CN105154560A - Fluorogenic quantitative PCR kit for ovarian cancer, detecting method and application - Google Patents
Fluorogenic quantitative PCR kit for ovarian cancer, detecting method and application Download PDFInfo
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- CN105154560A CN105154560A CN201510613041.2A CN201510613041A CN105154560A CN 105154560 A CN105154560 A CN 105154560A CN 201510613041 A CN201510613041 A CN 201510613041A CN 105154560 A CN105154560 A CN 105154560A
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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
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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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Abstract
The invention relates to the technical field of medical examinations, in particular to a fluorogenic quantitative PCR kit for ovarian cancer, a detecting method and an application. The kit comprises reverse transcription PCR liquid for ETV4, FOXM1 and/or LSR specificity mRNA reverse transcription, fluorogenic quantitative PCR liquid, a standard substance and a primer sequence. The detection kit is applied to detecting tumor specificity mRNA, and is high in detection sensitivity to the ovarian cancer, meanwhile, sample collection is convenient, and operation is easy and fast.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of fluorescent quantitative poly chain reaction (PCR) test kit for ovarian cancer and detection method and purposes.
Background technology
The ovarian cancer of commitment has higher resectability, but most of women is in late period when making a definite diagnosis, and therefore, the patient can survived 5 years only accounts for 44%.Ovarian cancer lacks specific clinical indication in early days, complicated classification is learned by tumor cell tissue, there is no effective shaker test at present and can make a definite diagnosis ovarian cancer, early detection mainly relies on the DNA quantitative analysis of malignant tumor of ovary and borderline tumor as diagnosis basis, method has chromosome analysis, quiescence cells art and flow cytometry, and method is complicated and sensitivity is low; Existing ovarian cancer mark HE4 and CA125 specificity are also very limited.Therefore, adopt tumour-specific mRNA to carry out diagnosis of ovarian cancer and have obvious advantage, a cancer cell may have hundreds of mRNA saltation zone to several thousand copies, compares DNA and tumor marker protein detects more reliably sensitive.
ETS is found in the gene order of bird marrow erythroblastosis virus E26 by U.S.'s Frederick National Cancer Institute molecular weight tumor laboratory.ETV4, as a member in ETS family PEA3 subfamily, was also once called as adenovirus E 1 A enhancer binding protein.FOXM1 is as a member in Fox transcription factor family, and its major function is by regulating cell to guarantee that cell mitogen is bred by the process of G1 phase to S phase, G2 phase to M phase.The FOXM1 of normal level controls Growth of Cells, and when cell regularly divides, FOXM1 has coordinated genetic material and has been assigned in two daughter cells.But when FOXM1 process LAN, the control of cell growth of this proteins lose, allows cell overpreading.FOXM1 not only can promote the formation of infantile tumour by improving ability of cell proliferation, can also strengthen transfer, the invasive ability of tumour at advanced tumor.The content of LSR (steatolysis activation lipoprotein receptor) shows the dependency with tumour equally.
Summary of the invention
The present invention needs the problem of prior art solved to be: the existing test kit based on DNA analysis that detects for ovarian cancer and device, complicated operation and sensitivity is low, existing based on ovarian cancer marker protein detect test kit and device, its specificity and sensitivity limited.
In order to solve the problem, the invention provides following technical scheme:
The invention provides a kind of PCR kit for fluorescence quantitative for ovarian cancer, comprise box body 1, box body 1 described in it comprises first area 11, second area 12 and the 3rd region 13, wherein, described first area 11 comprises standard QC 4, described second area 12 comprises primer pipe 5, and the 3rd described region 13 comprises the water pipe 6 for the Reverse transcription-PCR reaction solution pipe 2 of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Preferably, described standard QC 4 comprises the plasmid pipe containing ETV4, containing the plasmid pipe of FOXM1, and containing more than one or more in the plasmid pipe of LSR.
Preferably, described primer pipe 5 comprises ETV4 Auele Specific Primer pipe, FOXM1 Auele Specific Primer pipe, and more than one or more in LSR Auele Specific Primer pipe.
Preferably, described RT-PCR reaction solution pipe 2 is for including the reaction solution pipe of ThermoScript II, nucleic acid inhibitor, oligo (dT) primer, dNTPs and damping fluid.
Preferably, described fluorescence quantitative PCR reaction solution pipe 3 is for including the reaction solution pipe of fluorescence dye, archaeal dna polymerase, dNTPs and damping fluid.
Specifically, the invention provides following technical scheme:
The invention provides a kind of PCR kit for fluorescence quantitative for ovarian cancer, described test kit comprises for the reverse transcription PCR reaction solution of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution, standard substance and primer sequence.
Preferably, described reverse transcription PCR reaction solution comprises ThermoScript II, nucleic acid inhibitor, oligo (dT) primer, dNTPs and damping fluid.
Preferably, the concentration of described ThermoScript II is 30 ~ 80U/ μ l, the concentration of nucleic acid inhibitor is 3 ~ 8U/ μ l, the concentration of oligo (dT) primer is 50 ~ 80 μMs, the concentration of dNTPs is 1 ~ 3mM, and described damping fluid comprises the Tutofusin tris of 200 ~ 300mM (Tris-HCl), the Repone K (KCl) of 300 ~ 500mM, the magnesium chloride (MgCl of 10 ~ 30mM
2) and the dithiothreitol (DTT) (DTT) of 30 ~ 80mM.
Preferably, described fluorescence quantitative PCR reaction solution comprises fluorescence dye, archaeal dna polymerase, dNTPs and damping fluid.
Preferably, the concentration of described archaeal dna polymerase is 50 ~ 80U/ml, the concentration of dNTPs is 0.4 ~ 1mM, described damping fluid comprises the Tutofusin tris (Tris-HCl) of 80 ~ 120mM, the Repone K (KCl) of 100 ~ 200mM and the magnesium chloride (MgCl of 8 ~ 15mM
2).
Preferably, described standard substance comprise the plasmid containing ETV4, containing the plasmid of FOXM1, and containing more than one or more in the plasmid of LSR.
Preferably, described primer sequence comprises the primer sequence for ETV4 specific amplification, for the primer sequence of FOXM1 specific amplification, and for more than one or more in the primer sequence of LSR specific amplification.
Preferably, the described primer sequence for ETV4 specific amplification comprises primer 1 and primer 2, wherein the sequence of primer 1 is: (5 '-ggatacttggaccagcaagt-3 '), the sequence of primer 2 is: (5 '-gacttgatggcgatttgt-3 ');
Primer sequence for FOXM1 specific amplification comprises 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 comprises 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 described primer 1 for ETV4 is: 0.5 ~ 1 μM, and the concentration of primer 2 is 0.5 ~ 1 μM;
The concentration of the described primer 3 for FOXM1 is 0.5 ~ 1 μM, and the concentration of primer 4 is 0.5 ~ 1 μM;
The concentration of the described primer 5 for LSR is 0.5 ~ 1 μM, and the concentration of primer 6 is 0.5 ~ 1 μM.
Preferably, described test kit also comprises the water of nuclease free.
Present invention also offers a kind of detection method of the PCR kit for fluorescence quantitative for ovarian cancer, comprise the steps:
(1) reverse transcription reaction: sample this mRNA respectively, reverse transcription PCR reaction solution, obtains sample cDNA by reverse transcription reaction;
(2) quantitative fluorescent PCR reaction: add fluorescence quantitative PCR reaction solution, template cDNA, primer sequence respectively in reaction tubes, be obtained by reacting ETV4, FOXM1 and/or LSR by quantitative fluorescent PCR after mixing;
Wherein, described template cDNA comprises: sample cDNA prepared by step (1), the standard substance containing testing gene ETV4, FOXM1 and/or LSR.
Preferably, the reverse transcription reaction condition setting of described step (1) is 37 or 42 DEG C and hatches 20 ~ 30min; Then under being placed in 94 ~ 95 DEG C of conditions, reaction 5 ~ 10min.
Preferably, the amplification condition of the quantitative fluorescent PCR of described step (2) is set to 94 ~ 95 DEG C of denaturation 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 of 1 ~ 2min, 1 circulation.
Preferably, described sample comprises the uterine cervix being taken from tested individuality.
Present invention also offers test kit described above and the application of described detection method in the gene specific mRNA level in-site that detection is relevant to ovarian cancer.
ETV4 is a member of ETS family polyoma enhancer activator (PEA) 3 subfamily, there is complicated structure and function, by the analysis to ETV4 transcription factor mRNA level in-site, find that it has low-level expression in the healthy tissues of the multiple organ of human embryos phase and Adulthood.In the comparative study of healthy tissues and cancerous tissue, find there is notable difference between the two by the mRNA level in-site detecting ETV4.ETV4 is relevant with normal cell division, but when with other gene fusion, then occurs the abnormal activity of overexpression, and its process LAN causes the vicious transformation of cell, strengthens aggressive and the transitivity of tumour cell.
FOXM1 is a kind of hypotype of FOX transcription factor family, and its expression and transcriptional activation need the common participation of multiplefactor, multicellular signal path, and depend on cell cycle progression.The forming process of the expression level of FOXM1 and Normocellular increment, aging and organ is closely related, its unconventionality expression is then promoted the generation development of tumour by the progress etc. of the genomic stability of impact and mitotic division process and is shifted, and namely FOXM1 maintains in stem cell versatility in the existence of tumor stem cell and plays a significant role, find that the FOXM1 in the most of tumour of the mankind has higher expression level at present, relevant to multiple oncogenic signaling pathways.
San Diego, University of California medical college and Moores Cancer center scientist are by specific bioinformatics, analyzing two public genetic information databases---cancer gene collection of illustrative plates (TCGA) and gene organization's expression program (GTEx) identify in ovarian cancer cell contains LSRmRNA, proves that it is specific expressed in ovarian cancer tissue.
The specific mrna that the present invention is based on ovarian cancer detects, by research, creationaryly have chosen ETV4, FOXM1 and/or LSR gene strong with ovarian cancer dependency and detect, effectively for the specific detection of ovarian cancer, show strong specificity and high sensitivity.
Test kit of the present invention should be stored in-20 DEG C and following, reduces multigelation as far as possible.
The invention has the beneficial effects as follows:
(1) adopt tumour-specific mRNA to detect to diagnose early ovarian cancer, detect sensitiveer than DNA and tumor marker protein, can be used for early screening and the Treatment monitoring of ovarian cancer; Joint-detection is carried out to ETV4, FOXM1, LSR three relating to ovarian cancer simultaneously, sensitivity and specificity higher.
(2) carry out detection by quantitative by Real-Time Fluorescent Quantitative PCR Technique to ovarian cancer specific mrna, method is fast easy.
(3) sample easily gathers, and only need collect by the pap test of routine the ovarian cancer specific mrna diffused out from ovary in uterine cervix.
Below in conjunction with accompanying drawing and each embodiment, the present invention and Advantageous Effects thereof are described in detail.
Accompanying drawing explanation
Fig. 1 is the structural representation of the fluorescent quantificationally PCR detecting kit for ovarian cancer of embodiment one, wherein, 1 is box body, and 2 is RT-PCR reaction solution pipe, and 3 is fluorescence quantitative PCR reaction solution pipe, 4 is standard QC, 5 is primer pipe, and 6 is the water pipe of nuclease free, and 11 is first area, 12 is second area, and 13 is the 3rd region.
Embodiment
As mentioned above, the object of the invention is to: provide a kind of PCR kit for fluorescence quantitative for ovarian cancer and detection method and purposes, the highly sensitive and the high specific that realize ovarian cancer detect.
The invention provides a kind of detection kit of the quantitative fluorescent PCR for ovarian cancer, comprise box body 1, box body 1 described in it comprises first area 11, second area 12 and the 3rd region 13, wherein, described first area 11 comprises standard QC 4, described second area 12 comprises primer pipe 5, and the 3rd described region 13 comprises the water pipe 6 of the Reverse transcription-PCR reaction solution pipe 2 of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Specifically, the invention provides a kind of detection kit of the quantitative fluorescent PCR for ovarian cancer, comprise for the RT-PCR reaction solution of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution, standard substance and primer sequence.
Wherein, the primer sequence used in the embodiment of the present invention and comparative example and extension increasing sequence 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, 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, wherein the sequence of primer 11 is: (5 '-aagaagaagtggacact-3 '), the sequence of primer 12 is: (5 '-gcagcaccacaaacttct-3 ').
The extension increasing sequence utilizing above primer to obtain is respectively (wherein, the position of each Nucleotide in whole full length gene in sequence digitized representation extension increasing sequence below):
Accordingly, the extension increasing sequence of ETV4:
Accordingly, the extension increasing sequence of FOXM1:
Accordingly, the extension increasing sequence of LSR:
Accordingly, the extension increasing sequence of CD9 is as follows:
Accordingly, the extension increasing sequence of RAB11FIP4 is as follows:
Accordingly, the extension increasing sequence of FGFRL1 is as follows:
Below in conjunction with accompanying drawing and specific embodiment, the present invention is further detailed explanation.Wherein, in embodiment and comparative example, the producer of reagent used and instrument is as follows:
PCR instrument, U.S. Bio-Rad Bole PCR instrument T100 type, the U.S.;
Quantitative real time PCR Instrument, American AB I real-time fluorescence quantitative PCR instrument StepOne, the U.S.;
RTaseM-MLV, 5 × M-MLVBuffer, producer: precious biotechnology (Dalian) company limited;
Oligo (dT) primer, RNaseinhibitor and dNTPs, producer: Beijing DingGuo ChangSheng Biology Technology Co., Ltd;
Containing the standard plasmid pET28a-SUMO-ETV4 of ETV4, containing the standard plasmid pET28a-SUMO-FOXM1 of FOXM1, containing the standard plasmid pET28a-SUMO-LSR of LSR, containing the standard plasmid pET28a-SUMO-CD9 of CD9, containing the standard plasmid pET28a-SUMO-RAB11FIP4 of RAB11FIP4, standard plasmid pET28a-SUMO-FGFRL1 containing FGFRL1: all order from Miao Ling bio tech ltd, Wuhan, ETV4, FOXM1, LSR, CD9, RAB11FIP4, FGFRL1 of wherein containing in plasmid are the extension increasing sequence of above gene mentioned above;
greenI, producer: LifeTechnologies (AB & Invitrogen), the U.S.;
Taq DNA polymerase, producer: Sigma-Aldrich, the U.S.;
Coke diethyl phthalate stoste (DEPC stoste), producer: Sigma-Aldrich, the U.S.;
Without the deionized water (RNaseFreedH of RNase enzyme
2o): dH
2add DEPC stoste in O and be mixed with 0.1% (v/v) concentration, stir and autoclaving.
Embodiment one
One, test kit
Embodiment one provides a kind of fluorescent quantificationally PCR detecting kit for ovarian cancer, as shown in Figure 1, described test kit comprises box body 1 and the Reagent Tube for holding reaction reagent, described box body 1 is divided into first area 11, second area 12 and the 3rd region 13, so that the Reagent Tube of different volume size is deposited respectively, the 1/5th, three region 13 that wherein first area 11 and second area 12 have accounted for box body volume has respectively accounted for 3/5ths of described box body volume; Described first area 11 comprises standard QC 4, and described second area 12 comprises primer pipe 5, and described 3rd region 13 comprises the water pipe 6 for the RT-PCR reaction solution pipe 2 of ovarian cancer specific mrna reverse transcription, fluorescence quantitative PCR reaction solution pipe 3 and nuclease free.
Wherein, standard QC is by the plasmid control QC containing ETV4, containing the plasmid control QC of FOXM1, plasmid control QC containing LSR forms, its volume is respectively 500 μ l, and the standard plasmid 10 μ l containing testing gene ETV4 is housed respectively, containing the standard plasmid 10 μ l of testing gene FOXM1, containing the standard plasmid 10 μ l of testing gene LSR, concentration is respectively 50ng/ μ l.
Wherein, the volume of RT-PCR reaction solution pipe is 2ml, it is equipped with the reverse transcription reaction system of RT-PCR premixed liquid (RT-PCRMasterMix), by RTaseM-MLV, M-MLVBuffer, oligo (dT) primer (oligo (dT) primer), nucleic acid inhibitor (RNaseinhibitor) and dNTPs composition, 1ml altogether, wherein the concentration of RTaseM-MLV is 50U/ μ l, the concentration of oligo (dT) primer is 50 μMs, the concentration of RNaseinhibitor is the concentration of 5U/ μ l and dNTPs is 1mM, M-MLVBuffer comprises the Tris-HCl (pH8.3) of 250mM, the KCl of 375mM, the MgCl of 15mM
2with the DTT of 50mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescence dye is 2ml, and it is equipped with the quantitative fluorescent PCR reaction system of qPCR premixed liquid (qPCRMasterMix), by
greenI, Taq DNA polymerase, dNTPs and damping fluid (Buffer) form, altogether 1ml, wherein
greenI1000 × 0.2ml, the concentration of Taq DNA polymerase is the concentration of 60U/ml, dNTPs is the MgCl that 0.4mM, Buffer comprise the Tris-HCl (pH8.3) of 100mM, KCl and 10mM of 150mM
2.
Deionized water (the RNaseFreedH of nuclease free is housed in the water pipe of nuclease free
2o) be total to 1ml, its volume is 2ml.
Primer pipe is made up of ETV4 Auele Specific Primer pipe, FOXM1 Auele Specific Primer pipe and LSR Auele Specific Primer pipe, and its volume is respectively 1ml.
Wherein, ETV4 Auele Specific Primer pipe is equipped with Auele Specific Primer (ETV4primersset) the 100 μ l for the testing gene ETV4 that increases, wherein, the sequence of primer 1 is: (5 '-ggatacttggaccagcaagt-3 '), its concentration is 0.5 μM, the sequence of primer 2 is: (5 '-gacttgatggcgatttgt-3 '), its concentration is 0.5 μM.
FOXM1 Auele Specific Primer pipe is equipped with Auele Specific Primer (FOXM1primersset) the 100 μ l for the testing gene FOXM1 that increases, wherein, the sequence of primer 3 is: (5 '-caaacagcggaacaaact-3 '), its concentration is 0.5 μM, the sequence of primer 4 is: (5 '-tctgcttgattagactcct-3 '), its concentration is 0.5 μM.
LSR Auele Specific Primer pipe is equipped with Auele Specific Primer (LSRprimersset) the 100 μ l for the testing gene LSR that increases, wherein, the sequence of primer 5 is: (5 '-atatctgaaagcatgccct-3 '), its concentration is 0.5 μM, the sequence of primer 6 is: (5 '-tggaagaggatcaccacgt-3 '), its concentration is 0.5 μM.
Two, test kit detects
To detect the detection of detection with reference to ETV4 of ETV4, FOXM1 and LSR.
(1) test kit is used in accordance with the following steps:
1. reverse transcription reaction
Table 1 reverse transcription reaction system
| Sample RNA | 5μl |
| RT-PCR Master Mix | 2μl |
| RNase Free dH 2O | 3μl |
According to described in table 1, get a new ep pipe, then distinguish sample thief RNA5 μ l, RT-PCRMasterMix2 μ l, RNaseFreedH
2o3 μ l, utilizes PCR instrument to obtain sample cDNA, and wherein, reaction conditions is 37 DEG C and hatches 20min; Then under being placed in 95 DEG C of conditions, reaction 5min.After reaction terminates, the sample cDNA obtained taking-up is placed on ice chest for subsequent use.
2. quantitative fluorescent PCR reaction
Table 2 quantitative fluorescent PCR reaction system
According to shown in table 2, get new ep pipe, called after sample sets, standard substance group and negative control group, add 2 × qPCRMasterMix, template cDNA, ETV4primersset and RNaseFreedH successively respectively
2o, the add-on of often kind of reagent is as shown in table 2 respectively.Wherein, standard substance adopt RNaseFreedH
2o dilution is 5ng/ μ l, and negative control group adopts RNaseFreedH
2o.Evenly or with magnetic force vortice make sample mix even with liquid-transfering gun piping and druming, be then placed in quantitative real time PCR Instrument and react.Its program setting is, 95 DEG C of for2minutes, 1 circulation; 95 DEG C of for15seconds, 60 DEG C of for60seconds, 40 circulations; Last 95 DEG C of 1min, 1 circulation.After being provided with, preserve file, working procedure.
Fluorescent quantitation report the test:
Adopt software to analyze fluorescent quantitative PCR result, and markization calculate sampled data.
(1) if Ct value≤35 of test sample, can judgement sample be positive.
(2) if Ct value >=40 of test sample, can judgement sample be negative.
(3) if test sample 35 < Ct < 40, need to re-start detection operation to sample, if detected result Ct≤35 again, then judgement sample be the positive; If Ct value > 35, then judgement sample is negative.
Meanwhile, it should be noted that negative control should without amplification curve, the Ct value difference between multiple hole is different should within 0.5.
(2) test kit Performance Detection
(1) specificity: use this test kit to detect quality control product, three result displays, positive quality control product (standard plasmid containing ETV4) all detects the expression of mRNA, negative quality control product (RNaseFreedH
2o) do not detect expression, therefore, it is 100% that this law detects specific degree.
(2) detectability: get 0,1,10,100,1 × 10
3, 1 × 10
4, 1 × 10
5, 1 × 10
6standard plasmid (the copy number calculation formula: (6.02 × 10 containing ETV4 of individual copy
23) × (OD260 × extension rate × 50 × 10
-9)/(DNAlength × 660)=copies/ml), carry out PCR operation, determine that minimum detectability is 100 copies.
(3) clinical sample of test kit detects
Choose the clinical patient having confirmed as ovarian cancer of General Hospital of Tianjin Medical Univ. 48 example, the normal people that 47 examples do not have ovarian cancer.Adopt the very thin smear of pap test method to get the cell peeled off in the individual uterine cervix of test, then the mRNA in cell is extracted in employing (OligotexmRNA Mini Kit, QIAGEN).
Adopt this test kit to operate, experimental result is as shown in table 3 below:
Table 3 clinical sample detected result
Result shows, and 48 routine ovarian cancer patients samples carry out ETV4 detection, and 47 examples are positive, 1 example negative (ovarian cancer patients of numbering 19), positive coincidence rate 98%; It is positive that FOXM1 detects 47 examples, 1 example negative (ovarian cancer patients of numbering 24), positive coincidence rate 98%; It is positive that LSR detects 45 examples, 3 examples negative (numbering is respectively the ovarian cancer patients of 4,15,42), positive coincidence rate 94%.Normal pattern detection 47 example of 47 example is feminine gender, negative match-rate 100%.Prompting clinical detection ETV4, FOXM1, LSR can carry out auxiliary diagnosis to ovarian cancer, and it is higher that triple combination detects positive coincidence rate.
Embodiment two
Embodiment two is with the difference of embodiment one:
Standard QC in embodiment two is only the plasmid control QC containing FOXM1, and the standard plasmid 10 μ l containing testing gene FOXM1 is housed, and concentration is 50ng/ μ l.
Described primer pipe is FOXM1 Auele Specific Primer pipe, the Auele Specific Primer 100 μ l for the testing gene FOXM1 that increases is housed, is wherein respectively 0.8 μM for the primer 3 of FOXM1 specific amplification and the concentration of primer 4.
Wherein, the volume of RT-PCR reaction solution pipe is 2ml, it is equipped with the reverse transcription reaction system of RT-PCR premixed liquid (RT-PCRMasterMix), by RTaseM-MLV, M-MLVBuffer, oligo (dT) primer (oligo (dT) primer), nucleic acid inhibitor (RNaseinhibitor) and dNTPs composition, 1ml altogether, wherein the concentration of RTaseM-MLV is 30U/ μ l, the concentration of oligo (dT) primer is 60 μMs, the concentration of RNaseinhibitor is the concentration of 3U/ μ l and dNTPs is 2mM, M-MLVBuffer comprises the Tris-HCl (pH8.3) of 200mM, the KCl of 300mM, the MgCl of 30mM
2with the DTT of 80mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescence dye is 2ml, and it is equipped with the quantitative fluorescent PCR reaction system of qPCR premixed liquid (qPCRMasterMix), by
greenI, Taq DNA polymerase, dNTPs and damping fluid (Buffer) form, altogether 1ml, wherein
greenI1000 × 0.2ml, the concentration of Taq DNA polymerase is the concentration of 50U/ml, dNTPs is the MgCl that 1mM, Buffer comprise the Tris-HCl (pH8.3) of 80mM, KCl and 15mM of 100mM
2.
Wherein the detection method of test kit is as follows:
(1) test kit is used in accordance with the following steps:
1. reverse transcription reaction
Table 4 reverse transcription reaction system
| Sample RNA | 5μl |
| RT-PCR Master Mix | 2μl |
| RNase Free dH 2O | 3μl |
According to described in table 4, get a new ep pipe, then distinguish sample thief RNA5 μ l, RT-PCRMasterMix2 μ l, RNaseFreedH
2o3 μ l, utilizes PCR instrument to obtain sample cDNA, and wherein, reaction conditions is 42 DEG C and hatches 30min; Then under being placed in 94 DEG C of conditions, reaction 10min.After reaction terminates, the sample cDNA obtained taking-up is placed on ice chest for subsequent use.
2. quantitative fluorescent PCR reaction
Table 5 quantitative fluorescent PCR reaction system
According to shown in table 5, get new ep pipe, called after sample sets, standard substance group and negative control group, add 2 × qPCRMasterMix, template cDNA, FOXM1primersset and RNaseFreedH successively respectively
2o, the add-on of often kind of reagent is as shown in table 5 respectively.Wherein, standard substance adopt RNaseFreedH
2o dilution is 5ng/ μ l, and negative control group adopts RNaseFreedH
2o.Evenly or with magnetic force vortice make sample mix even with liquid-transfering gun piping and druming, be then placed in quantitative real time PCR Instrument and react.Its program setting is, 94 DEG C of for5minutes, 1 circulation; 94 DEG C of for20seconds, 55 DEG C of for40seconds, 50 circulations; Last 72 DEG C of 2min, 1 circulation.After being provided with, preserve file, working procedure.
Embodiment three
Embodiment three is with the difference of embodiment one:
Standard QC in embodiment three is only the plasmid control QC containing ETV4, and the standard plasmid 10 μ l containing testing gene ETV4 is housed, and concentration is 50ng/ μ l.
Described primer pipe is ETV4 Auele Specific Primer pipe, and the Auele Specific Primer 100 μ l for the testing gene ETV4 that increases is housed.Wherein, 1 μM is respectively for the primer 1 of ETV4 specific amplification and the concentration of primer 2.
Wherein, the volume of RT-PCR reaction solution pipe is 2ml, it is equipped with the reverse transcription reaction system of RT-PCR premixed liquid (RT-PCRMasterMix), by RTaseM-MLV, M-MLVBuffer, oligo (dT) primer (oligo (dT) primer), nucleic acid inhibitor (RNaseinhibitor) and dNTPs composition, 1ml altogether, wherein the concentration of RTaseM-MLV is 80U/ μ l, the concentration of oligo (dT) primer is 80 μMs, the concentration of RNaseinhibitor is the concentration of 8U/ μ l and dNTPs is 3mM, M-MLVBuffer comprises the Tris-HCl (pH8.3) of 300mM, the KCl of 500mM, the MgCl of 10mM
2with the DTT of 30mM.
Wherein, the volume of the fluorescence quantitative PCR reaction solution pipe containing fluorescence dye is 2ml, and it is equipped with the quantitative fluorescent PCR reaction system of qPCR premixed liquid (qPCRMasterMix), by
greenI, Taq DNA polymerase, dNTPs and damping fluid (Buffer) form, altogether 1ml, wherein
greenI1000 × 0.2ml, the concentration of Taq DNA polymerase is the concentration of 80U/ml, dNTPs is the MgCl that 0.8mM, Buffer comprise the Tris-HCl (pH8.3) of 120mM, KCl and 8mM of 200mM
2.
Wherein, reverse transcription reaction condition setting is 37 DEG C and hatches 30min; Then under being placed in 95 DEG C of conditions, reaction 10min.
Embodiment four
Embodiment four is with the difference of embodiment one:
Standard QC in embodiment four is only the plasmid control QC containing LSR, and the standard plasmid 10 μ l containing testing gene LSR is housed, and concentration is 50ng/ μ l.
Described primer pipe is LSR Auele Specific Primer pipe, and the Auele Specific Primer 100 μ l for the testing gene LSR that increases is housed.
Test kit in embodiment, does not comprise the water pipe of nuclease free, and the water of the nuclease free used in testing process can be prepared voluntarily.
Embodiment five
Embodiment five is with the difference of embodiment one:
Standard QC in embodiment five is by the plasmid control QC containing FOXM1, and form containing the plasmid control QC of LSR, standard plasmid 10 μ l containing testing gene FOXM1 is housed respectively, and containing the standard plasmid 10 μ l of testing gene LSR, concentration is respectively 50ng/ μ l.
Described primer pipe is made up of FOXM1 Auele Specific Primer pipe and LSR Auele Specific Primer pipe, and FOXM1 Auele Specific Primer pipe is equipped with the Auele Specific Primer 100 μ l for the testing gene FOXM1 that increases.
LSR Auele Specific Primer pipe, is equipped with the Auele Specific Primer 100 μ l for the testing gene LSR that increases.
Embodiment six
Embodiment six is with the difference of embodiment one:
Standard QC in embodiment six is by the plasmid control QC containing ETV4, and form containing the plasmid control QC of FOXM1, standard plasmid 10 μ l containing testing gene ETV4 is housed respectively, and containing the standard plasmid 10 μ l of testing gene FOXM1, concentration is respectively 50ng/ μ l.
Described primer pipe is made up of ETV4 Auele Specific Primer pipe and FOXM1 Auele Specific Primer pipe, and wherein, ETV4 Auele Specific Primer pipe is equipped with the Auele Specific Primer 100 μ l for the testing gene ETV4 that increases.
FOXM1 Auele Specific Primer pipe is equipped with the Auele Specific Primer 100 μ l for the testing gene FOXM1 that increases.
Embodiment seven
Embodiment seven is with the difference of embodiment one:
Standard QC in embodiment seven by the plasmid control QC containing ETV4, and forms containing the plasmid control QC of LSR, and the standard plasmid 10 μ l containing testing gene ETV4 is housed respectively, and containing the standard plasmid 10 μ l of testing gene LSR, concentration is respectively 50ng/ μ l.
Described primer pipe is made up of ETV4 Auele Specific Primer pipe and LSR Auele Specific Primer pipe, and wherein, ETV4 Auele Specific Primer pipe is equipped with the Auele Specific Primer 100 μ l for the testing gene ETV4 that increases.
LSR Auele Specific Primer pipe is equipped with the Auele Specific Primer 100 μ l for the testing gene LSR that increases.
Comparative example one
Comparative example one detects for ETV4 gene, is with the difference of embodiment one, and the program setting of quantitative fluorescent PCR reaction system is, 93 DEG C of for2minutes, 1 circulation, 93 DEG C of for15seconds, 60 DEG C of for60seconds, 40 circulations, last 93 DEG C of 10min, 1 circulation.Wherein in quantitative fluorescent PCR reaction system, the primer volume of ETV4 is 1 μ l, i.e. qPCRMasterMix10 μ l, template cDNA1ul, ETV4 primer 1 μ l, the water 8 μ l of nuclease free.
Reaction result shows, positive controls Ct value is 28, and higher, result is undesirable.
Comparative example two
Comparative example two detects for the mRNA of CD9 gene in ovarian cancer patients to be measured and normal sample according to the method for embodiment one
Comparative example three
Comparative example three detects for the mRNA of RAB11FIP4 gene in ovarian cancer patients to be measured and normal sample according to the method for embodiment one.
Comparative example four
Comparative example four detects for the mRNA of FGFRL1 gene in ovarian cancer patients to be measured and normal sample according to the method for embodiment one.
Wherein, the clinical sample detected result of comparative example two to four is in table 6.
The clinical sample detected result of table 6 comparative example one to three
The detected result of CD9 shows, in 48 routine ovarian cancer patients samples, 41 examples are positive, and 7 example feminine genders (numbering is respectively 6, and 13,20,25,32, the ovarian cancer patients of 42,45), positive coincidence rate 85.4%; 47 routine normal peoples, 44 examples are negative, 3 examples positive (numbering is respectively 64, the normal sample of 76,87), negative match-rate 93.6%.
The detected result of RAB11FIP4 shows, 48 routine ovarian cancer patients samples, and 40 examples are positive, and 8 example feminine genders (numbering is respectively 8, and 13,21,37,39,42,45, the ovarian cancer patients of 46), positive coincidence rate 83.3%; 47 routine normal peoples, 46 examples are negative, 1 example positive (being numbered the normal sample of 90), negative match-rate 97.8%.
The detected result of FGFRL1 shows, 48 routine ovarian cancer patients samples, and 41 examples are positive, and 7 example feminine genders (numbering is respectively 4, and 9,18,26,30, the ovarian cancer patients of 32,46), positive coincidence rate 85.4%; 47 routine normal peoples, 45 examples are negative, 2 examples positive (numbering is respectively 55, the normal sample of 57), negative match-rate 95.7%.
As can be seen from the detected result of table 6, the mRNA of CD9, RAB11FIP4, FGFRL1 tri-genes relevant to ovarian cancer is detected, detected result shows, there is false positive and the false negative of higher proportion in above three genes, the specific detection difference of ETV4, FOXM1 or LSR tri-genes that its specificity is relevant to ovarian cancer is larger.Prove specific detection ETV4, FOXM1 and/or LSR being used for ovarian cancer, its sensitivity and specificity high, and method is fast easy, can be applicable to early screening and the Treatment monitoring of ovarian cancer.
The foregoing is only present pre-ferred embodiments, and be not used in limitation the present invention, all amendments done within the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain all needing to be included in invention.
Claims (15)
1. for a PCR kit for fluorescence quantitative for ovarian cancer, it is characterized in that: described test kit comprises for the reverse transcription PCR reaction solution of ETV4, FOXM1 and/or LSR specific mrna reverse transcription, fluorescence quantitative PCR reaction solution, standard substance and primer sequence.
2. test kit according to claim 1, is characterized in that, described reverse transcription PCR reaction solution comprises ThermoScript II, nucleic acid inhibitor, oligo (dT) primer, dNTPs and damping fluid.
3. test kit according to claim 2, it is characterized in that, the concentration of described ThermoScript II is 30 ~ 80U/ μ l, the concentration of nucleic acid inhibitor is 3 ~ 8U/ μ l, the concentration of oligo (dT) primer is 50 ~ 80 μMs, the concentration of dNTPs is 1 ~ 3mM, and described damping fluid comprises the dithiothreitol (DTT) of the Tutofusin tris of 200 ~ 300mM, the Repone K of 300 ~ 500mM, the magnesium chloride of 10 ~ 30mM and 30 ~ 80mM.
4., according to the arbitrary described test kit of claim 1-3, it is characterized in that, described fluorescence quantitative PCR reaction solution comprises fluorescence dye, archaeal dna polymerase, dNTPs and damping fluid.
5. test kit according to claim 4, it is characterized in that, the concentration of described archaeal dna polymerase is the concentration of 50 ~ 80U/ml, dNTPs is 0.4 ~ 1mM, described damping fluid comprises the Tutofusin tris of 80 ~ 120mM, the Repone K of 100 ~ 200mM and the magnesium chloride of 8 ~ 15mM.
6. according to the arbitrary described test kit of claim 1-5, it is characterized in that, described standard substance comprise the plasmid containing ETV4, containing the plasmid of FOXM1, and containing more than one or more in the plasmid of LSR.
7. according to the arbitrary described test kit of claim 1-6, it is characterized in that, described primer sequence comprises the primer sequence for ETV4 specific amplification, for the primer sequence of FOXM1 specific amplification, and for more than one or more in the primer sequence of LSR specific amplification.
8. test kit according to claim 7, it is characterized in that, the described primer sequence for ETV4 specific amplification comprises primer 1 and primer 2, wherein the sequence of primer 1 is: (5 '-ggatacttggaccagcaagt-3 '), the sequence of primer 2 is: (5 '-gacttgatggcgatttgt-3 ');
Primer sequence for FOXM1 specific amplification comprises 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 comprises primer 5 and primer 6, and the sequence of primer 5 is: (5 '-atatctgaaagcatgccct-3 '), the sequence of primer 6 is: (5 '-tggaagaggatcaccacgt-3 ').
9. test kit according to claim 8, is characterized in that, the concentration of the described primer 1 for ETV4 is: 0.5 ~ 1 μM, and the concentration of primer 2 is 0.5 ~ 1 μM;
The concentration of the described primer 3 for FOXM1 is 0.5 ~ 1 μM, and the concentration of primer 4 is 0.5 ~ 1 μM;
The concentration of the described primer 5 for LSR is 0.5 ~ 1 μM, and the concentration of primer 6 is 0.5 ~ 1 μM.
10. the test kit according to claim 1-9, is characterized in that, described test kit also comprises the water of nuclease free.
The detection method of the PCR kit for fluorescence quantitative for ovarian cancer described in 11. claim 1-10, is characterized in that, comprise the steps:
(1) reverse transcription reaction: sample this mRNA respectively, reverse transcription PCR reaction solution, obtains sample cDNA by reverse transcription reaction;
(2) quantitative fluorescent PCR reaction: add fluorescence quantitative PCR reaction solution, template cDNA, primer sequence respectively in reaction tubes, be obtained by reacting ETV4, FOXM1 and/or LSR by quantitative fluorescent PCR after mixing;
Wherein, described template cDNA comprises: sample cDNA prepared by step (1), the standard substance containing testing gene ETV4, FOXM1 and/or LSR.
12. detection methods according to claim 11, is characterized in that, the reverse transcription reaction condition setting of described step (1) is 37 or 42 DEG C and hatches 20 ~ 30min; Then under being placed in 94 ~ 95 DEG C of conditions, reaction 5 ~ 10min.
13. detection methods according to claim 11 or 12, it is characterized in that, the amplification condition of the quantitative fluorescent PCR of described step (2) is set to 94 ~ 95 DEG C of denaturation 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 of 1 ~ 2min, 1 circulation.
14. according to the arbitrary described detection method of claim 10-13, and it is characterized in that, described sample comprises the uterine cervix being taken from tested individuality.
Test kit described in 15. claim 1-10 and the detection method described in claim 11-14 are detecting the application in the gene specific mRNA level in-site relevant to ovarian cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048037A (en) * | 2016-07-06 | 2016-10-26 | 上海市内分泌代谢病研究所 | Human circulating miR-122 detection method |
| 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 |
-
2015
- 2015-09-24 CN CN201510613041.2A patent/CN105154560B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| CHRISTIAN L. BARRETT ET AL.: "Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy", 《PNAS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048037A (en) * | 2016-07-06 | 2016-10-26 | 上海市内分泌代谢病研究所 | Human circulating miR-122 detection method |
| 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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