CN101691602A - Taqman fluorescence probe quantitative PCR method adopting plasmid constructed by tree shrew CD81 gene as standard substance - Google Patents
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Abstract
The invention relates to a method for detecting and estimating the change situation of CD81 molecule which is infected by hepatitis C virus (HCV) in vivo and in vitro, in particular to a Taqman fluorescence probe quantitative PCR detection method on the basis of using cloned tree shrew CD81 plasmid as standard substance, belongs to the molecular biology field. The method comprises the following steps: cloning tree shrew CD81 gene segment; designing reasonable probe and primer with Primers Express2.0 software on the cloned tree shrew CD81 gene segment; designing experiment to select annealing temperature (Tm value), primer concentration and probe concentration under optimal conditions; further adopting tree shrew CD81 plasmid as standard substance to perform 10 times concentration gradient dilution and building standard curve; verifying the repeatability, stability and sensitivity of the method of the invention; and finally detecting the change of CD81 molecule which is infected by hepatitis C virus (HCV) in vitro by the method of the invention. The method can detect the change situation of tree shrew CD81 accurately, fast and conveniently.
Description
Technical field:
The invention belongs to biology field, relate to a kind of detection and estimate tree shrew by the method for CD81 molecule changing conditions in HCV (hepatitis C virus) body, before and after the Infection in Vitro, in particular, relating to a kind of tree shrew CD 81 plasmid with the clone is standard substance, on this basis the detection method with the Taqman fluorescence probe quantitative PCR of Jian Liing.
Background technology:
Hepatitis C mainly by blood/body fluid communication, accounts for 70% of post-transfusion hepatitis due to being infected by hepatitis C virus (HCV).It is the major cause that causes chronic hepatopathy in the worldwide that HCV infects.WHO estimates that the whole world has 1.7 hundred million people to infect hepatitis C virus.Anti-HCV positive rate is 0.7%～3.1% in China's healthy population, about 3,800 ten thousand people [2].HCV infects and easily causes chronicity, and adult's acute infection of 50%～80% easily develops into chronic hepatitis, wherein 20%～30% will develop into liver cirrhosis.Have 1%～4% to develop into hepatocellular carcinoma every year in the liver cirrhosis patient.Yet, the medicine that does not up to the present also have relevant vaccine and effectively cure, most important reason is not have effective cell in vitro culture system and the small animal model that is fit to.Cause its chronicity infection mechanism unclear, result in the HCV vaccine development, clinical treatment is taken a step and is heavily fortified point.
Tree shrew (Tupaia belangeri, Tree shrew) is distributed in Yunnan Province of China, Guangxi in a large number, and South East Asia one band, and is small, easily raises, and breeding is fast.Because biology grade height, tree shrew is used as Primates very early and comprises the Human diseases animal model many viral susceptibles relevant with human diseases.Recent study is found, is used widely in the research of diseases such as nerve conduction, vision, diabetes, liver cancer.In addition, studies show that tree shrew not only can be infected in vivo by hepatitis C virus (HCV), and the primary hepatocyte of tree shrew is suitable can be external infected.So just provide good research platform for HCV chronic infection Study on Mechanism.
For HCV chronic infection Study on Mechanism, the research of its acceptor is again the maximum focus of research, the infection that studies show that HCV is by a plurality of receptor-mediated target cells that enter, these acceptors mainly comprise CD81, the I of B family type scavenger receptor (SR-BI), low density lipoprotein receptor (LDLr), between the specific for dendritic cells sexual cell adhesion molecule pounce on the plain molecule (DC-SIGN) of the nonconformity that obtains and lymphocyte specific intercellular adhesion molecule pounce on the plain molecule of nonconformity (LC-SIGN) that obtains and recently report HCV accessory receptor tight junction protein (Claudin-1) wherein CD81 during the HCV acceptor is studied, be considered to most probable a kind of principal recipient.
It belongs to CD81 four transmembrane proteins (teraspanin, TM4SF) superfamily member contains 4 and stride film and change film district and 2 extracellular regions, express in multiple tissue, have crucial biological function.People CD81 molecular weight is 25.8kD, its protein molecule has 236 amino-acid residues, there are 4 to stride film district (TM1-4), small one and large one two film outskirts (EC1 and EC2), its amino all is positioned at the cytolemma inboard with the C-terminal sequence, EC1 is striding between film district TM1 and the TM2, the little ring SEL high conservative of formation, and it has important effect for the cell surface expression of EC2 advantage.EC2 forms the diversity that different big ring LEL determines each species CD81 between TM3 and TM4, mediate the important effect that is combined with of CD81 and HCV.
Tree shrew is as a kind of possible new HCV animal model, in its body and external, the expression dependency of the infection of HCV and CD81 how, express on this aspect what similarities and differences is arranged as a kind of possible animal model with regard to CD81 with the physiognomy ratio, expression of CD81 relevant with other those acceptors again (such as Claudin-1) or the like, with regard to the research up to the present of these molecular biology aspects of tree shrew CD 81 seldom, yet wanting has clearer research to these aspects, more basic to the research of tree shrew CD 81 gene expression technology so.The real-time fluorescence quantitative PCR detection technique has realized the leap of PCR from qualitative to quantitative, wherein the method for Taqman probe especially high specificity, susceptibility height, good reproducibility, quantitatively accurately, characteristics such as speed is fast.Owing to do not find appropriate H CV small animal model now, and the acceptor of HCV and accessory receptor are not determined, cause the research that is considered to one of most important acceptor CD81 also to only limit to in the human CD81 study limitation, tree shrew is as a kind of suitable small animal model of HCV that probably becomes, and knows very few especially to its understanding of CD81.
Summary of the invention:
In view of the vacancy that exists in the prior art, purpose of the present invention just is to set up tree shrew CD 81 gene fluorescent quantitation Taqman PCR detection method.Set up that a kind of stability is strong, good reproducibility, tree shrew CD 81 detection method that susceptibility is high, from the aspect of acceptor, confirm that further tree shrew is as the suitable small animal model of HCV research.For the pharmacological agent of HCV chronic infection mechanism, HCV and the research of vaccine provide certain basis.
In order to realize above-mentioned purpose of the present invention, the present invention takes fluorescent quantitation Taqman round pcr, tree shrew CD 81 cDNA sequence according to the clone, design Auele Specific Primer and probe, set up the typical curve that is used to detect the tree shrew CD 81 gene expression amount, and verify its batch between, batch in, repeated problem and susceptibility between group, aspect organizing interior four.
Technical scheme provided by the invention is as follows: a kind of is the Taqman fluorescence probe quantitative PCR method of standard substance with the tree shrew CD 81 plasmid, comprise segmental clone of tree shrew CD 81 gene and order-checking, tree shrew CD 81 gene fragment with the clone is a target, design Taqman PCR primer and probe, contrived experiment finds annealing temperature, primer concentration, the concentration and probe concentration of primer under the optimal conditions, purification tree shrew CD 81 plasmid calculates CD81 gene fragment copy number, with 10 times of gradient dilutions, the preparation copy number is 10 with the tree shrew CD 81 plasmid that calculates
3～10
7The copies positive plasmid is set up typical curve, detects with the changing conditions of this detection architecture to the tree shrew primary hepatocyte sample CD81 of HCV Infection in Vitro.
Wherein, (1) PCR primer is two CD81 oligonucleotide primer F/R:
F：5’-ATGGGAGTGGAGGGCTGCACCAA-3’
R：5’-TCAGTACACGGAGCTGTTCCGGATG-3’
(2) be template with the RNA that extracts in the tree shrew liver, obtain tree shrew CD 81 cDNA fragment, this fragment purification and order-checking;
(3) behind this fragment purification, import in the PMD-18T plasmid, be converted into then among the competent escherichia coli cell JM-109 and increase by the T-A clone; Screening positive clone extracts clone's and carries out purifying, obtains the outer with reference to positive plasmid of tree shrew CD 81 molecular assay.
In addition, CD81 oligonucleotide primer and probe are:
CD81?FP：5’-GGAGGACTGCCACCAGAAGAT-3’
CD81?RP：5‘-CAGCACCATGCTCAGGATCA-3’
CD81?Probe：5‘(FAM)-AAGCTGTACCTCATCGGCATCGCG-(TAMRA)3’
This method comprises:
(1) determining of detection architecture annealing temperature,
(2) determining of detection architecture concentration and probe concentration and primer concentration,
(3) configuration 10
3～10
7The CD81 plasmid of copy number has been set up the typical curve of this detection architecture as template,
(4) batch between, batch in, between group, interior four levels of group verify the repeated and stable of this method system,
(5) utilize 10 times of gradient dilutions 10 of CD81 plasmid difference of determining concentration
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Copies obtains kinetic curve, detects the susceptibility of this detection architecture;
(6) utilize this detection architecture, CD81 gene in the tree shrew primary hepatocyte of different HCV infective doses is detected, determine the copy number of CD81 in the cell after the different infective doses.
The present invention is the foundation of the Taqman fluorescence probe quantitative PCR method of standard substance with the tree shrew CD 81 plasmid, and the step summary that it comprises is:
A is to segmental clone of tree shrew CD 81 gene and order-checking.
B is a target with clone's tree shrew CD 81 gene fragment, designs suitable primer and probe by Primer Express 2.0 softwares on this fragment.
The C contrived experiment obtains annealing temperature, primer concentration, the concentration and probe concentration of the primer under the optimal conditions and the plasmid of purifying calculates CD81 gene fragment copy number.
The tree shrew CD 81 plasmid of D to calculate, with 10 times of gradient dilutions, the preparation copy number is 10
3～10
7The copies positive plasmid is set up typical curve.
Repeatability, stability and the susceptibility of E checking present method.
F detects CD81 gene in the tree shrew primary hepatocyte sample of different HCV infective doses by this detection method.
Description of drawings:
Below in conjunction with accompanying drawing, further set forth the present invention with embodiments of the invention.Should be appreciated that these embodiment only are used to the present invention is described rather than limit the scope of the invention.
Fig. 1 is tree shrew HCV acceptor CD81 gene fragment RT-PCR electrophoresis detection result.
Fig. 2 is the full genome nucleotide sequence sequencing result of HCV acceptor CD81 in the tree shrew.
Fig. 3 is CD81 gene fragment amplification electrophoresis detection result under the different annealing temperature condition.
Fig. 4 is CD81 gene fragment amplification electrophoresis detection result under the different primer concentrations.
Fig. 5 is different probe concentration (50nm, 100nm, 150nm) amplification curve and Ct value.
Fig. 6 is with 1 * 10
3～1 * 10
7Copy number is typical curve and the linear relationship that template is set up.
Fig. 7 detects amplification curve for batch interior Taqman PCR repeatability.
Fig. 8 detects Taqman pcr amplification curve for repeatability between group.
Fig. 9 is the sample Taqman pcr amplification curve and the linear relationship of different concns.
Figure 10 is CD81 gene test result in the tree shrew primary hepatocyte sample of HCV infective dose.
Embodiment:
Embodiment 1: be the foundation of the Taqman fluorescence probe quantitative PCR method of standard substance with the tree shrew CD 81 plasmid:
The experimental technique of unreceipted actual conditions among the embodiment, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) or people such as Peng Xiuling, the genetically engineered experimental technique, condition described in (science tech publishing house), or the condition of advising according to manufacturer.The gene mapping of hepatitis C virus is edited medical molecular virology (Science Press, 2001) with reference to the document such as the Jin Qi that have reported.
To segmental clone of tree shrew CD 81 gene and order-checking:
1.PCR the amplification of primer design and PCR
The PCR primer be according to download among the GenBank 40 surplus the CD81 nucleotide sequence of a different plant species, by obtaining conserved sequence behind the sequence alignment, design therein and obtain, synthetic by U.S. genebase company.
From tree shrew hepatic tissue (100mg), extract RNA, add 5%RNas in (Promega) suspension with the 10mM DDT of 100 μ l with TrL20L System (GIBCO/BRL).The sample retention of 10 μ l is used for each RT-PCR at-80 ℃.
Single stage method RT-PCR reaction, 10 * One Step RNA PCR Buffer, 5 μ l, MgCl
2(25Mm) 10 μ l, dNTP Mixture (each 10mM) 5 μ l, RNase Inhibitor (40U/ μ l) 1 μ l, AMV RTase * L (5U/ μ l) 1 μ l, AMV-Optimized Taq (5U/ μ l) 1 μ l, peripheral upstream Auele Specific Primer (20 μ M) 1 μ l, peripheral downstream Auele Specific Primer (20 μ M) 1 μ l, the total RNA 1 μ l of tree shrew, RNase Fress dH
2O 24 μ l.With 50 μ l reaction systems, carry out the RT-PCR reaction by following condition: 50 ℃ of 30min, 94 ℃ of 2min; Carry out 94 ℃ of 30sec of 40 cyclic amplifications, 72 ℃ of 2min of 57 ℃ of 30sec then; 72 ℃ of 5min ,-20 ℃ of refrigerators are preserved standby.The amplified production size is 711bp, and 1% agarose gel electrophoresis detects (Fig. 1).
2.PCR the purifying of product
(BBI USA) finishes the purifying of PCR product to use DNA Gel Extraction Kit.The concrete operations step is as follows:
(1). prepare 1% sepharose, with above-mentioned steps 1 gained PCR product point sample, 80 volts of voltage electrophoresis 25 minutes.
(2). ultraviolet lamp downcuts down and contains target DNA agarose blob of viscose, puts into new 1.5ml eppendorf tube.
(3). the ratio in per 400 μ l/100mg sepharoses adds Binding Buffer, places 50～60 ℃ of water-baths 10 minutes, and glue is thoroughly melted.
(4). the sol solution that melts is transferred to cover be put in the purification column in the collection tube, room temperature was placed 2 minutes.Centrifugal 1 minute of 8000rpm.
(5). take off purification column, outwell the waste liquid in the collection tube, purification column is put into same collection tube, add 500 μ l Wash Solution, centrifugal 1 minute of 8000rpm room temperature.
(6). repeating step (5) is once.
(7). take off purification column, outwell the waste liquid in the collection tube, purification column is put into same collection tube, 12, centrifugal 15 seconds of 000rpm room temperature.
(8). purification column is put into a new 1.5ml eppendorf tube, add 30 μ lElution Buffer or water (pH＞7.0) in pillar film central authorities, room temperature or 37 ℃ were placed 2 minutes.
(9) .12, centrifugal 1 minute of 000rpm room temperature.The PCR product of purifying is stored in-70 ℃, and is stand-by.
3. purified product and pMD18-T carrier is connected and conversion
3.1 the preparation of recipient bacterium JM109 competent cell
(1). cultivate single bacterium colony of picking on 16-20 hour the fresh flat board of JM109 from 37 ℃, go in the 5ml LB nutrient solution, 37 ℃, the 150rpm shaking culture is spent the night.
(2). culture is gone in the 50ml LB nutrient solution, 37 ℃, 250rpm shaking culture 3 hours.
(3). culture is transferred in the centrifuge tube, placed on ice 15 minutes, make culture be cooled to 0 ℃.4 ℃ of 12000rpm centrifugal 10 minutes then, to reclaim cell.
(4). abandon supernatant, with the 0.1M CaCl of precooling
2The washed cell precipitation, centrifugal 10 minutes of 12000rpm; Use 10ml 0.1M CaCl then
2The suspension thalline, ice bath 30 minutes, 4 ℃ of 12000rpm centrifugal 10 minutes then, to reclaim cell.
(5). pour out supernatant liquor as far as possible.Use 4ml 0.1M CaCl then
2The solution re-suspended cell.
(6). can use after 12～24 hours in 4 ℃ of placements.The competent cell for preparing can directly use or add 30% glycerine packing and be stored in-70 ℃, and is stand-by.
3.2 purified product is connected and conversion with the pMD18-T carrier
Use the pMD18-T Vector (Dalian Bao Bio-Engineering Company) and the PCR product of step 2 purifying to carry out ligation: with 0.2ml PCR pipe 0.5 μ l pMD18-T Vector and 4.5 μ l PCR purified products to be mixed earlier, add 5 μ l ligation SolutionI again and mix, 16 ℃ connect 30 minutes or the connection of spending the night.
3.3 transformed competence colibacillus cell JM109
(1). 100 μ l JM109 competent cells and 10 μ l ligation liquid are mixed gently.
(2). ice bath 30 minutes, 42 ℃ of hot activations are 90 seconds then, and ice bath is 2 minutes again.
(3). add the LB liquid nutrient medium of 37 ℃ of preheatings of 400 μ l, cultivated 45 minutes in 37 ℃ of shaking table vibration 150rpm.
(4). get and cultivate the LB solid medium that the coating of bacterium liquid contains 100 μ g/mlAmp.
(5). culture dish was positioned over 37 ℃ of cultivations after 12-16 hour, and single bacterium colony that picking some (4-6/sample) grows carries out the PCR Rapid identification.
3.4 the screening of positive recombinant and evaluation
(1). the bacterium colony that picking grows on Amp+ selection substratum several (4-6/sample), place the LB liquid nutrient medium that contains 100 μ g/ml penicillin to cultivate 5ml earlier, 37 ℃ of shaken overnight are cultivated.
(2). get 500 μ l bacteria suspensions, 12000rpm abandoned supernatant with the precipitation thalline in centrifugal 1 minute, added 170 μ l STET lysates, resuspended thalline in precipitation.
(3). boiling lysis 45 seconds, centrifugal 10 minutes of 12000rpm carefully draws supernatant, abandons precipitation.
(4). as template, (YF2/YR2, C3/C4 or E1/E3) carries out pcr amplification and electrophoresis detection with the nested-PCR inner primer with this supernatant.The person is positive colony to detect the specific band.Select the LB liquid nutrient medium that positive colony places 50ml to contain 100 μ g/ml penicillin and carry out enlarged culturing.
3.5 the extraction of positive recombinant plasmid
Use MiniBEST Plasmid Purification Kit Ver.2.0 (Dalian Bao Bio-Engineering Company) to finish the extraction of positive recombinant plasmid.The concrete operations step is as follows:
(1). get the incubated overnight bacterium liquid of 1～4ml, 12, centrifugal 2 minutes of 000rpm abandons supernatant.
(2). bacterial precipitation fully suspends with the Solution I (containing RNase A1) of 250 μ l.
(3). the Solution II that adds 250 μ l spins upside down lightly and mixes 5～6 times, makes the abundant cracking of thalline, forms transparent liquid.
(4). add the Solution III of 4 ℃ of precoolings of 400 μ l, spin upside down lightly and mix 5～6 times, until forming consolidation aggegation piece, room temperature left standstill 2 minutes then.
(5). room temperature 12, centrifugal 5 minutes of 000rpm gets supernatant.
(6). the Spin Column in the test kit is placed on the Collection Tube.
(7). supernatant liquor in the aforesaid operations (5) is transferred among the Spin Column 12, and centrifugal 1 minute of 000rpm abandons filtrate.
(8). 500 μ l Rinse A are added among the Spin Column, 12, centrifugal 30 seconds of 000rpm abandons filtrate.
(9). 700 μ l Rinse B are added among the Spin Column, 12, centrifugal 30 seconds of 000rpm abandons filtrate.
(10). repetitive operation step (9).
(11). Spin Column is placed on the centrifuge tube of new 1.5ml, adds the Elution Buffer of 60 μ l in the centre of Spin Column film, room temperature left standstill 1 minute.
(12) .12, centrifugal 1 minute wash-out recombinant plasmid dna of 000rpm.
4. the nucleotide sequencing of recombinant plasmid
The nucleotide sequencing of all recombinant plasmids all entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to finish examining order (Fig. 2) in this research.
5.Taqman the foundation of PCR detection architecture
5.1Taqman the design of PCR primer probe sequence and synthetic
According to recombinant plasmid fragment 711bp sequence, utilize Primer express.2.0 design primer and probe, primer is crossed over intron, purpose fragment length 118bp.The sequence situation is as follows:
Tupaia-CD81?FP：5’-GGAGGACTGCCACCAGAAGAT-3’
Tupaia-CD81?RP：5‘-CAGCACCATGCTCAGGATCA’-3’
Tupaia-CD81?probe：5‘(FAM)-AAGCTGTACCTCATCGGCATCGCG-(TAMRA)3’
Primer and probe are synthetic by the precious biological company limited in Dalian.
5.2PCR determining of amplification and annealing temperature
Design 56 ℃ of annealing temperatures, 58 ℃, 60 ℃, 62 ℃ definite its optimum annealing temperatures respectively according to this primer and probe characteristics.Adopting following reaction system and condition, is that template applications PCR instrument increases with PMD-18T CD81 plasmid: Permix Taq enzyme Buffer 25ul, 2ul, template 5ul mix separately for DEPC treating water 16ul, Tupaia-CD81FP/RP.Cycle index is 40,94 ℃ of 2min; 94 ℃ of 30s, (56 ℃, 58 ℃, 60 ℃, 62 ℃) 30s, 72 ℃ of 1min, 40 circulations.72 ℃ of 5min amplifications; 4 ℃ standby.Amplified production 2.5% agarose electrophoresis detects.By the electrophoresis detection result as can be known: the amplified fragments that can both obtain 112bp under the different annealing temperature, but under 54 ℃ and 56 ℃ of conditions tangible non-specific amplification is arranged, electrophoretic band is clear under 60 ℃ of conditions, the band correct position does not have non-specific amplification, so this detection architecture is determined Tm=60 ℃ (Fig. 3).
5.3 determining of primer and concentration and probe concentration:
Primer concentration: with can detected template concentrations under the same terms minimum, the amplified production band is single, clear to be the primer choice criteria.With the CD81 TaqMan PCR upstream and downstream primer concentration of 100nm, 200nm, 300nm, 4 different concns of 400nm, being respectively applied for same concentration C D81 recombinant plasmid is template, and being divided into is 4 reaction train groups, carries out pcr amplification simultaneously.Reaction system is that 2 μ L, DEPC treating water 6 μ L, template 2.5 μ l mix separately for 25 μ L:Realtime PCR Master Mix, 12.5 μ l, Tupaia-CD81FP/RP.Loop parameter is 40, and cycle index is 40,94 ℃ of 2min; 94 ℃ of 30s, X ℃ of 30s, 72 ℃ of 1min, 40 circulations.72 ℃ of 5min amplifications; 4 ℃ standby.Amplified production 2.5% agarose electrophoresis detects.Electrophoresis showed under the different primer concentrations: be minimum concentration under the primer 2 00nm condition, this concentration purpose fragment (Fig. 4) that can well increase.
Concentration and probe concentration: thresholding (Ct) is minimum to circulate under the same template concentration, fluorescent signal is the probe choice criteria to the maximum than (Rn) activity.Adopt 50nm, 100nm, 3 concentration and probe concentration of 150nm, being respectively applied for same concentration C D81 recombinant plasmid is template, carries out TaqMan PCR and detects.Reaction system is that 2 μ L, Tupaia-CD81probe 0.5 μ L DEPC treating water 5.5 μ L, template 2.5 μ L mix separately for 25 μ L:Realtime PCR Master Mix12.5 μ L, Tupaia-CD81FP/RP.Loop parameter is 40, and cycle index is 40,94 ℃ of 2min; 94 ℃ of 30s, X ℃ of 30s, 72 ℃ of 1min, 40 circulations.72 ℃ of 5min amplifications; 4 ℃ standby.Observe amplification curve and examining report.Show that by different probe concentration amplification curve and Ct value this detection architecture Ct=8.45 value under the 100nm condition is minimum, the maximum amplification curve of Rn good (Fig. 5).
5.4 the purifying of plasmid and the mensuration of copy number: the purification step of plasmid is referring to the precious biological related kit specification sheets in Dalian.
Plasmid purification is surveyed its A
260/ A
280(1.8～2.0) calculate copy number according to following formula:
Copy number (copies/m)=plasmid concentration (ug/ul) * A Shi constant/plasmid molecule amount.The A Shi constant is 6.02 * 10
23The total length (bp) of molecular weight (the 649) * recombinant plasmid of plasmid molecule amount=one base pair.
5.5Taqman the foundation of PCR typical curve
With the plasmid of known copy number according to 1 * 10
4～1 * 10
7Copy number concentration is carried out 10 times of serial dilutions, obtain the PMD-18T CD81 positive plasmid concentration sample of 5 differing molecular content, carry out the TaqMan pcr analysis respectively, draw out the quantitative criterion curve according to the logarithmic value of the molecule number of sample in the reaction result and the corresponding relation of Ct value thereof.This detection architecture typical curve amplification curve standard as can be known from the graph, linear relationship is good, its slope-4.784, intercept 48.185, R
2=0.9996.Can get this detection architecture linear equations is: Ct=-4.784log (Copynumber)+48.185 (Fig. 6)
6.TaqMan the evaluation of PCR detection architecture
6.1 the evaluation of detection architecture repeatability, stability
This detection architecture repeatability, stability from batch between, batch in, the group between, the group interior four aspect contrived experiments verify.
Between batch: the same concentration recombinant plasmid that disposes between different batches, 10 times of gradient dilution copy numbers are 10
3～10
7Different time detects 3 times (per 5 days once) between each batch of copies, calculates their variation coefficient of curve correlation coefficient separately respectively.Crossing different batches is 0.155% every the variation coefficient of the relation conefficient of five days bioassay standard curves, little much smaller than 5% visible this detection architecture variation coefficient, accuracy height (seeing Table 1).
The same concentration recombinant plasmid that disposes between table 1 different batches is the variation coefficient of curve correlation coefficient separately
In batch: with the finite concentration recombinant plasmid of a collection of configuration, 10 times of gradient dilution copy numbers are 10
3～10
7Copies detects 6 times simultaneously, calculates the variation coefficient of their curve correlation coefficients.By the standard deviation calculated with 6 groups of amplification curve CT values in the sets of batches is 0.232, and the variation coefficient is 1.079%, the little good reproducibility of the variation coefficient (Fig. 7).
Between group: to the high, medium and low different concns (10 of recombinant plasmid
8Copies, 10
6Copies, 10
4Copies) each repeats to detect simultaneously for 3 times, calculates their Ct value variation coefficient separately.The variation coefficient of three detected result Ct values is respectively 0.816%, 0.582%, 0.308%, the little good reproducibility of the variation coefficient (Fig. 8).
In the group: to the finite concentration recombinant plasmid, with-20 ℃ of preservations heavily inspection every 30 days.Calculate their Ct value variation coefficient.Twice experiment Ct value variation coefficient is respectively 1.343% and 1.079% all less than 5%, this detection architecture stability, good reproducibility (seeing Table 2) in the group.
The t value variation coefficient of table 2 finite concentration recombinant plasmid
6.2 the evaluation of detection architecture susceptibility
The standard recombinant plasmid that calculates is done 10 times of gradient dilutions, and copy number is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Copies obtains kinetic curve, is the susceptibility that lower limit detects this detection architecture with Ct value＞35 circulation.By amplification curve diagram as can be known this detection architecture can detect 10
2Copy number rank (Fig. 9).
6.3 the CD81 gene detects in the tree shrew primary hepatocyte sample
Different HCV infective doses 1 * 10
6, 5 * 10
5, 3 * 10
5, 1 * 10
5, 5 * 10
4, 3 * 10
4, 1 * 10
4, 5 * 10
3, 3 * 10
3, 1 * 10
5The metainfective sample of tree shrew primary hepatocyte of copies carries out the detection of CD81 gene.As can be known along with the increase of the infective dose of HCV, the expression of CD81 is the variation of decline (Figure 10) by amplification curve diagram.
1.CD81 two oligonucleotide primer sequence F/R of gene fragment pcr amplification:
F：5’-ATGGGAGTGGAGGGCTGCACCAA-3’
R：5’-TCAGTACACGGAGCTGTTCCGGATG-3’
2. be that CD81 oligonucleotide primer and probe sequence are in the Taqman probe PCR method of standard substance with the tree shrew CD 81 plasmid:
CD81?FP：5’-GGAGGACTGCCACCAGAAGAT-3’
CD81?RP：5‘-CAGCACCATGCTCAGGATCA-3’
CD81?Probe：5‘(FAM)-AAGCTGTACCTCATCGGCATCGCG-(TAMRA)3’
3. the full genome nucleotide sequence sequencing result of HCV acceptor CD81 in the tree shrew
001?ATGGGAGTGG?AGGGCTGCAC?CAAGTGCATC?AAGTACCTGC?TCTTCGTCTT?CAACTTCGTC
061?TTCTGGCTGG?CCGGCAGGTG?GATCCTAGGT?GTGGCCCTTT?GGCTCCGGCA?TGATCCACAG
121?ACCACCAACC?TCCTCTATTT?GGAGCTGGGA?GACCGACCTG?CACCCAATAC?CTTCTACGTA
181?GGCATCTACA?TCCTCATTGC?CGTGGGTGCC?GTGATGATGT?TCGTGGGCTT?CCTGGGCTGC
241?TACGGGGCCA?TCCAGGAGTC?CCAGTGCCTG?CTGGGGACGT?TCTTCACCTG?CCTGGTGATC
301?CTCTTTGCCT?GTGAGGTGGC?CGCCGGCATC?TGGGGCTTTG?TCAACAAGGA?CCAGATCGCC
361?AAGGACGTGA?AGCAGTTCTA?CGACCAGGCC?CTACAGCAGG?CTGTGGTGGA?TGACGAAGCC
421?AACAACGCCA?AGGCCGTGGT?GAAGACTTTC?CACGAGACGC?TCAACTGCTG?TGGTTCTGGC
481?ACGCTGTTCA?CCCTGACCAC?CTCAGTGCTG?AAGAACAACC?TGTGTCCCTC?AGGCAGCAAC
541?GTCATCAGCA?ACTTGTTTAA?GGAGGACTGC?CACCAGAAGA?TAGATGACCT?CTTCTCTGGG
601?AAGCTGTACC?TCATCGGCAT?CGCGGCCATC?GTGGTCGCTG?TGATCATGAT?CTTTGAGATG
661?ATCCTGAGCA?TGGTGCTGTG?CTGTGGCATC?CGGAACAGCT?CCGTGTACTG?A
Claims (4)
1. a plasmid that makes up with tree shrew CD 81 gene is the Taqman fluorescence probe quantitative PCR method of standard substance, comprise segmental clone of tree shrew CD 81 gene and order-checking, made up the PMD-18T-CD81 plasmid by the T-A clone technology, tree shrew CD 81 gene fragment with the clone is a target, design Taqman PCR primer and probe, contrived experiment finds annealing temperature, primer concentration, the concentration and probe concentration of primer under the optimal conditions, purify this plasmid and calculate CD81 gene fragment copy number, with 10 times of gradient dilutions, the preparation copy number is 10 with the plasmid that calculates
3～10
7The copies positive plasmid is set up typical curve, detects with the changing conditions of this detection architecture to the tree shrew primary hepatocyte sample CD81 of HCV Infection in Vitro.
2. according to claim 1 is the Taqman probe PCR method of standard substance with the tree shrew CD 81 plasmid, it is characterized in that:
(1) the PCR primer is two CD81 oligonucleotide primer F/R:
F：5’-ATGGGAGTGGAGGGCTGCACCAA-3’
R：5’-TCAGTACACGGAGCTGTTCCGGATG-3’
(2) be template with the RNA that extracts in the tree shrew liver, obtain tree shrew CD 81 cDNA fragment, this fragment purification and order-checking;
(3) behind this fragment purification, be connected in the PMD-18T plasmid, be converted into then among the competent escherichia coli cell JM-109 and increase by the T-A clone; Screening positive clone extracts clone's and carries out purifying, obtains the outer with reference to positive plasmid of tree shrew CD 81 molecular assay.
3. according to claim 1 is the Taqman probe PCR method of standard substance with the tree shrew CD 81 plasmid, it is characterized in that CD81 oligonucleotide primer and probe are:
CD81FP：5’-GGAGGACTGCCACCAGAAGAT-3’
CD81RP：5‘-CAGCACCATGCTCAGGATCA-3’
CD81Probe：5‘(FAM)-AAGCTGTACCTCATCGGCATCGCG-(TAMRA)3’
4. be the Taqman probe PCR method of standard substance according to claim 1 with the tree shrew CD 81 plasmid, it is characterized in that:
(1) determining of detection architecture annealing temperature,
(2) determining of detection architecture concentration and probe concentration and primer concentration,
(3) configuration 10
3～10
7The CD81 plasmid of copy number has been set up the typical curve of this detection architecture as template,
(4) batch between, batch in, between group, interior four levels of group verify the repeated and stable of this method system,
(5) utilize 10 times of gradient dilutions 10 of CD81 plasmid difference of determining concentration
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Copies obtains kinetic curve, detects the susceptibility of this detection architecture;
(6) utilize this detection architecture, CD81 gene in the tree shrew primary hepatocyte of different HCV infective doses is detected, determine the copy number of CD81 in the cell after the different infective doses.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146838A (en) * | 2013-03-28 | 2013-06-12 | 中国医学科学院医学生物学研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) method of TAQMAN probe with standard plasmids built by tree shrew IL-6 (interleukin 6) |
| CN103276055A (en) * | 2013-03-28 | 2013-09-04 | 中国医学科学院医学生物学研究所 | TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance |
| CN105087828A (en) * | 2015-08-24 | 2015-11-25 | 昆明理工大学 | Fluorogenic quantitative PCR (polymerase chain reaction) detecting method for A type rotavirus |
| CN106939300A (en) * | 2017-04-26 | 2017-07-11 | 中国医学科学院医学生物学研究所 | Tree shrew immortalized liver cell cell line and its construction method and application |
| CN108537004A (en) * | 2018-03-19 | 2018-09-14 | 昆明理工大学 | A method of identifying high yield Radix Notoginseng using smallRNA high-flux sequence abundance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103146838A (en) * | 2013-03-28 | 2013-06-12 | 中国医学科学院医学生物学研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) method of TAQMAN probe with standard plasmids built by tree shrew IL-6 (interleukin 6) |
| CN103276055A (en) * | 2013-03-28 | 2013-09-04 | 中国医学科学院医学生物学研究所 | TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance |
| CN103146838B (en) * | 2013-03-28 | 2014-08-27 | 中国医学科学院医学生物学研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) method of TAQMAN probe with standard plasmids built by tree shrew IL-6 (interleukin 6) |
| CN103276055B (en) * | 2013-03-28 | 2015-02-18 | 中国医学科学院医学生物学研究所 | TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance |
| CN105087828A (en) * | 2015-08-24 | 2015-11-25 | 昆明理工大学 | Fluorogenic quantitative PCR (polymerase chain reaction) detecting method for A type rotavirus |
| CN106939300A (en) * | 2017-04-26 | 2017-07-11 | 中国医学科学院医学生物学研究所 | Tree shrew immortalized liver cell cell line and its construction method and application |
| CN108537004A (en) * | 2018-03-19 | 2018-09-14 | 昆明理工大学 | A method of identifying high yield Radix Notoginseng using smallRNA high-flux sequence abundance |
| CN108537004B (en) * | 2018-03-19 | 2021-07-16 | 昆明理工大学 | Method for identifying high-yield pseudo-ginseng by utilizing smallRNA high-throughput sequencing abundance |
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