CN106399555A - Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection - Google Patents
Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as a standard substance and a detection kit for real-time fluorescent quantitative PCR detection. The special standard substance is used for drawing standard curves, real-time fluorescent quantitative PCR detection is performed on an HER2 gene of a to-be-detected sample and a reference gene, and the detection method is used for clinical targeted therapy of breast cancer and stomach cancer. The invention further provides a plasmid vector containing a reference gene segment and a to-be-detected target gene segment in an equal proportion and taken as the detection standard substance. The invention also provides the real-time fluorescent quantitative PCR kit for detecting the amplification state of the HER2 gene. The kit comprises a PCR reaction system for detecting the amplification state of the HER2 gene, the standard substance and negative and positive controls. The detection method and the detection kit have the advantages of being convenient to use, high in detection efficiency, cost-saving and the like. Great significance in diagnosis of HER2 positive breast cancer as well as research and development, quality control and clinical application of breast cancer resistant drugs is realized.
Description
Technical field
The invention belongs to biomedicine technical field is and in particular to a kind of real-time fluorescence quantitative PCR detection method and its mark
Quasi- product and detection kit.
Background technology
Copy number variation (Copy Number Variation, CNV) is the important component part of gene structure variation, by
Genome occurs to reset and leads to, and the copy number referring generally to the genome large fragment that length is more than 1kb increases or reduces, main
Show as disappearance and the repetition of sub- microscopic level.There is close relation with tumor in CNV, Disklin etc. finds 1q21.1 site
CNV in close relations with neuroblastoma；And the CNV of tumor-related gene can be very big to the generation of tumor and transfer generation
Impact, such as the CNV on GSTM1 gene can lead to the onset risk of bladder cancer to increase；CNV on antioncogene WWOX can lead
Cause the activity decrease of antioncogene, make pulmonary carcinoma be more easy to occur.The targeted therapy detecting for some tumor patients to related CNV
It is critical that, the related gene copy number difference that doctor can be directed to patient takes personalized treatment meanss, for example logical
Cross the detection to HER2 gene copy number (HER2 gene relative quantification) to judge whether it expands is clinical treatment transitivity mammary gland
Whether cancer is suitable for the crucial indication of targeted drug Herceptin.
Breast carcinoma is the harm topmost malignant tumor of women's health, has observed in primary breast cancer patients
Patient's HER2 overexpression of 25%-30%.HER2 (ErbB-2, human epidermal growth
Factor receptor-2, HER2) gene, i.e. c-erbB-2 gene, it is positioned on chromosome 17q12-21.32, coding is relatively
Molecular mass is 185000 transmembrane receptor sample albumen, has tyrosine kinase activity.In the case of normal human, HER2 is singly to copy
Shellfish gene, but in breast carcinoma and gastric cancer tumor cell, often there is chromosome heterogeneity phenomenon to occur, such as No. 17 chromosome quantitatives
Increase, on more commonly No. 17 chromosomes, the DNA sequence of HER2 gene and its upstream and downstream 300-1000M is inserted through gene rearrangement
Enter thus to lead to the copy number of HER2 gene to increase on other chromosomes, and then lead to tumor cell surface HER2 albumen mistake
Expression.From 1997, food and medicine surveillance authority of U.S. priority have approved Herceptin (Herceptin trade name) for HER2
The breast carcinoma of overexpression, the metastatic breast cancer of HER2 overexpression, the treatment of the metastatic gastric carcinoma of HER2 overexpression.Herceptin
It is a kind of recombinant humanized monoclonal antibody, the cell of human epidermal growth factor receptor-2 (HER2) can be specifically acted on
Outer position, can suppress the propagation of the tumor cell of HER2 overexpression.Herceptin has become clinical HER2 positive breast at present
The first-line drug of cancer targeted therapy, nearly 8,000,000,000 dollars of global annual sales amount.Clinical trial certificate HER2 overexpression patient accepts
Herceptin therapeutic effect is more notable.American Society of Clinical Oncology and Chinese medical case association issue《Breast cancer treatment refers to
South》And《Breast carcinoma HER2 guide detection》All clearly propose to carry out HER2 detection before breast carcinoma targeted therapy, correct inspection
Survey and the evaluation HER2 protein expression of breast carcinoma and gene amplification state to the clinical treatment of breast carcinoma and Index for diagnosis to closing weight
Will, it is directed not only to patient if appropriate for the targeted therapy for HER2, and Endocrine treatment, the selection of chemotherapy regimen and pre-
Assess directive function afterwards.
At present, the HER2 detection method of FDA accreditation be divided into SABC (IHC) and in situ hybridization (ISH include FISH,
CISH, SISH) two classes.IHC is the paraffin section of formalin fix to be carried out on protein level using immune chromogenic reaction
HER2 overexpression detects, and ISH is then the nucleic probe using special marking and HER2 in fixing nucleus on paraffin section
Gene DNA target sequence hybridizes, in nucleic acid level, the amplification situation of detection HER2 gene, and also need when IHC testing result is 2+
To carry out FISH detection further to make a definite diagnosis.Above two class method complex operations in Clinical detection, take longer, expensive
(clinical charge 2500-3000 unit), is difficult to be standardized in general visiting laboratory.
Real-time fluorescence quantitative PCR has that linear detection range is big, detection speed is fast, flux is high, closing detection is pollution-free, clever
Many advantages, such as sensitivity is high, has had many clinical practices in terms of nucleic acid copies quantitation.At present should by quantitative fluorescent PCR
Detection for gene copy number variation mainly has two kinds of relative quantitation method.The first detects examination with the gene amplification of Roche HER2
Agent box (only limit scientific research uses, and does not apply for clinical license) is the Δ Δ Ct method of representative, its Cleaning Principle be according to HER2 gene with
The copy number difference that the Ct value difference different (being Δ Δ Ct after the Δ Ct correction through sample for reference) of reference gene amplified reaction compares,
And the difference of amplification efficiency is difficult to eliminate and PCR equipment also has difference between hole between the system of actually distinct PCR reaction, therefore
And the method is difficult to meet the requirement to stability, precision for the clinical HER2 detection.The reference gene selected additionally, due to it
Gastrin there is also amplification phenomenon in part patients with gastric cancer, therefore after Herceptin increased gastric cancer indication, this reagent
Box has exited market.Its two be with match silent fly your relative standard curve method as representative of generation, obtained really using standard curve
Amplification efficiency value, can correction result to a certain extent.Concrete grammar is a series of genes of interest using variable concentrations gradients
And after standard curve drawn by reference gene standard substance (plasmid, genomic DNA or chemistry become one of sequence), calculate sample respectively
Genes of interest in this, reference gene are with respect to the copy number of respective standard substance.But the absolute quantitation difficulty due to standard substance itself
Height, standard concentration range of error is larger so that genes of interest is difficult to control to the concentration ratio relation of reference gene, can cause
Error in experimental result.
Content of the invention
The present inventor is directed in existing gene copy number relative quantification detection method due to standard substance itself absolutely
, standard concentration range of error high to quantitative difficulty is larger, leads to genes of interest and the concentration ratio relation of reference gene to be difficult to
Control, thus the experimental result error causing and detection method are complicated, expend time cost higher the problems such as, there is provided a kind of
Easy, efficient real-time fluorescence quantitative PCR detection method and its standard substance and detection kit.
In order to solve above-mentioned technical problem, one of technical scheme that the present invention takes is：A kind of gene copy number is relatively fixed
The real-time fluorescence quantitative PCR detection method of amount, this real-time fluorescence quantitative PCR detection method comprises the following steps：
(1) reference gene is connected with genes of interest fragment equal proportion to be measured, is cloned on same plasmid vector, build
A kind of standard substance being simultaneously used for reference gene and genes of interest augmentation detection to be measured, and by gained standard substance according to concentration
It is simultaneously used for after gradient dilution drawing the standard curve of reference gene and genes of interest to be measured；
(2) after sample to be tested carries out real-time fluorescence quantitative PCR amplification with the described standard substance of step (1) are synchronized, purpose base
Because calculating respective copy number with reference gene according to respective standard curve, calculate genes of interest and the reference base of sample to be tested
Because of copy number ratio, obtain final product the copy number relative quantification testing result of genes of interest.
Wherein step (1) is：Reference gene is connected with genes of interest fragment equal proportion to be measured, is cloned into same plasmid
On carrier, build a kind of standard substance being simultaneously used for reference gene and genes of interest augmentation detection to be measured, and by gained mark
Quasi- product are simultaneously used for drawing the standard curve of reference gene and genes of interest to be measured according to after Concentraton gradient dilution.
Wherein said reference gene is this area routine reference gene, and preferably house-keeping gene is it is therefore preferable to the expansion of RPPH1
Increase region, wherein said plasmid vector is this area conventional plasmid carrier, preferably PCR cloning vehicle it is therefore preferable to TA
Cloning carrier, described TA cloning carrier is preferably pMD18-T carrier.The nucleotide sequence of wherein said standard substance is such as
SEQ ID NO in sequence table:Shown in 6.Described equal proportion is preferably 1：1.Due to genes of interest to be measured in standard substance and ginseng
Ratio according to gene is 1:1, solve the concentration ratio of genes of interest and reference gene in current relative standard curve method's application
The unmanageable problem of relation, improves the reliability of HER2 gene amplification detection.
Step (2) is：After sample to be tested carries out real-time fluorescence quantitative PCR amplification with the described standard substance of step (1) are synchronized,
Genes of interest and reference gene calculate respective copy number according to respective standard curve, calculate the genes of interest of sample to be tested with
Reference gene copy number ratio, obtains final product the copy number relative quantification testing result of genes of interest.
Wherein said genes of interest to be measured is the conventional genes of interest to be measured in this area, the preferably spy of tumor or disease
Specific gene, more preferably for the amplification region of HER2 gene, described fluorescent quantitative PCR is that this area conventional fluorescent is quantitative
PCR amplification method, described fluorescent quantitative PCR method is preferably multichannel fluorescent quantitative PCR, more preferably for bilateral
Road fluorescent quantitative PCR.
Wherein said standard curve is this area conventional fluorescent quantitative pcr amplification method gained standard curve, described standard
The method for drafting of curve is this area routine method for drafting.Term " copy number " of the present invention is this area conventional sense, that is,
Refer to number in a certain biological genome for certain gene.
In order to solve above-mentioned technical problem, the two of technical scheme that the present invention takes is：One kind is used for gene copy number phase
Standard substance to the detection of quantitative real-time fluorescence quantitative PCR, described standard substance are that a kind of equal proportion comprises reference gene fragment and treats
Survey the plasmid vector of genes of interest fragment.
Wherein said reference gene is the conventional reference gene in this area, preferably house-keeping gene, more preferably for
RPPH1 gene.Wherein said genes of interest to be measured is the conventional genes of interest to be measured in this area, preferably tumor or disease
Specific gene, more preferably for HER2 gene.Wherein said plasmid vector is plasmid vector commonly used in the art, preferably
PCR cloning vehicle, more preferably for TA cloning carrier, described TA cloning carrier is preferably pMD18-T carrier.Wherein
The nucleotide sequence of described standard substance is preferably as SEQ ID NO in sequence table:Shown in 6.
In order to solve above-mentioned technical problem, the three of technical scheme that the present invention takes is：A kind of real-time fluorescence quantitative PCR inspection
Test agent box, this real-time fluorescence quantitative PCR detection kit includes：Taq enzyme, PCR reaction buffer, dNTP mixed liquor, purpose
Gene and each self-corresponding specificity upstream and downstream primer of reference gene and fluorescent probe, standard substance, negative control and the positive are right
According to.
Wherein said genes of interest is preferably HER2 gene, and its corresponding specific forward primer sequence is preferably such as
SEQ ID NO:Shown in 7, its corresponding specific Down Stream primer is preferably as SEQ ID NO:Shown in 8, its corresponding probe is
This area conventional fluorescent probe, preferably Taqman fluorescence probe.Described probe is preferably：It is connected with fluorescence at 5 ' ends to send out
Light group, is connected with fluorescent quenching group at 3 ' ends, and described transmitting group is preferably this area conventional fluorescent transmitting group, excellent
Selection of land is HEX, and described fluorescent quenching group is preferably TARMA, and the sequence of described fluorescent probe is preferably as SEQ in sequence table
ID NO:Shown in 11.
Wherein said reference gene is preferably RPPH1 gene, and its corresponding specific forward primer sequence is preferably such as
SEQ ID NO:Shown in 9, its corresponding specific Down Stream primer is preferably as SEQ ID NO:Shown in 10, its corresponding probe is
This area conventional fluorescent probe, preferably Taqman fluorescence probe.Described probe is preferably connected with fluorescence radiation base at 5 ' ends
Group, is connected with fluorescent quenching group at 3 ' ends, and described transmitting group is preferably this area conventional fluorescent transmitting group, preferably
For FAM, described fluorescent quenching group is preferably TARMA, and the sequence of described fluorescent probe is preferably as SEQ ID in sequence table
NO:Shown in 12.
Wherein said standard substance are this area conventional criteria product, and preferably equal proportion comprises reference gene RPPH1 gene
And the pMD18-T carrier of genes of interest HER2 gene to be measured, SEQ ID NO in its sequence such as sequence table:Shown in 6.
Wherein said Taq enzyme is this area routine Taq enzyme, preferably hot start Taq polymerase；PCR reaction buffer is this
Field Standard PCR reaction buffer, the formula of described PCR reaction buffer is this area conventional formulation, preferably commercially available,
Such as when the buying hot start Taq polymerase 10X PCR buffer of supporting offer, wherein said dNTP mixed liquor is that this area is conventional
DNTP mixed liquor.
Wherein said negative control and positive control are this area Routine control product, and wherein negative control is preferably HER2
It is 1 with RPPH1 genetic fragment ratio：1 single plasmid, wherein HER2 are preferably with RPPH1 genetic fragment concentration
5E6copies/μL.Positive control is preferably made up of the plasmid of the target gene comprising different proportion and crt gene, wherein
HER2 and RPPH1 amplified fragments ratio is preferably 4：1, wherein RPPH1 genetic fragment concentration is preferably 5E6copies/ μ L,
HER2 genetic fragment concentration is preferably 2E7copies/ μ L.
The each composition of described PCR amplification system is final concentration of：Primer concentration corresponding to genes of interest, reference gene is preferably
For 0.14-0.18 μM, more preferably for 0.16 μM；Preferably 0.06-1.0 μM of concentration and probe concentration, more preferably for 0.08 μM；Taq enzyme
Consumption is preferably 0.02-0.06U, and more preferably for 0.04U, described Taq enzyme is more preferably hot start Taq polymerase.
Present invention also offers real-time fluorescence quantitative PCR detection kit as above is in detection testing sample HER2 base
Purposes during gene-amplification state.Testing sample is this area routine testing sample, is preferably comprised：Flesh tissue, paraffin embedding
Tissue and peripheral blood reclaim the samples such as cell.
On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, obtain final product each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are all commercially available.
The positive effect of the present invention is：The present invention provide standard substance be by comprise genes of interest HER2 gene and
The nucleotide sequence of reference gene RPPH1 gene is according to 1:The plasmid that 1 ratio is formed by connecting.This standard substance can solve the problem that at present relatively
In standard curve method application, genes of interest and the unmanageable problem of concentration ratio relation of reference gene, improve HER2 gene and expand
Increase the reliability of detection, can correctly detect and evaluate HER2 gene amplification state in breast cancer tissue or cell.
The real-time fluorescence quantitative PCR detection method that the present invention provides carries out HER2 gene and ginseng using dual pathways fluorescent PCR
According to the synchronous amplification of gene, detection flux can be significantly improved, testing cost is greatly reduced.Meanwhile, inspection provided by the present invention
Test agent box has the easy and simple to handle, advantage such as good stability, repeated height, detection efficiency height.The present invention is for the HER2 positive
The diagnosis of breast carcinoma, resists research and development, quality control or even the clinical practice of this disease medicament all to have great importance.
Brief description
Fig. 1 is the plasmid map that the embodiment of the present invention 3 builds gained standard substance, and arrow indication is in carrier and inserts gene
The position of PCR primer sequence.
Fig. 2 is real-time fluorescence quantitative PCR amplification.Wherein, Fig. 2 (A) is with the embodiment of the present invention 3 gained plasmid control
Product are template, and single channel carries out the amplification curve of HER2 gene amplification, and Fig. 2 (B) is with the embodiment of the present invention 3 gained plasmid control
Product are template, and single channel carries out the amplification curve of RPPH1 gene amplification, and Fig. 2 (C) is the embodiment of the present invention 3 gained plasmid control
The amplification curve diagram of product dual pathways synchronous amplification HER2 and RPPH1.
Fig. 3 is the standard curve of HER2 and RPPH1 being drawn according to Fig. 2 (C).
Fig. 4 is HER2 the and RPPH1 gene amplification curve of sample described in embodiment 5.Wherein Fig. 4 (A) is that numbering is
The gene amplification curve of 1520438 paraffin-embedded tissue, Fig. 4 (B) is the gene of the paraffin-embedded tissue that numbering is 1601415
Amplification curve.
Fig. 5 is described real-time fluorescence quantitative PCR detection method schematic diagram.
Specific embodiment
Below example is used for being provided on how to implement for those skilled in the art and uses the present invention
Full disclosure and description, and the invention scope that these examples are not intended to inventor is thought limits, also non-meaning
The experiment referring to hereafter is all experimentss being carried out and is only enforceable experiment.The reality of unreceipted actual conditions in experimental example
Proved recipe method, generally according to normal condition or according to the condition proposed by manufacturer.
Embodiment 1 Human cell line genome DNA extraction
The K562 cell line that the genomic DNA that plasmid standard of the present invention builds comes from In vitro culture (is bought certainly
ATCC company).
Introduce cell line dna extraction step taking the Allprep DNAmini KIT using Qiagen company as a example：
(1) cell number is counted, if cell number ＜ 5 × 106, add 350 μ L Buffer RLT Plus；If cell
Number≤1 × 107, add 600 μ L Buffer RLT Plus (if volume is excessive, extracting by several times).
(2) transfer in Allprep DNA adsorption column after mixing and (be placed in 2mL collecting pipe), high speed centrifugation 30 seconds.
(3) change a new 2mL collecting pipe, add 500 μ L Buffer AW1, high speed centrifugation was outwelled in collecting pipe after 15 seconds
Liquid.
(4) 500 μ L Buffer AW2, high speed centrifugation 2 minutes are added.
(5) change a new collecting pipe (1.5mL is with cover), add 100 μ L Buffer EB, room temperature is placed 1 minute.The most at a high speed from
The heart affords DNA in 1 minute.
Embodiment 2 mankind's paraffin-embedded tissue genome DNA extraction
The present invention test sample for paraffin-embedded human mammary cancerous tissue (from the attached length of The 2nd Army Medical College
Extra large hospital).
Introduce sample DNA extraction step taking the GeneRead DNA FFPE KIT using Qiagen company as a example：
(1) being organized in 1.5mL centrifuge tube on scraping paraffin section.
(2) add 160 μ L deparaffnize solution, be simply centrifuged after vortex 10s, 56 DEG C of incubation 3min.
(3) add after restoring balance at room temperature freshly prepared 100 μ L incubation buffering liquids/Proteinase K Solution (specifically by
55 μ L no RNase water, 25 μ L FTB buffer, 20 μ L E.C. 3.4.21.64s are formulated).After vortex centrifugal, after 56 DEG C are incubated 1 hour,
Directly proceed to 90 DEG C and continue incubation 1 hour.
(4) after simple centrifugation, take limpid bottom liquid, enter in new centrifuge tube, add 115 μ L no RNase water mixing.
(5) add 35 μ L UNG enzymes to be vortexed latter 50 DEG C to be incubated 1 hour.
(6) add 2 μ L ribonuclease A (100mg/ml) mixing after simple centrifugation, after being incubated 2 minutes under room temperature, add
250 μ L AL buffer, add 250 μ L ethanol after the concussion that is vortexed at a high speed, are centrifuged after the concussion that is vortexed at a high speed again.
(7) proceed to lysate (if volume is higher than 700 μ L, graded proceeds to) toward in QIAampMinElute post, note not
Tube wall to be touched.High speed centrifugation, after 1 minute, removes all lysates.
(8) add high speed centrifugation 1 minute after 500 μ L cleaning buffer solutions 1, remove all buffer.
(9) add high speed centrifugation 1 minute after 500 μ L cleaning buffer solutions 2, remove all buffer.
(10) transfer in new collecting pipe, high speed centrifugation 1 minute is to remove all residual solution.
(11) transfer in new 1.5mL centrifuge tube, uncap, add 20-40 μ L eluent (Deca is at the center of film).
(12), after incubation 1-5 minute under room temperature, high speed centrifugation obtained genomic DNA after 1 minute.
Embodiment 3 prepares particular criteria product
1st, the preparation of carrier
TA cloning vehicle pMD18-T is purchased from promega company.
2nd, the preparation of Insert Fragment
2.1st, the preparation of genes of interest and reference gene amplification region
Prepare the genetic fragment of genes of interest and reference gene using PCR method, pcr template is to extract through embodiment 1
Cell genomic dna, reaction system is as shown in table 1 (PCR reaction system 1).
The reaction system (50 μ L/ pipes, PCR reaction system 1) of target gene fragment prepared by table 1, PCR amplification
Add forward and reverse primer sequence outside HER2 in PCR amplification system, HER2 genetic fragment can be prepared；Amplification
Add forward and reverse primer sequence outside RPPH1 in system, RPPH1 genetic fragment can be prepared, described sequence is as shown in table 2,
PCR amplification condition is as shown in table 3.
Table 2, the Outside primer of PCR reaction system and connection primer sequence
Table 3, the PCR amplification condition of preparation genetic fragment
2.2 genetic fragments connecting genes of interest and reference gene
Reclaim after target fragment using agarose gel DNA QIAquick Gel Extraction Kit (purchased from Tiangeng company), using PCR method even
Connect HER2 genetic fragment and RPPH1 genetic fragment, be prepared into Insert Fragment, pcr amplification primer thing and connection primer sequence such as table 2 institute
Show, target sequence connects PCR reaction system as shown in table 4 (PCR reaction system 2), and PCR amplification condition is as shown in table 5.
Table 4, target sequence connect PCR reaction system (50 μ L, PCR reaction system 2)
Table 5, target sequence connect PCR system reaction condition
Reclaim target fragment using agarose gel QIAquick Gel Extraction Kit.
3rd, the structure of carrier and identification
By TA clone, Insert Fragment is connected into pMD18-T carrier (purchased from promega company), described TA cloning process
Refer toCarrier system product description.The new plasmid building is carried out in e. coli jm109 bacterial strain in a large number
Amplification, after carrying out blue white macula screening, picking monoclonal send sequencing, as standard substance after sequencing is correct.The nucleotide of gained standard substance
SEQ ID NO in sequence such as sequence table:Shown in 6.
Fig. 1 is the plasmid map that the present invention builds gained standard substance, and arrow indication is carrier insertion gene PCR product sequence
The position of row.
The comparison of the single, double channel fluorescence quantitative pcr amplification result of embodiment 4
1st, the single channel fluorescent quantitative PCR result of genes of interest and reference gene
1.1st, single channel quantitative fluorescent PCR reaction template is the standard substance of embodiment 3 preparation, right for blank with distilled water
According to.
1.2nd, single channel quantitative fluorescent PCR reaction system is as shown in table 6 to (PCR reaction system 3), PCR reaction condition such as table 8
Shown.
Table 6, single channel quantitative fluorescent PCR reaction system (25 μ L/ pipes, PCR reaction system 3)
Need in HER2 gene by fluorescence quantitative PCR amplification system to add forward and reverse primer and HER2 probe inside HER2；
Need in RPPH1 fluorescent quantitative PCR system add RPPH1 inside forward and reverse primer and RPPH1 probe, described primer and
The sequence of probe is as shown in table 7.In described probe, the fluorescent emission group that labelling HER2 gene by fluorescence probe 5 ' is held is HEX, 3 '
End fluorescent quenching group be TARMA, the fluorescent emission group that labelling RPPH1 gene by fluorescence probe 5 ' is held be FAM, 3 ' hold glimmering
Optical quenching group is TARMA.
The nucleotide sequence of table 7, inner primer and fluorescent probe
Table 8, quantitative fluorescent PCR system reaction condition
Fluorescent quantitative PCR result is as shown in Fig. 2 wherein：In Fig. 2 (A), 8 curves of rising are with variable concentrations
The plasmid standard of (4 times of gradient dilutions) is template, and the amplification carrying out HER2 gene single channel fluorescent quantitative PCR gained is bent
Line, from left to right plasmid standard concentration be followed successively by 3.2E8copies/ μ L, 8E7copies/ μ L, 2E7copies/ μ L, 5E6,
1.25E6copies/ μ L, 3.12E5copies/ μ L, 7.81E4copies/ μ L, the plasmid standard of 1.95E4copies/ μ L
Amplification curve.In Fig. 2 (B), 8 curves of rising are with the plasmid standard of variable concentrations (4 times of gradient dilutions) as template, enter
The amplification curve of row RPPH1 gene single channel fluorescent quantitative PCR gained, from left to right plasmid standard concentration be followed successively by
3.2E8copies/μL、8E7copies/μL、2E7copies/μL、5E6、1.25E6copies/μL、3.12E5copies/μL、
7.81E4copies/μL、1.95E4copies/μL.
2nd, the dual pathways fluorescent quantitative PCR result of genes of interest and reference gene
2.1 quantitative fluorescent PCR reaction template are the standard substance of embodiment 3 preparation, with distilled water as blank.
2.2nd, needed for the reaction of dual pathways quantitative fluorescent PCR, primer and probe sequence are as shown in table 7, quantitative fluorescent PCR system
Reaction condition as shown in table 8, dual pathways quantitative fluorescent PCR reaction system is as shown in table 9 (PCR reaction system 4).
Table 9, dual pathways quantitative fluorescent PCR reaction system (25 μ L/ pipes, PCR reaction system 4)
Standard substance gained CT value result according to serial dilutions draws standard curve.Fig. 2 (C) is to be implemented with the present invention
Example 3 gained particular criteria product are template, the amplification curve diagram of dual pathways quantitative fluorescent PCR synchronous amplification HER2 and RPPH1 gained.
Plasmid standard concentration be followed successively by 3.2E8copies/ μ L, 8E7copies/ μ L, 2E7copies/ μ L, 5E6,
1.25E6copies/μL、3.12E5copies/μL、7.81E4copies/μL、1.95E4copies/μL.In in figure, solid line generation
The amplification curve of table HER2 gene, dotted line represents the amplification curve of RPPH1 gene, from left to right 4 times of ladders of plasmid standard concentration
Degree reduces.
3rd, the comparison of single, double channel fluorescence quantitative pcr amplification result
Can be seen that accuracy and the single-pass of dual pathways fluorescent quantitative PCR detection method acquired results from result shown in Fig. 2
Road detecting system highly consistent, and have raising detection flux, save testing cost advantage.
HER2 gene amplification state in embodiment 5 dual pathways fluorescence quantitative PCR detection mankind's paraffin-embedded tissue sample
1st, quantitative fluorescent PCR reaction template is the breast carcinoma sample genomic dna that embodiment 2 is extracted, with distilled water as sky
White comparison, it is 1 that negative control prepares gained HER2 and RPPH1 genetic fragment ratio for embodiment 3：1 single plasmid, HER2 with
RPPH1 amplified fragments concentration is 5E6copies/ μ L；Positive reference substance is made up of two kinds of different plasmids, adjustment HER2 with
RPPH1 amplified fragments ratio is 4：1, wherein RPPH1 amplified fragments concentration is 5E6copies/ μ L, and HER2 amplified fragments concentration is
2E7copies/μL.
2nd, quantitative fluorescent PCR reaction the primer and fluorescent probe sequence, amplification reaction condition and reaction system referring to
Table 7, table 8, table 9 content.Carry out HER2 and RPPH1 gene test using dual pathways fluorescence quantifying PCR method simultaneously.HER2 expands
Need in system to add HER2-F-2 and HER2-R-2 primer and its fluorescent probe.Need in RPPH1 amplification system to add
RPPH1-F-2 and RPPH1-R-2 primer and its fluorescent probe.
3rd, draw standard curve
As shown in Fig. 2 (C), with the plasmid standard containing equal proportion genes of interest and reference gene genetic fragment as mould
Plate, after carrying out the amplification curve that dual pathways fluorescence quantitative PCR detection synchronously obtains HER2 and RPPH1 gene, can draw further
Obtain the standard curve of corresponding gene.Fig. 3 is the standard curve of HER2 and RPPH1 being drawn according to Fig. 2 (C), wherein black side
What block curve represented is the standard curve of HER2 gene, and what white square curve represented is the standard curve of RPPH1 gene.
4th, utilize the amplification state of HER2 gene in gene copy number relative quantification Evaluation of detection methods sample
According to the standard curve drawn, HER2 and RPPH1 gene copy number can be obtained respectively, so by calculate HER2 with
The ratio relation of RPPH1 gene copy number realizes the relative quantification to HER2 gene copy number, and Fig. 5 is described real time fluorescent quantitative
PCR detection method schematic diagram.
Fig. 4 is two sample HER2 and RPPH1 gene amplification curve in the present embodiment.Wherein Fig. 4 (A) is that numbering is
The gene amplification curve of 1520438 paraffin-embedded tissue, its CT value is 22.57；Fig. 4 (B) is the paraffin that numbering is 1601415
The gene amplification curve of investing tissue, its Ct value is 27.42.The CT value of reference gene RPPH1 is respectively 25.33 and 27.75, point
Do not tried to achieve after respective copy number by corresponding calibration curve formula (Fig. 3), obtain HER2/RPPH1 and be respectively 7.86 (HER2 expansions
Increase) and 1.72 (HER2 does not expand).
5th, Δ Δ Ct method is utilized to assess the amplification state of HER2 gene in sample
As indicated above, by real-time fluorescence quantitative PCR detection method, it is obtained in that mesh in unknown sample and check sample
Gene and reference gene Ct value, the amplification times of HER2 can be calculated afterwards according to formula 1,2,3.Described formula 1,2,3
As follows：
Formula 1：Δ Ct=Ct genes of interest-Ct reference gene
Formula 2：Δ Δ Ct=Δ Ct unknown sample-Ct check sample；
Formula 3：Multiple change=2-ΔΔCt.
To comprise HER2 and RPPH1 genetic fragment ratio for 1 in the present embodiment：1 single plasmid is as check sample, right
Described originally carry out quantitative fluorescent PCR detection by quantitative in the same old way, the Average Ct values according to HER2 and RPPH1 obtain average delta Ct control sample
This value；
When assessing the HER2 gene amplification situation of unknown sample, analytical procedure is：
Firstth, pass through qPCR detection by quantitative, after obtaining genes of interest and the Ct value of reference gene, Δ is obtained according to formula 1
Ct unknown sample, Δ Ct check sample；
Secondth, utilize formula 2 and existing Δ Ct unknown sample value and Δ Ct check sample value, calculate after Δ Δ Ct, that is,
Multiple variation relation can be obtained according to formula 3.
Amplification situation using the HER2 gene to 50 samples for the Δ Δ Ct method is estimated, and specifically tests and analyzes result
It is shown in Table 10.
6th, pattern detection interpretation of result
The present embodiment has carried out the relative quantification detection of HER2 gene to 50 breast carcinoma paraffin wax embedded tissue samples, and with
Fluorescence in situ hybridization (FISH), the testing result of SABC (IHC) compare.
Sample processing method of the present invention is with reference to the GeneRead DNA FFPE KIT operation instruction of Qiagen company, primer
Sequence, amplification system and amplification condition, with reference to content described in table 7,8,9, are expanded on ABI7500 quantitative real time PCR Instrument,
Using two kinds of HER2 gene relative quantification detection methods, amplification is estimated simultaneously.IHC detection antibody purchase in this experiment
From Abcam company, FISH detectable is purchased from Abbott Laboratories Vysis, and experimental implementation is carried out with reference to description.Two kinds of present invention offer
HER2 gene amplification detection method and fluorescence in situ hybridization (FISH), the testing result of SABC (IHC), as shown in table 10.
HER2 the and RPPH1 gene copy number proportionate relationship of 10,50 paraffin-embedded tissue samples of table and its fluorescence immunoassay
In situ hybridization and the testing result of SABC
IHC is to carry out HER2 albumen mistake on protein level using the paraffin section immunity chromogenic reaction of formalin fix
Detection of expression, and FISH is then by fluorescently-labeled nucleic probe and fixing endonuclear HER2 gene on paraffin section
DNA target sequence hybridize to detect its gene amplification situation on chromosome.Also need to into one when IHC testing result is for 2+
Step carries out FISH detection and makes a definite diagnosis.
Found after the result of analytical table 10 gained, two kinds of HER2 that 30 FISH positive sample, the present invention are provided
The detection of gene relative quantification detection method is all judged as that HER2 amplification is positive；To 20 FISH negative sample, the present invention provides
Two kinds of HER2 gene relative quantification detection method detections are all judged as that HER2 amplification is negative, show, two kinds of sides that the present invention provides
The result that method obtains is highly consistent with clinical goldstandard FISH result.
Traditional IHC detection and FISH detection method not only complex operation in Clinical detection, expensive, and consume
When longer (IHC detection need 1-2 working day, FISH detection method then needs 4-5 working day) it is impossible to be standardized.With
The method of the invention is compared, and not only sensitivity is relatively low, and also non-specific false positive.
The whole detection process of this method can complete in 3-5 hour, achievable full-automatic operation；In addition this method adopts
Dual channel detection, easy and simple to handle, detection while once can achieve 40-50 sample, and FISH is entirely manual operations, once
Detection sample is difficult to more than 10 person-portions；Last this method is easy and simple to handle, thus good stability, its repeatability be much better than IHC and
FISH detects.Therefore this method can become the new standard of clinical HER2 gene test completely, has huge economic worth.
It should be understood that after the above having read the present invention, those skilled in the art can make various to the present invention
Change or change, these equivalent form of values equally fall within the application appended claims limited range.
Sequence table
<110>The three lives, state was good for Pharmaceutical（Shanghai）Limited company
<120>A kind of real-time fluorescence quantitative PCR detection method and its standard substance and detection kit
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Claims (10)
1. a kind of real-time fluorescence quantitative PCR detection method of gene copy number relative quantification is it is characterised in that described real-time fluorescence
Quantitative PCR detecting method comprises the following steps：
(1) reference gene is connected with genes of interest fragment equal proportion to be measured, is cloned on same plasmid vector, build a kind of
Reference gene and the standard substance of genes of interest augmentation detection to be measured can be simultaneously used for, and by gained standard substance according to Concentraton gradient
It is simultaneously used for after dilution drawing the standard curve of reference gene and genes of interest to be measured；
(2) sample to be tested with the described standard substance of step (1) are synchronized carry out real-time fluorescence quantitative PCR amplification after, genes of interest with
Reference gene calculates respective copy number according to respective standard curve, and the genes of interest calculating sample to be tested is copied with reference gene
Shellfish number ratio, obtains final product the copy number relative quantification testing result of genes of interest.
2. real-time fluorescence quantitative PCR detection method as claimed in claim 1 is it is characterised in that the described reference gene of step (1)
For RPPH1 gene, described genes of interest to be measured is HER2 gene, and described equal proportion is 1:1；Described plasmid vector is cloned for PCR
Carrier, SEQ ID NO in the nucleotide sequence such as sequence table of described standard substance:Shown in 6.
3. real-time fluorescence quantitative PCR detection method as claimed in claim 1 is it is characterised in that the described fluorescent quantitation of step (2)
PCR expands as dual pathways fluorescent quantitative PCR.
4. a kind of standard substance of the real-time fluorescence quantitative PCR detection for gene copy number relative quantification are it is characterised in that described
Standard substance are the plasmid vectors that a kind of equal proportion comprises reference gene and genes of interest fragment to be measured.
5. standard substance as claimed in claim 4 it is characterised in that described reference gene be RPPH1 gene, described purpose to be measured
Gene is HER2 gene, and described equal proportion is 1：1；Described plasmid vector is PCR cloning vehicle, the nucleotide sequence of described standard substance
As SEQ ID NO in sequence table:Shown in 6.
6. a kind of real-time fluorescence quantitative PCR detection kit is it is characterised in that this real-time fluorescence quantitative PCR detection kit bag
Include：PCR reaction buffer, Taq enzyme, dNTP mixed liquor, genes of interest and each self-corresponding specific primer of reference gene and
Fluorescent probe, standard substance, negative control and positive control.
7. real-time fluorescence quantitative PCR detection kit as claimed in claim 6 is it is characterised in that described genes of interest is
HER2 gene, SEQ ID NO in its corresponding specific forward primer sequence such as sequence table:Shown in 7, under its corresponding specificity
Trip primer such as SEQ ID NO in sequence table:Shown in 8, SEQ ID NO in its corresponding probe sequence such as list:Shown in 11.
8. real-time fluorescence quantitative PCR detection kit as claimed in claim 6 is it is characterised in that described reference gene is
RPPH1 gene, SEQ ID NO in its corresponding specific forward primer sequence such as sequence table:Shown in 9, its corresponding specificity
SEQ ID NO in downstream primer such as sequence table:Shown in 10, SEQ ID NO in its corresponding probe sequence such as sequence table:12 institutes
Show.
9. real-time fluorescence quantitative PCR detection kit as claimed in claim 6 is it is characterised in that described standard substance are to wait ratio
Example comprises reference gene RPPH1 gene and the pMD18-T carrier of genes of interest HER2 gene to be measured, in its sequence such as sequence table
SEQ ID NO:Shown in 6.
10. real-time fluorescence quantitative PCR detection kit as claimed in claim 6 is it is characterised in that in each amplified reaction,
Final concentration of 0.14-0.18 μM of primer corresponding to described genes of interest, reference gene, corresponding probe is final concentration of
0.06-1.0 μM, the consumption of Taq enzyme is 0.02-0.06U.
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| PCT/CN2017/073336 WO2018086263A1 (en) | 2016-11-10 | 2017-02-13 | Real-time fluorescent quantitative pcr detection method, and standard sample and detection kit thereof |
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