CN1683564A - Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction - Google Patents
Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction Download PDFInfo
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Abstract
The composite quantitative PCR process of detecting bird flu virus and Newcastle disease virus has the operation steps of: designing and synthesizing fluorescently labeling primer specific for bird flu virus and Newcastle disease virus based on the nucleoprotein gene of bird flu virus and the fusion protein gene of Newcastle disease virus, acquiring sample and extracting its RNA, preparing composite PCR liquid, performing reverse transcription and PCR amplification in quantitative PCR instrument, and final data analysis and result judgment with relevant software. The present invention completes reverse transcription and PCR amplification in the same system, can amplifying two kinds of target genes and performs real-time monitoring of two fluorescent signals. The present invention has high specificity, high sensitivity, high detection efficiency, and low cost.
Description
Affiliated technical field
The invention belongs to the detection technique of bird infective virus, is a kind ofly to judge whether bird or other products infect the new method of avian influenza virus and Avian pneumo-encephalitis virus.
Background technology
Bird flu and newcastle disease are to involve global two kinds of great bird transmissible diseases.
(Avian Influenza Virus AIV) belongs to orthomyxoviridae family, Influenza Virus to avian influenza virus, is the sub-thread minus-stranded rna virus, and the spiral symmetry has cyst membrane, has the active glycoprotein of the hemagglutinin of containing and neuraminidase fine prominent on the cyst membrane.Influenza virus has 3 type that antigenicity is different: A, B and C type.All avian influenza virus all belong to the A C-type virus C, can cause bird from respiratory system to multiple symptoms such as serious general septicemia.Antigenic difference according to hemagglutinin and neuraminidase is divided into 15 HA hypotypes and 9 NA hypotypes with avian influenza virus (AIV) strain, and the AIV blood serum subtype of China's report mainly is H9, H5, H7 and H14.Because AIV nucleic acid is segmented, gene is very easily undergone mutation or is reset, and the height variability of AIV makes to the control of bird flu very difficult.The clinical symptom of bird flu and pathological change the different differences that show such as kind, age, course of disease length, accompanying infection and infection strain virulence because of infecting fowl, and the atypism symptom changes with cuing open to examine.This makes that the detection of avian influenza virus is very difficult.
Avian pneumo-encephalitis virus (Newcastle Disease Virus, NDV) can cause bird a kind of with respiratory tract, gastrointestinal mucosal is hemorrhage is the acute infectious disease of classical symptom.Avian pneumo-encephalitis virus belongs to Paramyxoviridae, paramyxovirus subfamily, Rubulavirus.Avian paramyxoviruses has 9 serotypes, i.e. PMV-1 to PMV-9, and wherein Avian pneumo-encephalitis virus (NDV) is the main member of avian paramyxoviruses 1 type (APMV-1).Test shows have more than 250 kind of birds to be infected by NDV, and the consequence of its harm and initiation is extremely serious.Classify it as I class eqpidemic disease in the animal epidemic catalogue of Ministry of Agriculture's issue in 1999, OIE (OIE) classifies it as category-A eqpidemic disease.
The present invention is before making, and to the inspection and quarantine method of avian influenza virus and Avian pneumo-encephalitis virus, OIE (OIE) stipulates that its method comprises: isolation identification, agar gel immunodiffusion test, blood clotting and the hemagglutination-inhibition test etc. of virus.
The separation and the evaluation of virus: the chicken embryo that at first sample is seeded to specific pathogen free, virus can be bred along with the growth of chicken embryo, after a week, extract the allantoic fluid of chicken embryo, carry out blood clotting and hemagglutination-inhibition test, thereby can judge and detect in the sample whether contain virus.The advantage of this method is highly sensitive, but the cycle is long, need 21 days approximately, and workload is big, needs lot of manpower and material resources.
Agar gel immunodiffusion test: carry out antigen mixture is analyzed in nineteen forty-six by Oudin the earliest.Ultimate principle is in the gel of certain pore size, when antigen and antibody molecule meet, forms immune complex, if both ratios are suitable, then composite molecular weight increases, and particle increases, and no longer continues diffusion and produces precipitation line.The formed precipitation of different antigen has position separately, thereby antigen can be separated.The advantage of this method is simple, does not need specific apparatus, but because antibody molecule is always not man-to-man with combining of antigenic determinant, and, because factors such as ionic strength, pH cause the specificity of this method lower.
Blood clotting and hemagglutination-inhibition test: its ultimate principle is some virus or viral hemagglutinin, can optionally make the red corpuscle generation aggegation of several animals, and the erythrocytic phenomenon of this aggegation is called blood clotting.Add specific antibody in the suspension of virus, and the amount of this antibody is when being enough to suppress virion or its hemagglutinin, then the acceptor of erythrocyte surface just can not directly contact with virion or its hemagglutinin.At this moment erythrocytic agglutination phenomenon just is suppressed, and becomes hemagglutination inhibition reaction.The advantage of this method be simple fast, but because its detects is antiviral antibody, and antibody must could produce in the quite a while after infection, therefore, the range of application of this method is restricted.
Aforesaid method because of susceptibility, specificity, make a definite diagnosis problems such as overlong time or environmental pollution, be difficult to satisfy demand quick, responsive, special, economic in the foreign trade.
Technology contents
The objective of the invention is to set up a kind of sensitivity, special, accurate detection method; and be applied to the inspection and quarantine of avian influenza virus and Avian pneumo-encephalitis virus; to improve susceptibility, the specificity of detection method; shorten detection time; reduce and detect cost, the development of protection and promotion China's livestock industry and foreign trade.
The present invention finishes by following technological operation scheme:
Design and synthesize the specificity fluorescent labeled primer that detects bird flu and Avian pneumo-encephalitis virus, gather sample and extract the RNA of sample, then prepare composite PCR reaction liquid, afterwards reaction solution is put into the quantitative PCR instrument and carried out reverse transcription and pcr amplification reaction, the ultimate analysis data, carry out the result and judge, and carry out the specificity analyses and the sensitivity analysis of detection method.
Design and synthesize the fluorescent primer that detects avian influenza virus and Avian pneumo-encephalitis virus: be the sequences Design of the antigen-4 fusion protein gene (F) of nucleoprotein (NP) gene according to avian influenza virus and Avian pneumo-encephalitis virus, and carried out fluorescent mark; Primer and mark fluorescent are respectively:
Avian influenza virus: positive strand primer: 5 '-GCTTGTCCTC TGGTATGTGG C-3 '
The minus strand primer: 5 '-GACCAGGGAG AAGACGCAAC TGCTGGC[JOE]-3 '
Avian pneumo-encephalitis virus: positive strand primer: 5 '-GAGCATTGCG GCAACCAATG AA-3 '
The minus strand primer: 5 '-CACGGTCCAA TTCTCGCGCC G[FAM] G-3 '.
The sample of gathering: being brush,throat or cloaca swab, muscle or the internal organs of live-bird, also can be serum, blood plasma and freezing tissue transudate.
Extract the RNA of institute's analyzing samples: adopt Trizol lysate lysed sample, the chloroform extracting, the method for isopropanol precipitating prepares the RNA of sample.
Prepare compound RT-PCR reaction solution: promptly mix various PCR reacted constituents, comprise the RT-PCR damping fluid, bird flu primer, newcastle disease primer, RNA solution, RNA enzyme inhibitors, water.Blending means is for adding in the PCR reaction tubes mixing with a certain amount of above-mentioned each composition.Composite PCR reaction liquid comprises two layers of meaning: the first, comprise ThermoScript II and archaeal dna polymerase in the RT-PCR damping fluid, and can in an individual system, carry out reverse transcription and pcr amplification reaction; The second, in a reaction system, added bird flu primer and newcastle disease primer, nucleoprotein (NP) gene of the avian influenza virus that can increase respectively and the antigen-4 fusion protein gene (F) of Avian pneumo-encephalitis virus.
Reaction solution is put into the quantitative PCR instrument carry out reverse transcription and pcr amplification reaction.Adopt the two channels quantitative real time PCR Instrument,, start reverse transcription and pcr amplification reaction through starting shooting, open SDS application software, newly-built reaction file, set fluorescent primer, the reaction parameter supervisor being set.
Data analysis and result judge: be to utilize quantitative analysis software SDS 2.1 analytical data, result of determination.Method is: be that judgment basis judges to detect in the sample whether contain avian influenza virus and Avian pneumo-encephalitis virus with the melting curve.
With the melting curve is judging criterion: if melting curve has been located absorption peak at 75.5 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains avian influenza virus; If melting curve has been located absorption peak at 78 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains Avian pneumo-encephalitis virus; If melting curve 75.5 ℃ ± 1 ℃ locate and 78 ℃ ± 1 ℃ locate all to have absorption peak, then two kinds of viruses all contain; If all do not have absorption peak two positions, then two kinds of viruses do not contain.
Carry out the specificity analyses and sensitivity analysis of detection method: the RNA that extracts the different strains of NDV, the different strains of AIV and IBV, IBDV respectively carries out composite quantitative RT-PCR reaction, to determine the specificity of detection method.Obtain to contain the segmental RNA of purpose by in-vitro transcription, carry out 10 times of serial dilutions, the quantitative RT-PCR reaction draws typical curve by software analysis, to determine the sensitivity of detection method.
The present invention and prior art contrast are characterized in:
1, employed primer is single fluorescent mark, and it can save cost than two fluorescently-labeled Taqman probes.Its principle is that the firsts and seconds structure of oligonucleotide is influential to the fluorescent emission performance of institute's bonded fluorescence dye, and primer has autofluorescence cancellation performance, and after the pairing of it and target sequence, its fluorescence will recover.
2, this reaction is the compound detection system, comprises two layers of meaning: the first, comprise ThermoScript II and archaeal dna polymerase in the RT-PCR reaction buffer, and can in an individual system, carry out reverse transcription and pcr amplification reaction; The second, in a reaction system, added bird flu primer and newcastle disease primer, nucleoprotein (NP) gene of the avian influenza virus that can increase respectively and the antigen-4 fusion protein gene (F) of Avian pneumo-encephalitis virus.
3, easy to be quick.Utilize this method,, only need 4 hours from extracting nucleic acid result's judgement to the end.Do not need amplification aftertreatment and the result of traditional RT-PCR to judge, thereby avoided environmental pollution.
4, highly sensitive.This system is can detected AIV gene copy number minimum to be 40, and the NDV gene copy number is minimum to be 79.
5, the result judges accurately, the reliability height.During judged result, as judging criterion, the specificity of PCR product decision Tm value is specific, therefore can accurately judge to detect in the sample whether contain avian influenza virus and Avian pneumo-encephalitis virus, gets rid of because the false positive results that non-specific amplification causes by melting curve.
Description of drawings:
Fig. 1: the avian influenza virus positive, the negative melting curve that detects sample of Avian pneumo-encephalitis virus
The melting curve of 1:JOE wavelength, the melting curve of 2:FAM wavelength, corresponding Tm value is 75.5 ℃
Fig. 2: the melting curve of avian influenza virus feminine gender, Avian pneumo-encephalitis virus positive detection sample
The Tm value of the melting curve correspondence of the melting curve 2:JOE wavelength of 1:FAM wavelength is 78 ℃
Fig. 3: the melting curve of the avian influenza virus positive, Avian pneumo-encephalitis virus positive detection sample
The Tm value of the melting curve correspondence of the melting curve 2:FAM wavelength of 1:JOE wavelength is respectively 75.5 ℃, 78 ℃
Fig. 4: avian influenza virus feminine gender, the negative melting curve that detects sample of Avian pneumo-encephalitis virus
The Tm value of the melting curve correspondence of the melting curve 2:FAM wavelength of 1:JOE wavelength is respectively 73 ℃, 75 ℃
Fig. 5: the test pattern of the specific detection of this method
1,2,3,4,5,6,7 are respectively: NDV-L, and NDV-I, NDV-S, NDV-PY,
NDV-CY, AIV-H5, " S " type amplification curve of AIV-H9; 8,9 are respectively non-" S " type amplification curve of IBV, IBDV.
Fig. 6: this method detects the test pattern of the sensitivity of avian influenza virus
1,2,3,4,5,6,7 are respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Dilution in-vitro transcription AIV purpose segmental " S " type amplification curve.
Fig. 7: this method detects the test pattern of the sensitivity of Avian pneumo-encephalitis virus
1,2,3,4,5,6,7 are respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Dilution in-vitro transcription NDV purpose segmental " S " type amplification curve.
Embodiment
Be described in detail the technological method that composite quantitative RT-PCR of the present invention detects avian influenza virus and Avian pneumo-encephalitis virus in conjunction with the accompanying drawings below by embodiment:
It at first is the sequence of the antigen-4 fusion protein gene (F) of nucleoprotein (NP) gene according to avian influenza virus and Avian pneumo-encephalitis virus, design and synthesize the specificity fluorescent labeled primer, gather sample afterwards and extract the RNA of sample, then prepare composite PCR reaction liquid, again composite PCR reaction liquid is put into the quantitative PCR instrument and carry out reverse transcription and pcr amplification, the ultimate analysis data are carried out result's judgement, carry out the specificity analyses and the sensitivity analysis of detection method.
1, design detects the primer of avian influenza virus and Avian pneumo-encephalitis virus
From the sequence (seeing the nucleotides sequence tabulation) of ncbi genbank acquisition avian influenza virus NP gene (GENBANK AF509118) and F gene of NDV strain (GENEBANK AF322052), by the online design software of LUX primer (network address is www.invitrogen.com/lux) the design LUX primer of InVitrogen company.The principle of LUX primer is that the firsts and seconds structure of oligonucleotide is influential to the fluorescent emission performance of institute's bonded fluorescence dye, and primer has autofluorescence cancellation performance, and after the pairing of it and target sequence, its fluorescence will recover.The key step of design primer is:
1), registration: use name, unit, address, E-mail address, the phone information of answering the registered user first.
2), list entries: with the sequence (seeing sequence table) of avian influenza virus NP gene (seeing sequence table) and F gene of NDV strain, name respectively, and it is added in the sequence frame.As required, parameter area is set, the magnitude range that amplified fragments is set in this example is 50-200bp, and the annealing temperature Tm of primer is 60-70 ℃.
3), the design alternative fluorescent dye primer: the fluorescent dye primer of software design be positioned at operation interface the top, and with title, sequence, the position in target sequence, the point penalty information of primer.Wherein, point penalty is represented the expection performance of this primer in the PCR reaction, and the point penalty value is low more, and primer is good more.
4), the non-marked primer of selection and fluorescent dye primer collocation: each fluorescent dye primer all has corresponding non-marked primer.According to test objective, it is right to choose a non-marked primer and labeled primer composition primer.
5), select fluorescent mark: for the bird flu primer, mark is selected JOE, and for the newcastle disease primer, mark is selected FAM.
6), confirm the primer purchase order, give Invitrogen company synthetic.
Through above-mentioned design procedure, choose two pairs of primers, be respectively: (seeing the nucleotides sequence tabulation)
Avian influenza virus: positive strand primer: 5 '-GCTTGTCCTC TGGTATGTGG C-3 '
The minus strand primer: 5 '-GACCAGGGAG AAGACGCAAC TGCTGGC[JOE]-3 '
Avian pneumo-encephalitis virus: positive strand primer: 5 '-GAGCATTGCG GCAACCAATG AA-3 '
The minus strand primer: 5 '-CACGGTCCAA TTCTCGCGCC G[FAM] G-3 '
2, gnotobiosis is gathered sample
Detecting sample is brush,throat or cloaca swab, muscle or the internal organs of live-bird, also can be serum, blood plasma and freezing tissue transudate.
1), the sampling equipment: scissors, tweezers, syringe, 1.5mL Eppendorf centrifuge tube, must with they through 121 ℃ of autoclaving 15min and the oven dry.
2), the detection sample is live-bird, when getting the cloaca swab, the cloaca that swab is goed deep into animal turns around, the sticking ight soil of getting.Swab is put into the test tube thorough washing that fills 2mL PBS (containing antibiotic), on tube wall, extract liquid then.Gained solution changes in the aseptic centrifuge tube standby.
3), brush,throat: when getting brush,throat, swab is goed deep into larynx mouth and the last cleft palate of animal and scrape back and forth several times, get the throat juice, put into the test tube thorough washing that fills 2mL PBS (containing antibiotic), on tube wall, extract liquid.Gained solution changes in the aseptic centrifuge tube standby.
4), muscle or internal organs: muscle or internal organs
Get 5g detection sample to be checked and in the mortar of cleaned, sterilization and oven dry, fully grind, add 10mL PBS mixing, get supernatant and change in the aseptic centrifuge tube standby.For making the representativeness that detects sample stronger, also can get 50g detection sample to be checked, add the aseptic PBS of 50mL, after handling with the Bagmixer homogenizer, it is standby to get supernatant.
5), serum, blood plasma or freezing tissue transudate
If detecting sample is serum, blood plasma or freezing tissue transudate, then directly be taken in the Eppendorf centrifuge tube with asepsis injector, number standby.
3, the extraction of sample rna
Get n (n=sample number+1 pipe negative control+1 pipe positive control) 1.5mL centrifuge tube of sterilizing, add 600 μ L Trizol lysates, add each 200 μ L of sample to be tested, negative control, positive control then respectively, add 200 μ L chloroforms again, 5 seconds of concussion mixing on the vortex mixer; In 4 ℃, the centrifugal 15min of 12000rpm.Draw supernatant liquor 500 μ L, add the Virahol of 400 μ L-20 ℃ precoolings, put upside down mixing.In 4 ℃, the centrifugal 15min of 12000rpm removes supernatant gently; Add 600 μ L, 75% washing with alcohol precipitation, and drying at room temperature 3min.Add 20 μ L DEPC water, obtain RNA solution ,-20 ℃ of preservations standby (note: the RNA of this moment is subject to the RNA enzyme liberating most, please carries out pcr amplification in 2 hours).
4, preparation composite PCR reaction liquid
PCR is reacted various compositions take out, the centrifugal 5sec of 2000rpm from refrigerator.Add in a reaction tubes afterwards: RT-PCR damping fluid 12.5 μ L (include archaeal dna polymerase, ThermoScript II, MgCl
2, AmpErase UNG, dNTPs, dUTP), 200nM bird flu primer; 200nM newcastle disease primer; RNA enzyme inhibitors 0.6 μ L; RNA solution 4 μ L; Make up water to 25 μ L.Various compositions are blown and beaten mixing with the rifle head.Composite PCR reaction liquid comprises two layers of meaning: the first, comprise ThermoScript II and archaeal dna polymerase in the RT-PCR damping fluid, and can in an individual system, carry out reverse transcription and pcr amplification reaction; The second, in a reaction system, added bird flu primer and newcastle disease primer, nucleoprotein (NP) gene of the avian influenza virus that can increase respectively and the antigen-4 fusion protein gene (F) of Avian pneumo-encephalitis virus.
5, carry out reverse transcription and pcr amplification reaction
Two channels fluorescent PCR instrument is used in reverse transcription and pcr amplification reaction and fluoroscopic examination, is example with ABIPrism 7900HT type fluorescent primer quantitative PCR instrument, and concrete operations have following steps:
1), start, and open the SDS application software.
2), new files, fill in hereof and detect the sample title, (reporter group of AIV FLuorescent primer is selected JOE for use, and quenching group selects None to duplicate the fluorescent primer file; The reporter group of NDV FLuorescent primer is selected FAM for use, and quenching group selects None).
3), use fluorescent primer: in the square frame in Well Inspector dialog box under the Use item of fluorescent primer tabulation, choose bird flu, two fluorescent primers of newcastle disease, then instrument is surveyed two kinds of fluorescent signals automatically.
4), reaction parameter is set: the reverse transcription reaction parameter is: 48 ℃ of 30min; 95 ℃ of 10min of PCR reaction parameter; Carry out 95 ℃ of 15sec afterwards, melting curve analysis (95 ℃ of 15sec, 60 ℃ of 15sec, 95 ℃ of 15sec) is carried out in 40 circulations of 62 ℃ of 1min at last.
5), start PCR: click the Open/Close button at the Instrument window, eject specimen holder (on the right side of main frame).Sample panel is inserted pallet, click the Open/Close button again, the sample door is closed in specimen holder automatic drawing back and pass.Begin experiment by Start, have specimen holder to put in place during beginning, work such as scanner head puts in place, intensifications of heat lid, wait occurs promptly beginning commencement of commercial operation after the Remain Time some time.
6, data analysis and result judge
Experiment finishes, and utilizes SDS software analysis result.In SDS software, can select automatically or manual definite Ct value, if manual definite, the input threshold value is selected from and moves or the manual baseline that is provided with earlier.The starting point of baseline (Baseline) and terminal point determine that principle is: because 95 ℃ of fluorescent signals that high temperature caused increase, starting point will be avoided the front several cycles, generally is located at the 6th circulation; Terminal point will be located at the front that signal obviously increases the place.According to the data of this experiment, baseline is set at 3-15 circulation, clicks and analyzes key, checks amplification curve, melting curve etc. afterwards in the result.
The result judges: be that judgment basis judges to detect in the sample whether contain avian influenza virus and Avian pneumo-encephalitis virus with the melting curve.Wherein, quality control standard is:
The bird flu quality control standard: positive control detected result melting curve has been located single absorption peak (Fig. 1) at 75.5 ℃ ± 1 ℃, and negative control detected result melting curve locates there is not absorption peak at 75.5 ℃ ± 1 ℃.
The newcastle disease quality control standard: positive control detected result melting curve has been located single absorption peak (Fig. 2) at 78 ℃ ± 1 ℃, and negative control detected result melting curve locates there is not absorption peak at 78 ℃ ± 1 ℃.
Have only quality control standard to set up, can judge and detect in the sample whether contain avian influenza virus and Avian pneumo-encephalitis virus.
Detect sample: if melting curve has been located absorption peak at 75.5 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains avian influenza virus.If melting curve has been located absorption peak at 78 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains Avian pneumo-encephalitis virus.If melting curve 75.5 ℃ ± 1 ℃ locate and 78 ℃ ± 1 ℃ locate all to have absorption peak, then two kinds of viruses all contain (Fig. 3); If all do not have absorption peak two positions, then two kinds of viruses do not contain (Fig. 4).
7, the specific assay of detection method
Extract NDV-L respectively, NDV-I, NDV-S, NDV-PY, NDV-CY, AIV-H5, the RNA of AIV-H9, IBV, IBDV (shown in embodiment 3) carries out composite quantitative RT-PCR reaction.25ul reaction system main component is as follows: RT-PCR reaction solution 12.5 μ L; 200nM bird flu primer, 200nM newcastle disease primer; RNasin 0.6 μ L; RNA solution 4 μ L; Replenish DEPC water to 25 μ L, carry out the quantitative PCR reaction, the line data analysis of going forward side by side (shown in the 5th, the 6th step of embodiment).The results are shown in Figure 5.Each strain of NDV has typical amplification curve, and AIV-H5, has " S " type amplification curve at AIV-H9, and IBV, IBDV tangible " S " type amplification curve not.
8, the sensitivity determination of detection method
1), the structure of pGEM-N in-vitro transcription carrier
AIV gene and NDV gene masculine plasmid purpose fragment and pGEM-4z carrier be double digestion respectively: add ultrapure water 50 μ L in every pipe, 10 * K buffer, 10 μ L, Bam HI 2.5 μ L, Eco RI 2.5 μ L, add positive plasmid purpose fragment 35 μ L and pGEM carrier 35 μ L respectively, cumulative volume 100 μ L.37 ℃ act on 3 hours.Enzyme is cut the product electrophoresis and is identified, reclaims test kit purifying purpose band with glue respectively.
Connect: 10 * T4 DNA ligase buffer, 1.0 μ L, ligase 1.0 μ L, N protein coding gene 1.0 μ L, pGEM-4Z 3.5 μ L, ultrapure water 3.5 μ L, cumulative volume 10 μ L.16 ℃ are spent the night.Transform DH5 α competent cell, coat on the ammonia benzyl flat board, cultivate 18 hours single bacterium colonies of picking for 37 ℃, enlarged culturing upgrading grain, Bam HI and Eco RI double digestion are identified, positive plasmid called after pGEM-N.
2), the purifying of goal gene in-vitro transcription and transcription product
At ambient temperature, add following material in order: transcribe and optimize 5 * buffer 10uL, 100nmDTT 5 μ L, RNase 1.25 μ L, rATP, rGTP, each 2.5 μ L of rUTP, rCTP (10 times of mother liquor dilutions) 6 μ L, dna profiling 14 μ L, T7 RNA polymerase 1 μ L, ddH2O 4.75 μ L, 37 ℃-40 ℃ reactions of total system 50uL 1.5h.Sucking-off 5uL from each pipe is used for electrophoresis and identifies all the other-80 ℃ of preservations.
The component of positive control is as follows: transcribe and optimize 5 * buffer, 4 μ L, 100nm DTT 2 μ L, RNase 0.5 μ L, each 1 μ L of rATP, rGTP, rUTP and rCTP, pGEM positive control template 1 μ L, T7 RNA polymerase 1 μ L, ddH2O 7.5 μ L, total system 20 μ L.
Add RQ1 RNase Free Dnase, make template ribonucleic acid concentration reach 1 μ L/ μ g, 37 ℃ of effect 15min, the saturated phenol of TE of 1 times of volume of adding: chloroform: primary isoamyl alcohol (25: 24: 1), whirling 1min, the centrifugal 2min of 12000r/m.Water intaking adds the chloroform of 1 times of volume: primary isoamyl alcohol (24: 1), whirling 1min, the centrifugal 2min of 12000r/m in another EP pipe.Water intaking adds the 7.5M ammonium acetate of 0.5 times of volume, the dehydrated alcohol of 2.5 times of volumes in another EP pipe.Put the centrifugal 20min of 30min.12000r/m for-70 ℃.Carefully abandon supernatant, wash precipitation with 70% ethanol, drying.With 20 μ L TE or water dissolution RNA ,-70 ℃ of preservations are standby.OD with nucleic acid-protein analysis-e/or determining in-vitro transcription product
260Absorption value.
3), the sensitivity of fluorescence RT-PCR reaction to determine to detect
With in-vitro transcription contain the segmental RNA of purpose with 10 times of serial dilutions, carry out quantitative RT-PCR and detect, with the sensitivity of determining to detect.25ul reaction system main component is as follows: RT-PCR damping fluid (2 times) 12.5 μ L; 200nM bird flu primer; 200nM newcastle disease primer; RNA enzyme inhibitors 0.6 μ L; RNA solution 4 μ L; Replenish DEPC water to 25 μ L.The amplification curve of AIV, NDV is seen Fig. 6, Fig. 7 respectively.
According to formula RNA=OD
260* extension rate * 40 * 10
-6G calculates the quality (being made as A) of every μ L transcribe rna, again according to the molecular weight (B) of RNA, calculates the copy number (A/B) of RNA in every μ L transcribe rna solution.
The typical curve of avian influenza virus is: Ct value=-3.39 1gX+43.18; Coefficient R=0.973;
The typical curve of Avian pneumo-encephalitis virus is: Ct value=-2.88 1gX+42.97; Coefficient R=0.963.
With Ct value=35 is threshold value, calculates the sensitivity that detects avian influenza virus and Avian pneumo-encephalitis virus and is respectively 40,79 virus copies.
The nucleotides sequence tabulation
＜110〉Qingdao Entry-Exit Inspection and Quarantine Bureau
＜120〉composite quantitative polyase chain reaction detects the method for bird flu and Avian pneumo-encephalitis virus
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＜213〉Avian pneumo-encephalitis virus (Avulavirus sp.)
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<211>1497
<212>RNA
＜213〉avian influenza virus (Orthomyxoviridae sp.)
<220>
<221>CDS
<222>(1)...(1497)
<400>2
atggcgtctc?aaggcaccaa?acgatcttat?gaacagatgg?aaactggtgg?agaacgccag 60
aatgccactg?agatcagagc?atctgtcgga?agaatggttg?gtggaattgg?gaggttttat 120
atacagatgt?gcactgaact?caaactcagt?gactatgaag?ggaggctgat?tcagaacagc 180
ataacaatag?agagaatggt?tctctctgca?tttgatgaaa?ggaggaacaa?atacctggaa 240
gaacatccca?atgcggggaa?agacccaaag?aaaactgggg?gtccaatcta?ccgaaggaga 300
gacgggaagt?gggtgagaga?gctgattctg?tatgataaag?aggagatcag?gaggatttgg 360
cgccaagcga?acaatggaga?agatgcaact?gctggtctca?ctcatctgat?gatctggcat 420
tccaatctaa?atgatgccac?ataccagagg?acaagagctc?tcgtgcgtac?tgggatggac 480
cccagaatgt?gttctctaat?gcaaggatca?actctcccga?ggagatctgg?agctgcaggt 540
gcagcagtaa?agggagtcgg?aacgatggtg?atggaactaa?ttcggatgat?aaagcgaggg 600
attaatgatc?ggaatttctg?gagaggtgaa?aatgggcgaa?gaacgaggat?tgcatatgag 660
agaatgtgca?acatcctcaa?agggaaattc?caaacagcag?cacaaagagc?aatgatggac 720
caggtacggg?aaagcaggaa?tcctgggaat?gctgaaattg?aagatctcat?ctttctggca 780
cggtctgcac?tcatcctgag?aggatcagtg?gctcacaagt?cctgcttgcc?tgcttgtgtg 840
tacgggcttg?ctgtagccag?tggatatgac?tttgagagag?aagggtattc?tctggtcggg 900
attgatccct?ttcgtctgct?gcaaaacagc?caagtcttca?gtctaattag?accaaatgag 960
aacccagcac?ataaaagtca?actggtatgg?atggcatgcc?attctgcagc?atttgaagat 1020
ctgagggtct?caagtttcat?cagagggaca?agaatagttc?caaggggaca?actatctact 1080
agaggagttc?aaattgcttc?aaatgagaac?atggatacaa?tggactccaa?cactctagaa 1040
ctgagaagca?gatattgggc?tataagaacc?aggagtggag?gaaacaccaa?tcagcagaga 1200
gcatctgcag?gacaaatcag?tgtgcagcct?actttctcgg?tacagagaaa?tcttcccttc 1260
gaaagagcga?ccattatggc?ggcatttaca?gggaatacag?agggcagaac?atctgacatg 1320
aggactgaaa?tcataagaat?gatggagagc?gccagaccag?aagatgtgtc?tttccagggg 1380
cggggagtct?tcgagctctc?ggacgaaaag?gcaacgaacc?cgatcgtgcc?ttcctttgac 1440
atgagtaatg?aaggatctta?cttcttcgga?gacaatgcag?aggagtttga?gaattaa 1497
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require design according to PCR reaction, the positive strand primer of the avian influenza virus that is used to increase.
<400>3
GCTTGTCCTC?TGGTATGTGG?C 21
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require design according to PCR reaction, the minus strand primer of the avian influenza virus that is used to increase.
<400>4
GACCAGGGAG?AAGACGCAAC?TGCTGGC 27
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require design according to PCR reaction, the positive strand primer of the Avian pneumo-encephalitis virus that is used to increase.
<400>5
GAGCATTGCG?GCAACCAATG?AA 22
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require design according to PCR reaction, the minus strand primer of the Avian pneumo-encephalitis virus that is used to increase.
<400>6
CACGGTCCAA?TTCTCGCGCC?G 21
Claims (4)
1. a composite quantitative polyase chain reaction detects the method for bird flu and Avian pneumo-encephalitis virus, the technological operation program that it is characterized in that it is: design and synthesize the specificity fluorescent labeled primer that detects bird flu and Avian pneumo-encephalitis virus, gather sample and extract the RNA of sample, then prepare composite PCR reaction liquid, afterwards reaction solution is put into the quantitative PCR instrument and carried out reverse transcription and pcr amplification reaction, the ultimate analysis data, carry out the result and judge, and carry out the specificity analyses and the sensitivity analysis of detection method.
2. a kind of composite quantitative polyase chain reaction according to claim 1 detects bird flu and Avian pneumo-encephalitis virus method, it is characterized in that described primer is the sequences Design of the antigen-4 fusion protein gene (F) of nucleoprotein (NP) gene according to avian influenza virus and Avian pneumo-encephalitis virus, and carried out fluorescent mark; Primer and mark fluorescent are respectively:
Avian influenza virus: positive strand primer: 5 '-GCTTGTCCTC TGGTATGTGG C-3 '
The minus strand primer: 5 '-GACCAGGGAG AAGACGCAAC TGCTGGC[JOE]-3 '
Avian pneumo-encephalitis virus: positive strand primer: 5 '-GAGCATTGCG GCAACCAATG AA-3 '
The minus strand primer: 5 '-CACGGTCCAA TTCTCGCGCC G[FAM] G-3 '.
3. a kind of composite quantitative polyase chain reaction according to claim 1 detects bird flu and Avian pneumo-encephalitis virus method, it is characterized in that described preparation composite PCR reaction liquid, promptly mix various PCR reacted constituents, comprise RT-PCR damping fluid, bird flu primer and newcastle disease primer, RNA enzyme inhibitors, RNA template and water; Composite PCR reaction liquid comprises two layers of meaning: the first, comprise ThermoScript II and archaeal dna polymerase in the RT-PCR damping fluid, and can in an individual system, carry out reverse transcription and pcr amplification reaction; The second, in a reaction system, added bird flu primer and newcastle disease primer, nucleoprotein (NP) gene of the avian influenza virus that can increase respectively and the antigen-4 fusion protein gene (F) of Avian pneumo-encephalitis virus.
4. a kind of composite quantitative polyase chain reaction according to claim 1 detects bird flu and Avian pneumo-encephalitis virus method, it is characterized in that described analytical data, carry out result's judgement, be to be judging criterion with the melting curve, if melting curve has been located absorption peak at 75.5 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains avian influenza virus; If melting curve has been located absorption peak at 78 ℃ ± 1 ℃, and the absorption peak strength of signal judges then that greater than 1/3 of positive control absorption peak signal detecting sample contains Avian pneumo-encephalitis virus; If melting curve 75.5 ℃ ± 1 ℃ locate and 78 ℃ ± 1 ℃ locate all to have absorption peak, then two kinds of viruses all contain; If all do not have absorption peak two positions, then two kinds of viruses do not contain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510042527 CN1683564A (en) | 2005-03-01 | 2005-03-01 | Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510042527 CN1683564A (en) | 2005-03-01 | 2005-03-01 | Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction |
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|---|---|---|---|
| CN 200510042527 Pending CN1683564A (en) | 2005-03-01 | 2005-03-01 | Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317861A (en) * | 2019-07-18 | 2019-10-11 | 江苏宏微特斯医药科技有限公司 | A kind of kit detecting pathogen |
| CN110358815A (en) * | 2019-07-18 | 2019-10-22 | 江苏宏微特斯医药科技有限公司 | Method and its kit a kind of while that detect multiple target nucleic acids |
-
2005
- 2005-03-01 CN CN 200510042527 patent/CN1683564A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317861A (en) * | 2019-07-18 | 2019-10-11 | 江苏宏微特斯医药科技有限公司 | A kind of kit detecting pathogen |
| CN110358815A (en) * | 2019-07-18 | 2019-10-22 | 江苏宏微特斯医药科技有限公司 | Method and its kit a kind of while that detect multiple target nucleic acids |
| CN110317861B (en) * | 2019-07-18 | 2023-04-07 | 江苏宏微特斯医药科技有限公司 | Kit for detecting pathogen |
| CN110358815B (en) * | 2019-07-18 | 2023-05-23 | 江苏宏微特斯医药科技有限公司 | Method for simultaneously detecting multiple target nucleic acids and kit thereof |
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