation need multiple-factor, many cells letter
The common participation of number access, and depend on cell cycle progression.The increment of the expression and normal cell of FOXM1, aging and
The forming process of organ is closely related, and its unconventionality expression then pass through influence genome stability and mitosis process into
Exhibition etc. promotes the occurrence and development and transfer of tumour, and FOXM1 is in terms of the presence of tumor stem cell maintains stem cell versatility
It plays a significant role, the FOXM1 expression all with higher in the most of tumours of the mankind is had now been found that, with a variety of tumorigenesis
Signal pathway is related.
University of California Santiago medical college and Moores Cancer center scientist are calculated by specific bioinformatics
Method analyzes two public hereditary information databases --- cancer gene map (TCGA) and gene organization's expression program (GTEx) mirror
It makes and contains LSR mRNA in ovarian cancer cell, it was demonstrated that it is specific expressed in ovarian cancer tissue.
The present invention is based on the specific mrnas of oophoroma to be detected, creative to have chosen and oophoroma by research
Strong ETV4, FOXM1 and/or LSR gene of correlation is detected, and is effectively used for the specific detection of oophoroma, is shown
Strong specificity and high sensitivity.
Kit of the invention should be stored in -20 DEG C and hereinafter, reduce multigelation to the greatest extent.
The beneficial effects of the invention are as follows：
(1) early ovarian cancer is diagnosed using tumour-specific mRNA detection, is examined than DNA and tumor marker protein
Survey is sensitiveer, can be used for the early screening and Treatment monitoring of oophoroma；Simultaneously to being related to ETV4, FOXM1, LSR of oophoroma
Three carries out joint-detection, and sensitivity and specificity are higher.
(2) quantitative detection is carried out to oophoroma specific mrna by Real-Time Fluorescent Quantitative PCR Technique, method is easy to be fast
Speed.
(3) sample easily acquires, and only need to collect the oophoroma diffused out from ovary in cervix by conventional pap test
Specific mrna.
With reference to the accompanying drawing with each specific embodiment, the present invention and its advantageous effects are described in detail.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the fluorescent quantificationally PCR detecting kit for oophoroma of embodiment one, wherein 1 is
Box body, 2 react liquid pipe for RT-PCR, and 3 be fluorescence quantitative PCR reaction solution pipe, and 4 be standard quality control, and 5 be primer pipe, and 6 be free nucleic acid
The water pipe of enzyme, 11 be first area, and 12 be second area, and 13 be third region.
Specific embodiment
As described above, it is an object of the invention to：A kind of PCR kit for fluorescence quantitative for oophoroma and detection are provided
Method and purposes realize the high sensitivity and high specific detection of oophoroma.
The present invention provides a kind of detection kit of quantitative fluorescent PCR for oophoroma, including box body 1, described in
Box body 1 include first area 11, second area 12 and third region 13, wherein the first area 11 include standard items
Pipe 4, the second area 12 include primer pipe 5, and the third region 13 includes ETV4, FOXM1 and/or LSR specificity
The Reverse transcription-PCR of mRNA reverse transcription reacts the water pipe 6 of liquid pipe 2, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Specifically, the present invention provides a kind of detection kits of quantitative fluorescent PCR for oophoroma, including it is used for
The RT-PCR reaction solution of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution, standard items and draw
Object sequence.
Wherein, the primer sequence and extension increasing sequence used in the embodiment of the present invention and comparative example are as follows：
Primer sequence for testing gene ETV4 is respectively primer 1 and primer 2, and the sequence of primer 1 is：(5'-
Ggatacttggaccagcaagt-3 '), the sequence of primer 2 is：(5'-gacttgatggcgatttgt-3')；
Primer sequence for testing gene FOXM1 is respectively primer 3 and primer 4, and the sequence of primer 3 is：(5'-
Caaacagcggaacaaact-3 '), the sequence of primer 4 is：(5'-tctgcttgattagactcct-3')；
Primer sequence for testing gene LSR is respectively primer 5 and primer 6, and the sequence of primer 5 is：(5'-
Atatctgaaagcatgccct-3 '), the sequence of primer 6 is：(5'-tggaagaggatcaccacgt-3').
Primer sequence for testing gene CD9 is respectively primer 7 and primer 8, and the sequence of primer 7 is：(5'-
Catcaaatacctgctgtt-3 '), the sequence of primer 8 is：(5'-ttaatcacctcatccttgt-3')
Primer sequence for testing gene RAB11FIP4 is respectively primer 9 and primer 10, and the sequence of primer 9 is：(5'-
Gtgatgtcaccgcagcttgat-3 '), the sequence of primer 10 is：(5'-gtctgaggtcgtcatggat-3').
Primer sequence for testing gene FGFRL1 is respectively primer 11 and primer 12, and wherein the sequence of primer 11 is：
The sequence of (5 '-aagaagaagtggacact-3 '), primer 12 is：(5'-gcagcaccacaaacttct-3').
The extension increasing sequence obtained using the above primer is respectively that (wherein, the subsequent number of sequence represents every in extension increasing sequence
Position of a nucleotide in whole gene overall length)：
Correspondingly, the extension increasing sequence of ETV4：
Correspondingly, the extension increasing sequence of FOXM1：
Correspondingly, the extension increasing sequence of LSR：
Correspondingly, the extension increasing sequence of CD9 is as follows：
Correspondingly, the extension increasing sequence of RAB11FIP4 is as follows：
Correspondingly, the extension increasing sequence of FGFRL1 is as follows：
The present invention is described in further detail with specific embodiment with reference to the accompanying drawing.Wherein, embodiment and right
The producer of reagent used in ratio and instrument is as follows：
PCR instrument, U.S.'s Bio-Rad Bole's PCR instrument T100 type, the U.S.；
Fluorescence quantitative PCR instrument, American AB I real-time fluorescence quantitative PCR instrument StepOne, the U.S.；
RTase M-MLV, 5 × M-MLV Buffer, producer：Precious bioengineering (Dalian) Co., Ltd；
Oligo (dT) primer, RNase inhibitor and dNTPs, producer：Beijing ancient cooking vessel state prosperity biotechnology is limited
Responsible company；
Standard plasmid pET28a-SUMO-ETV4 containing ETV4, the standard plasmid pET28a-SUMO-FOXM1 containing FOXM1,
Standard plasmid pET28a-SUMO-LSR containing LSR, the standard plasmid pET28a-SUMO-CD9 containing CD9, the mark containing RAB11FIP4
Quasi- plasmid pET28a-SUMO-RAB11FIP4, the standard plasmid pET28a-SUMO-FGFRL1 containing FGFRL1：It orders from Wuhan
Miao Ling Biotechnology Co., Ltd, ETV4, FOXM1, LSR, CD9, RAB11FIP4, the FGFRL1 wherein contained in plasmid are upper
The extension increasing sequence for the above gene that text is mentioned；
Green I, producer：Life Technologies (AB&Invitrogen), the U.S.；
Taq archaeal dna polymerase, producer：Sigma-Aldrich, the U.S.；
Coke diethyl phthalate stoste (DEPC stoste), producer：Sigma-Aldrich, the U.S.；
Deionized water (RNase Free dH without RNase enzyme2O)：dH2DEPC stoste is added in O and is configured to 0.1% (v/
V) concentration stirs evenly simultaneously high pressure sterilization.
Embodiment one
One, kit
Embodiment one provides a kind of fluorescent quantificationally PCR detecting kit for oophoroma, as shown in Figure 1, described
Kit includes box body 1 and the Reagent Tube for holding reaction reagent, and the box body 1 divides for first area 11, second area 12
With third region 13, in order to which the Reagent Tube of different volume sizes is stored respectively, wherein first area 11 and second area
12 have accounted for 1/5th of box body volume respectively, and third region 13 has accounted for 3/5ths of the box body volume；Described
One region 11 includes standard quality control 4, and the second area 12 includes primer pipe 5, and the third region 13 includes being used for oophoroma
The RT-PCR of specific mrna reverse transcription reacts the water pipe 6 of liquid pipe 2, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Wherein, standard quality control is by the plasmid control quality control containing ETV4, the plasmid control quality control containing FOXM1, the plasmid containing LSR
Standard quality control composition, volume is respectively 500 μ l, is respectively provided with the 10 μ l of standard plasmid of the ETV4 containing testing gene, contains base to be measured
Because of the 10 μ l of standard plasmid of standard plasmid 10 the μ l, the LSR containing testing gene of FOXM1, concentration is respectively 50ng/ μ l.
Wherein, it is 2ml that RT-PCR, which reacts the volume of liquid pipe, is equipped with RT-PCR premixed liquid (RT-PCR Master Mix)
Reverse transcription reaction system, by RTase M-MLV, M-MLV Buffer, oligo (dT) primer (oligo (dT) primer), core
Sour enzyme inhibitor (RNase inhibitor) and dNTPs composition, total 1ml, wherein the concentration of RTase M-MLV is 50U/ μ l,
The concentration of oligo (dT) primer is 50 μM, and the concentration of RNase inhibitor is that the concentration of 5U/ μ l and dNTPs are 1mM, M-
MLV Buffer includes the Tris-HCl (pH8.3) of 250mM, the MgCl of the KCl of 375mM, 15mM2With the DTT of 50mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescent dye is 2ml, and qPCR premixed liquid is housed
The quantitative fluorescent PCR reaction system of (qPCR Master Mix), byGreen I, Taq archaeal dna polymerase, dNTPs
It is formed with buffer (Buffer), total 1ml, whereinThe concentration of Green I1000 × 0.2ml, Taq archaeal dna polymerase
Concentration for 60U/ml, dNTPs is 0.4mM, and Buffer includes the KCl and 10mM of the Tris-HCl (pH8.3) of 100mM, 150mM
MgCl2。
Deionized water (the RNase Free dH of nuclease free is housed in the water pipe of nuclease free2O) total 1ml, volume are
2ml。
Primer pipe is made of ETV4 specific primer pipe, FOXM1 specific primer pipe and LSR specific primer pipe,
Volume is respectively 1ml.
Wherein, ETV4 specific primer pipe is equipped with the specific primer (ETV4primers for expanding testing gene ETV4
Set) 100 μ l, wherein the sequence of primer 1 is：(5 '-ggatacttgg accagcaagt-3 '), concentration are 0.5 μM, primer
2 sequence is：(5 '-gacttgatggcgatttgt-3 '), concentration are 0.5 μM.
FOXM1 specific primer pipe is equipped with the specific primer (FOXM1primers for expanding testing gene FOXM1
Set) 100 μ l, wherein the sequence of primer 3 is：(5 '-caaacagcggaacaaact-3 '), concentration are 0.5 μM, primer 4
Sequence is：(5 '-tctgcttgattagactcct-3 '), concentration are 0.5 μM.
LSR specific primer pipe is equipped with the specific primer (LSR primers set) for expanding testing gene LSR
100 μ l, wherein the sequence of primer 5 is：(5 '-atatctgaaagcatgccct-3 '), concentration are 0.5 μM, the sequence of primer 6
It is classified as：(5 '-tggaagaggatcaccacgt-3 '), concentration are 0.5 μM.
Two, kit detects
For detecting ETV4, the detection of FOXM1 and LSR refer to the detection of ETV4.
(1) kit is used in accordance with the following steps：
1. reverse transcription reaction
1 reverse transcription reaction system of table
| Sample RNA | 5μl |
| RT-PCR Master Mix | 2μl |
| RNase Free dH2O | 3μl |
According to a new ep pipe described in table 1, is taken, then separately sampled 5 μ l, RT-PCR Master Mix of product RNA, 2 μ l,
RNase Free dH23 μ l of O, obtains sample cDNA using PCR instrument, wherein reaction condition is 37 DEG C of incubation 20min；Then it sets
Under the conditions of 95 DEG C, 5min is reacted.Obtained sample cDNA taking-up is placed in after reaction spare on ice chest.
2. quantitative fluorescent PCR reacts
2 quantitative fluorescent PCR reaction system of table
According to shown in table 2, new ep is taken to manage, is respectively designated as sample sets, standard item group and negative control group, successively plus
Enter 2 × qPCR Master Mix, template cDNA, ETV4primers set and RNase Free dH2O, every kind of reagent plus
It is as shown in table 2 to enter amount difference.Wherein, standard items use RNase Free dH2O is diluted to 5ng/ μ l, and negative control group uses
RNase Free dH2O.It uniformly or with magnetic force vortice is uniformly mixed sample with liquid-transfering gun piping and druming, it is fixed to be subsequently placed in fluorescence
It is reacted in amount PCR instrument.Its program setting is 95 DEG C of for 2minutes, 1 circulation；95 DEG C of for 15seconds, 60 DEG C of for
60seconds, 40 circulations；Last 95 DEG C of 1min, 1 circulation.After being provided with, file is saved, runs program.
The report of fluorescent quantitation result：
Fluorescent quantitative PCR result is analyzed using software, and marking calculates sample data.
(1) if value≤35 Ct of test sample, it can determine whether sample for the positive.
(2) if value >=40 Ct of test sample, it can determine whether sample for feminine gender.
(3) if 35 < Ct < 40 of test sample, need to re-start sample detection operation, if testing result again
Ct≤35, then judgement sample is the positive；If Ct value > 35, judgement sample is feminine gender.
Simultaneously, it should be noted that negative control should be without amplification curve, and Ct value difference between multiple holes is different should be within 0.5.
(2) kit performance detection
(1) specific：Quality-control product is detected using this kit, three times the results show that positive quality control product (contains ETV4
Standard plasmid) detect the expression of mRNA, negative quality-control product (RNase Free dH2O it) does not detect to express, therefore, this
It is 100% that method, which detects specificity,.
(2) detection limit：Take 0,1,10,100,1 × 103、1×104、1×105、1×106The standard containing ETV4 of a copy
Plasmid (copy number calculation formula：(6.02×1023) × (OD260 × extension rate × 50 × 10-9)/(DNA length×
660)=copies/ml), PCR operation is carried out, determines that minimum detectability is 100 copies.
(3) the clinical sample detection of kit
It chooses 48 clinics of General Hospital of Tianjin Medical Univ. and has confirmed that the patient for oophoroma, 47 do not have ovarian cancer just
Ordinary person.The cell tested and peeled off in individual cervix is taken with very thin smear using pap test method, then uses (Oligotex
MRNA Mini Kit, QIAGEN) extract cell in mRNA.
It is operated using this kit, experimental result is as shown in table 3 below：
3 clinical sample testing result of table
The results show that 48 ovarian cancer patients samples carry out ETV4 detection, 47 positives, 1 feminine gender (ovary of number 19
Cancer patient), positive coincidence rate 98%；FOXM1 detects 47 positives, and 1 feminine gender (ovarian cancer patients of number 24), the positive meets
Rate 98%；LSR detects 45 positives, 3 feminine genders (number is respectively 4,15,42 ovarian cancer patients), positive coincidence rate 94%.
It is feminine gender, negative match-rate 100% that 47 normal samples, which detect 47,.Prompt clinical detection ETV4, FOXM1, LSR can be to ovum
Nest cancer carries out auxiliary diagnosis, and it is higher that triple combination detects positive coincidence rate.
Embodiment two
Embodiment two and embodiment one the difference is that：
Standard quality control in embodiment two is only the plasmid control quality control containing FOXM1, the mark equipped with the FOXM1 containing testing gene
Quasi- 10 μ l of plasmid, concentration are 50ng/ μ l.
The primer pipe is FOXM1 specific primer pipe, equipped with the specific primer for expanding testing gene FOXM1
100 μ l, wherein being respectively 0.8 μM for the primer 3 of FOXM1 specific amplification and the concentration of primer 4.
Wherein, it is 2ml that RT-PCR, which reacts the volume of liquid pipe, is equipped with RT-PCR premixed liquid (RT-PCR Master Mix)
Reverse transcription reaction system, by RTase M-MLV, M-MLV Buffer, oligo (dT) primer (oligo (dT) primer), core
Sour enzyme inhibitor (RNase inhibitor) and dNTPs composition, total 1ml, wherein the concentration of RTase M-MLV is 30U/ μ l,
The concentration of oligo (dT) primer is 60 μM, and the concentration of RNase inhibitor is that the concentration of 3U/ μ l and dNTPs are 2mM, M-
MLV Buffer includes the Tris-HCl (pH8.3) of 200mM, the MgCl of the KCl of 300mM, 30mM2With the DTT of 80mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescent dye is 2ml, and qPCR premixed liquid is housed
The quantitative fluorescent PCR reaction system of (qPCR Master Mix), byGreen I, Taq archaeal dna polymerase, dNTPs
It is formed with buffer (Buffer), total 1ml, whereinGreen I 1000 × 0.2ml, Taq archaeal dna polymerase it is dense
Degree is 50U/ml, and the concentration of dNTPs is 1mM, and Buffer includes the KCl and 15mM of the Tris-HCl (pH8.3) of 80mM, 100mM
MgCl2。
Wherein the detection method of kit is as follows：
(1) kit is used in accordance with the following steps：
1. reverse transcription reaction
4 reverse transcription reaction system of table
| Sample RNA | 5μl |
| RT-PCR Master Mix | 2μl |
| RNase Free dH2O | 3μl |
According to a new ep pipe described in table 4, is taken, then separately sampled 5 μ l, RT-PCR Master Mix of product RNA, 2 μ l,
RNase Free dH23 μ l of O, obtains sample cDNA using PCR instrument, wherein reaction condition is 42 DEG C of incubation 30min；Then it sets
Under the conditions of 94 DEG C, 10min is reacted.Obtained sample cDNA taking-up is placed in after reaction spare on ice chest.
2. quantitative fluorescent PCR reacts
5 quantitative fluorescent PCR reaction system of table
According to shown in table 5, new ep is taken to manage, is respectively designated as sample sets, standard item group and negative control group, successively plus
Enter 2 × qPCR Master Mix, template cDNA, FOXM1primers set and RNase Free dH2O, every kind of reagent
Additional amount difference is as shown in table 5.Wherein, standard items use RNase Free dH2O is diluted to 5ng/ μ l, and negative control group uses
RNase Free dH2O.It uniformly or with magnetic force vortice is uniformly mixed sample with liquid-transfering gun piping and druming, it is fixed to be subsequently placed in fluorescence
It is reacted in amount PCR instrument.Its program setting is 94 DEG C of for 5minutes, 1 circulation；94 DEG C of for 20seconds, 55 DEG C of for
40seconds, 50 circulations；Last 72 DEG C of 2min, 1 circulation.After being provided with, file is saved, runs program.
Embodiment three
Embodiment three and embodiment one the difference is that：
Standard quality control in embodiment three is only the plasmid control quality control containing ETV4, the standard equipped with the ETV4 containing testing gene
10 μ l of plasmid, concentration are 50ng/ μ l.
The primer pipe is ETV4 specific primer pipe, equipped with the specific primer for expanding testing gene ETV4
100μl.Wherein, the concentration for the primer 1 of ETV4 specific amplification and primer 2 is respectively 1 μM.
Wherein, it is 2ml that RT-PCR, which reacts the volume of liquid pipe, is equipped with RT-PCR premixed liquid (RT-PCR Master Mix)
Reverse transcription reaction system, by RTase M-MLV, M-MLV Buffer, oligo (dT) primer (oligo (dT) primer), core
Sour enzyme inhibitor (RNase inhibitor) and dNTPs composition, total 1ml, wherein the concentration of RTase M-MLV is 80U/ μ l,
The concentration of oligo (dT) primer is 80 μM, and the concentration of RNase inhibitor is that the concentration of 8U/ μ l and dNTPs are 3mM, M-
MLV Buffer includes the Tris-HCl (pH8.3) of 300mM, the MgCl of the KCl of 500mM, 10mM2With the DTT of 30mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescent dye is 2ml, and qPCR premixed liquid is housed
The quantitative fluorescent PCR reaction system of (qPCR Master Mix), byGreen I, Taq archaeal dna polymerase, dNTPs
It is formed with buffer (Buffer), total 1ml, whereinGreen I 1000 × 0.2ml, Taq archaeal dna polymerase it is dense
Degree is 80U/ml, and the concentration of dNTPs is 0.8mM, Buffer include the Tris-HCl (pH8.3) of 120mM, 200mM KCl and
The MgCl of 8mM2。
Wherein, reverse transcription reaction condition setting is 37 DEG C of incubation 30min；It is subsequently placed under the conditions of 95 DEG C, reacts 10min.
Example IV
Example IV and embodiment one the difference is that：
Standard quality control in example IV is only the plasmid control quality control containing LSR, the standard matter equipped with the LSR containing testing gene
10 μ l of grain, concentration are 50ng/ μ l.
The primer pipe is LSR specific primer pipe, equipped with 100 μ of specific primer for expanding testing gene LSR
l。
Kit in embodiment does not include the water pipe of nuclease free, and the water for the nuclease free used in detection process can
Voluntarily to be prepared.
Embodiment five
Embodiment five and embodiment one the difference is that：
Standard quality control in embodiment five is by the plasmid control quality control containing FOXM1, and the plasmid control quality control group containing LSR
At being respectively provided with the 10 μ l of standard plasmid of standard plasmid 10 the μ l, the LSR containing testing gene of the FOXM1 containing testing gene, concentration difference
For 50ng/ μ l.
The primer pipe is made of FOXM1 specific primer pipe and LSR specific primer pipe, FOXM1 specific primer
Pipe is equipped with the 100 μ l of specific primer for expanding testing gene FOXM1.
LSR specific primer pipe, equipped with the 100 μ l of specific primer for expanding testing gene LSR.
Embodiment six
Embodiment six and embodiment one the difference is that：
Standard quality control in embodiment six is by the plasmid control quality control containing ETV4, and the plasmid control quality control containing FOXM1
Composition, is respectively provided with the 10 μ l of standard plasmid of standard plasmid 10 the μ l, the FOXM1 containing testing gene of the ETV4 containing testing gene, concentration
Respectively 50ng/ μ l.
The primer pipe is made of ETV4 specific primer pipe and FOXM1 specific primer pipe, wherein ETV4 specificity
Primer pipe is equipped with the 100 μ l of specific primer for expanding testing gene ETV4.
FOXM1 specific primer pipe is equipped with the 100 μ l of specific primer for expanding testing gene FOXM1.
Embodiment seven
Embodiment seven and embodiment one the difference is that：
Standard quality control in embodiment seven is by the plasmid control quality control containing ETV4, and the plasmid control quality control group containing LSR
At being respectively provided with the 10 μ l of standard plasmid of standard plasmid 10 the μ l, the LSR containing testing gene of the ETV4 containing testing gene, concentration difference
For 50ng/ μ l.
The primer pipe is made of ETV4 specific primer pipe and LSR specific primer pipe, wherein ETV4 specificity is drawn
Property management is equipped with the 100 μ l of specific primer for expanding testing gene ETV4.
LSR specific primer pipe is equipped with the 100 μ l of specific primer for expanding testing gene LSR.
Comparative example one
Comparative example is first is that detect ETV4 gene, the difference is that, quantitative fluorescent PCR is anti-with embodiment one
The program setting for answering system is 93 DEG C of for 2minutes, 1 circulation, 93 DEG C of for 15 seconds, 60 DEG C of for
60seconds, 40 circulations, last 93 DEG C of 10min, 1 circulation.Wherein in quantitative fluorescent PCR reaction system ETV4 primer
Volume is 1 μ l, i.e. 10 μ l of qPCR Master Mix, 1 μ l of template cDNA 1ul, ETV4 primer, the 8 μ l of water of nuclease free.
Reaction result shows that positive controls Ct value is 28, higher, as a result undesirable.
Comparative example two
Comparative example two according to embodiment one method for CD9 gene in ovarian cancer patients to be measured and normal sample mRNA
It is detected
Comparative example three
Comparative example three is according to the method for embodiment one for RAB11FIP4 gene in ovarian cancer patients to be measured and normal sample
MRNA detected.
Comparative example four
Comparative example four is according to the method for embodiment one for FGFRL1 gene in ovarian cancer patients to be measured and normal sample
MRNA is detected.
Wherein, the clinical sample testing result of comparative example two to four is shown in Table 6.
The clinical sample testing result of 6 comparative example one to three of table
The testing result of CD9 shows in 48 ovarian cancer patients samples, 41 positives, 7 feminine genders (number is respectively 6,
13,20,25,32,42,45 ovarian cancer patients), positive coincidence rate 85.4%；47 normal persons, 44 feminine genders, 3 positives
(number is respectively 64,76,87 normal sample), negative match-rate 93.6%.
The testing result of RAB11FIP4 shows 48 ovarian cancer patients samples, 40 positives, 8 feminine gender (number difference
It is 8,13,21,37,39,42,45,46 ovarian cancer patients), positive coincidence rate 83.3%；47 normal persons, 46 feminine genders, 1
Example is positive (normal sample that number is 90), negative match-rate 97.8%.
The testing result of FGFRL1 shows 48 ovarian cancer patients samples, 41 positives, 7 feminine genders (number is respectively 4,
9,18,26,30,32,46 ovarian cancer patients), positive coincidence rate 85.4%；47 normal persons, 45 feminine genders, 2 positives (are compiled
Number be respectively 55,57 normal sample), negative match-rate 95.7%.
It can be seen that from the testing result of table 6 to tri- genes of CD9, RAB11FIP4, FGFRL1 relevant to oophoroma
MRNA detected, testing result show three above gene there are the false positive of higher proportion and false negative, specificity
The specific detection difference of tri- genes of ETV4, FOXM1 or LSR relevant to oophoroma is larger.Prove by ETV4, FOXM1 and/
Or LSR is used for the specific detection of oophoroma, sensitivity and specificity are high, and method is easy quickly, can be applied to ovary
The early screening and Treatment monitoring of cancer.
The foregoing is merely present pre-ferred embodiments, are not used to the limitation present invention, all in spirit and original of the invention
The modifications, equivalent substitutions and improvements etc. done within then are required within the protection scope of invention.
Claims (8)
1. a kind of PCR kit for fluorescence quantitative for oophoroma, it is characterised in that：The kit include for ETV4,
Reverse transcription PCR reaction solution, fluorescence quantitative PCR reaction solution, standard items and the primer sequence of FOXM1 and LSR specific mrna reverse transcription
Column；
Wherein, the primer sequence includes the primer sequence for ETV4 specific amplification, for FOXM1 specific amplification
Primer sequence, and the primer sequence for LSR specific amplification；
The primer sequence for ETV4 specific amplification includes primer 1 and primer 2, and wherein the sequence of primer 1 is：5'-
The sequence of ggatacttggaccagcaagt-3 ', primer 2 is：5'-gacttgatggcgatttgt-3'；
Primer sequence for FOXM1 specific amplification includes primer 3 and primer 4, and wherein the sequence of primer 3 is：5'-
The sequence of caaacagcggaacaaact-3 ', primer 4 is：5'-tctgcttgattagactcct-3'；
Primer sequence for LSR specific amplification includes primer 5 and primer 6, and the sequence of primer 5 is：5'-
The sequence of atatctgaaagcatgccct-3 ', primer 6 is：5'-tggaagaggatcaccacgt-3'；
The concentration of the primer 1 is：0.5~1 μM, the concentration of primer 2 is 0.5~1 μM；
The concentration of the primer 3 is 0.5~1 μM, and the concentration of primer 4 is 0.5~1 μM；
The concentration of the primer 5 is 0.5~1 μM, and the concentration of primer 6 is 0.5~1 μM；
The standard items include the plasmid of ETV4, the plasmid of FOXM1 and the plasmid of LSR；
The extension increasing sequence of the ETV4 gene contained in plasmid, sequence are as follows：
The extension increasing sequence of the FOXM1 gene contained in plasmid, sequence are as follows：
The extension increasing sequence of the LSR gene contained in plasmid, sequence are as follows：
Wherein, the subsequent number of sequence represents position of the extension increasing sequence nucleotide in whole gene overall length.
2. kit according to claim 1, which is characterized in that the reverse transcription PCR reaction solution include reverse transcriptase,
Nucleic acid inhibitor, oligodT primer, dNTPs and buffer.
3. kit according to claim 2, which is characterized in that the concentration of the reverse transcriptase is 30~80U/ μ l,
The concentration of nucleic acid inhibitor is that the concentration of 3~8U/ μ l, oligodT primer is 50~80 μM, and the concentration of dNTPs is 1~3mM,
The buffer includes the chlorine of the trishydroxymethylaminomethane of 200~300mM, the potassium chloride of 300~500mM, 10~30mM
Change the dithiothreitol (DTT) of magnesium and 30~80mM.
4. kit according to claim 1 to 3, which is characterized in that the fluorescence quantitative PCR reaction solution includes
Fluorescent dye, archaeal dna polymerase, dNTPs and buffer.
5. kit according to claim 4, which is characterized in that the concentration of the archaeal dna polymerase is 50~80U/ml,
The concentration of dNTPs is 0.4~1mM, and the buffer includes the trishydroxymethylaminomethane of 80~120mM, 100~200mM's
The magnesium chloride of potassium chloride and 8~15mM.
6. kit according to claim 1-3, which is characterized in that the kit further includes nuclease free
Water.
7. kit according to claim 4, which is characterized in that the kit further includes the water of nuclease free.
8. kit according to claim 5, which is characterized in that the kit further includes the water of nuclease free.
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| CN106755406A (en) * | 2016-12-22 | 2017-05-31 | 亚能生物技术（深圳）有限公司 | A kind of oophoroma detects product and kit |
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| Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy;Christian L. Barrett et al.;《PNAS》;20150526;摘要，E3051页第1段-E3054页最后1段，图1,表S2 * |
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