CN103834747B - The method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking - Google Patents
The method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking Download PDFInfo
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Abstract
The method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking, comprises H1N1 virus hemagglutinin gene is carried out to RT-PCR amplification; Pcr amplification product is carried out to pyrophosphoric acid order-checking, judge whether the cracking site of Influenza A H1N1 HA gene has sudden change; Wherein pyrophosphoric acid order-checking employing SQA pattern, nucleotides application of sample order are AGCT. By by the HA gene cracking site sequence comparison of assay and H1N1virus type strain, the sudden change of the HA gene cracking site that detects H1N1virus that can pin-point accuracy. The present invention for definite virus virulence, pathogenic and host range provides simply, experimental program efficiently, avoid the related loaded down with trivial details experimental procedure of full gene sequencing, can also accurately grasp viral variation direction, conveniently it is monitored, the isolation infection sources cuts off route of transmission, stops the further expansion of epidemic situation.
Description
Technical field
The present invention relates to a kind of method that detects H1N1virus pathogenicity, be specifically related to a kind of based on pyrophosphoric acid order-checkingThe method that detects H1N1virus pathogenicity, belongs to virus detection techniques field.
Background technology
The Influenza A H1N1 of in March, 2009 outburst is another great public health event after SARS, highly pathogenic bird flu,Cause huge fear to the people of other countries. Influenza A virus is the segmented RNA virus of sub-thread minus strand, and antigen easily occursDrift, antigen conversion and genetic recombination, especially sudden change and the restructuring of the outer membrane protein hemagglutinin (HA) of virus, can form variousMutant strain or recombinant strain, cause viral virulence and the variation of host range, thereby cause influenza pandemic in various degree. Blood clottingCan element (HA) be cut into HA1 and HA2 determines the pathogenic key factor of influenza virus, and the variation of cracking site can be drawnPlay the variation of pathogenicity, the sequence in analytical pyrolysis site, studies its evolution laws and can more in depth understand H1N1 by let usPathogenic, virulence is big or small, host range.
The amino acid sequence at cracking site place is one of factor determining influenza virus pathogenicity. General highly pathogenic influenza virus existsNear cracking site, there is continuous more than 4 basic amino acid (RRRR), and the HA cracking site place one of low pathogenicity influenza virusAs just only have single basic amino acid (R). The multiple continuous basic amino acids in HA cracking site place of highly pathogenic influenza virus,Can be by the cracking of multiple protein enzyme institute, thus virus can be in host's different tissues cell growth and breeding, cause host's whole bodyInfection, exhaustion also cause death. And the HA protein cleavage site of low pathogenicity influenza virus can only be thin by minority tissues such as respiratory tractsProtease institute cracking in born of the same parents, thereby can only in host's respiratory tract, breed, temperate infection caused. The cracking site of HA albumenSequence signature to viral host range, tissue tropism with pathogenicly have a critical impact, be virus in body, propagate mustWant condition]. Low pathogenicity influenza virus is likely by the multiple basic amino acid in the connection peptide between HeHA2 district of HA1 districtGene mutation strengthens virulence, becomes highly pathogenic Strain, and therefore, cracking site sequence analysis and the monitoring of HA are to weigh very muchWant
In order to strengthen the control to H1N1 fast propagation and to avoid its eruption and prevalence, develop quick, reliable high-throughout detection virusMutating technology is the focus of research, and traditional full-automatic DNA sequencing technology based on Sanger method is detecting viral gene sudden changeThere is shortcoming time-consuming, effort. Pyrophosphoric acid sequencing technologies is short segment DNA gene sequence analysis of new generation, can be quick, accurateCarry out really, in real time short dna sequence analysis, detect flux high, simple to operation, programmable, utilizes pyrophosphoric acid order-checkingThe research that technology detects the cracking site of Influenza A H1N1 HA gene has no report.
Summary of the invention
The object of the invention is to provide a kind of method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking, its usePyrophosphoric acid sequence measurement detects the sequence method of H1N1virus hemagglutinin (HA) cracking site, judges hemagglutinin (HA)Cracking site whether undergo mutation, cause the variation of viral virulence, thereby cause influenza pandemic in various degree.
Can determine the pathogenic key factor of influenza virus, cracking position owing to hemagglutinin (HA) be cut into HA1 and HA2The variation of point can cause the variation of pathogenicity, and therefore technical conceive of the present invention is for H1N1virus cracking positionPoint carries out pyrophosphoric acid order-checking and detects. The method that detection type H1N1 influenza virus pathogenicity of the present invention changes, first adopts RT-PCRFragment (comprising cracking site sequence) in amplification H1N1 viral hemagglutinin (HA) gene, subsequently with pyrophosphoric acid PCR sequencing PCR to amplificationFragment accurately checks order, thereby has set up one cracking site sudden change fast and effectively detection method. For determining virus poisonPower, pathogenic and host range provides simply, experimental program efficiently, avoided the related loaded down with trivial details experiment of full gene sequencingStep, can also accurately grasp viral variation direction, conveniently it is monitored, and the isolation infection sources cuts off route of transmission, stopsThe further expansion of epidemic situation.
Cardinal principle of the present invention is:
Pyrophosphoric acid sequencing technologies
1) pyrophosphoric acid order-checking (Pyrosequencing) technology is brand-new a kind of DNA sequence analysis technology, can be fast, accuratelyOne section of shorter target fragment is measured on ground. This technology need not be carried out electrophoresis, and DNA fragmentation also need not fluorescence labeling, and operation is letter veryJust.
2) pyrophosphoric acid sequencing technologies is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, general principle asUnder: by 1 specific sequencing primer and the combination of single stranded DNA template, then add enzymatic mixture (to comprise DNAPolymerase, ATPSulfurylase, Luciferase and Apyrase) and substrate mixture (comprising APS and Luciferin).In reaction system, add a kind of dNTP, if it can just with the next base pairing of DNA profiling, can be poly-at DNAUnder the effect of synthase, add 3 ' end of sequencing primer to, discharge the pyrophosphoric acid group of molal quantity simultaneously. In ATP sulphationUnder the effect of enzyme, the PPi of generation can be with APS in conjunction with forming ATP; Under the catalysis of luciferase, the ATP of generation againCan, with fluorescein in conjunction with forming oxyluciferin, produce visible ray simultaneously. By CCD optical system can obtain one specialDetected peaks, the height of peak value and the base number that matches be directly proportional. Remaining dNTP and residual a small amount of in reaction systemATP degrades under the effect of Apyrase. Add another kind of dNTP, 2nd～4 step reactions are repeated, according to acquisitionPeak value figure can read DNA sequence dna information accurately. The height of each peak value just becomes with the nucleotides number mixing in reactionRatio. Then add lower a kind of dNTP, continue the synthetic of DNA chain.
3) in pyrophosphoric acid order-checking operating system, have 2 patterns and can carry out the detection of base mutation, i.e. sequence analysis pattern(SequenceAnalysis, SQA) and SNP pattern (SingleNucleotidepolymorphism, SNP).Wherein the nucleotides application of sample of SQA pattern order be circular order application of sample method, by circulate repeatedly add A, G, C, T tetra-Kind of nucleotides to be to detect the sequence of sample to be checked, the once accurate base of interpretation 40bp left and right of this method, by measured sequence andInterpretation sudden change situation voluntarily after known array comparison. The nucleotides application of sample order of SNP pattern is particular order application of sample method, i.e. systemAccording to the application of sample order of the site to be checked of setting and the site automatic product nucleus thuja acid of sequence around, only the base of Single locus is becomeChange and detect, whole sequence is not measured. For different detected objects, need to grope different pyrophosphoric acid order-checking inspectionsSurvey method.
The method of the detection H1N1virus pathogenicity based on pyrophosphoric acid order-checking of the present invention, is characterized in that comprising followingStep:
Step 1, carries out RT-PCR amplification to H1N1 viral hemagglutinin (HA) gene;
Step 2, carries out pyrophosphoric acid order-checking to pcr amplification product, judges whether the cracking site of Influenza A H1N1 HA gene has prominentBecome; Wherein pyrophosphoric acid order-checking employing SQA pattern, nucleotides application of sample order are AGCT.
In above-mentioned steps 1, H1N1 viral hemagglutinin (HA) gene is carried out to twice PCR amplification, wherein RT-PCR expands for the first timeIncrease taking sample H1N1virus RNA extract as template, increase for the second time taking RT-PCR amplified production for the first time as mouldPlate.
The above-mentioned for the first time PCR primer that amplification is used has sequence shown in H1F and H1R, primer amplification fragment length 950bp,And comprise cracking site sequence,
The one PCR primer sequence is as follows: H1F:ACGTGTTACCCAGGAGATTTC, H1R:TCTTTACCYACTRCTGTGAA
For the cracking site design pyrophosphoric acid sequencing primer of Influenza A H1N1 HA gene;
The above-mentioned for the second time PCR primer that amplification is used has with biotin labeling, and this primer is at H1N1virus bloodSolidifying element (HA) gene order is high conservative and unique; Used amplification is used for the second time PCR primer sequence and order-checking are drawnThing sequence refers to table 1
Table 1.HA gene cracking site sequencing primer and secondary amplification are used PCR primer
The concentration of the above-mentioned for the first time PCR primer that amplification is used and the PCR primer that amplification is used is for the second time 50nml/L.
In above-mentioned steps 1, RT-PCR amplified reaction process comprises the following steps:
Taking sample H1N1virus RNA extract as template, adopt one-step method to carry out RT-PCR amplification, amplification 950bpThe HA1 genetic fragment of H1N1virus, RT-PCR reaction condition is: 50 DEG C of reverse transcription 30min; 94 DEG C of denaturations2min; 94 DEG C of 15s, 55 DEG C of 30s, 68 DEG C of 1min, 45 circulations; 68 DEG C of 5min. Getting 5 μ lPCR products, to carry out agarose solidifyingGel electrophoresis qualification detects amplification.
By assay is compared with the HA gene cracking site sequence of H1N1virus type strain, can be highly accurateThe sudden change of the true HA gene cracking site that detects H1N1virus, can be conveniently by its application in clinical researchAmong detecting.
The HA1 gene cracking site type strain sequence of H1N1virus type strain is in table 2
Composition and the position of table 2. H1N1virus type strain cracking site amino acid and gene
2. pair pcr amplification product carries out pyrophosphoric acid order-checking, judges whether the cracking site of Influenza A H1N1 HA gene has prominentBecome; Wherein pyrophosphoric acid order-checking adopts SQA pattern, nucleotides application of sample order to be AGCT, specific as follows:
(1) take turns pcr amplification product by second and be fixed on magnetic bead,
(2) magnetic bead is adsorbed on vacuum sample translator, then vacuum sample translator is put into the slight vibration of 70% ethanol 5 seconds,Then move on in sex change buffer solution and aspirate 5 seconds, make DNA sex change to obtain the single stranded DNA template of purifying, finally move on to washingIn buffer solution, clean 5～10 seconds, wash loose single stranded DNA, thereby obtain the single stranded DNA as sequencing template,
(3) primer hybridization: first in the pyrophosphoric acid order-checking each hole of microwell plate, add annealing buffer 50 μ L, then add sequence sequencing primer,The minimum requirements concentration of sequencing primer is 1 μ mol/L, and maximum concentration is 0.2mmol/L, then by vacuum sample translator from PCR plateMove into pyrophosphoric acid order-checking microwell plate, close vavuum pump, by mixed to the single stranded DNA template that purifying is good and sequence order-checking thing primer,At 80 DEG C, sex change 2 minutes, is then cooled to room temperature, makes primer and template annealing hybridization;
The sequence of sequencing primer: 5-GTTACTTTGGCCGTT-3
(4) pyrophosphoric acid order-checking: utilize sequenator and kit, add successively enzyme, substrate and 4 kinds of dNTPs in agent bin, selectSQA detecting pattern, carry out sequencing reaction with the base addition sequence of A, G, C, T sequential loop application of sample 16 times; Pass through CCDOptical system detects each application of sample afterproduct, to obtain special detected peaks, can read standard according to the peak value figure obtainingTrue DNA sequence dna information,
(5) pyrophosphoric acid is checked order compared with the HA1 gene cracking site sequence of assay and H1N1virus type strain, and then judge whether the cracking site of Influenza A H1N1 HA gene has sudden change.
The HA1 gene cracking site that utilizes pyrophosphoric acid sequencing technologies to carry out H1N1virus detects compared with other Molecular DetectionMethod has unique advantage, for definite virus virulence, pathogenic and host range provides simply, experimental program efficiently,Avoid the related loaded down with trivial details experimental procedure of full gene sequencing, can also accurately grasp viral variation direction, conveniently it has been supervisedSurvey, the isolation infection sources cuts off route of transmission, stops the further expansion of epidemic situation.
Brief description of the drawings
Fig. 1 is the HA genetic fragment RT-PCR augmentation detection result of the present invention to H1N1virus.
Wherein, M swimming lane: DNA mark, other swimming lanes: HA genetic fragment RT-PCR product
Fig. 2 is the HA gene cracking site pyrophosphoric acid result with SQA pattern order-checking H1N1virus.
Detailed description of the invention
By the following examples content of the present invention is described in further detail.
1, RNA extracts
The template that the MDCK passage cell infecting with fresh propagative viruses extracts as RNA, the High that adopts Roche company to producePureViralRNAKit extracts viral RNA, and concrete operations are undertaken by kit description, the RNA of extraction immediately forRT-PCR increases or puts-80 DEG C of preservations.
2, RT-PCR reaction amplification HA1 genetic fragment
Carry out first round amplification with thering is the PCR primer of sequence shown in H1F and having with the PCR primer of sequence shown in H1R, to carryThe viral RNA of getting is template. In 50 μ L reaction systems, the concentration of first primer the best is 50nmol/L. RT-PCR reactionCondition is: 50 DEG C of reverse transcription 30min; 94 DEG C of denaturation 2min; 94 DEG C of 15s, 55 DEG C of 30s, 68 DEG C of 1min, 45 circulations; 68 DEG C5min. Get 5 μ lPCR products and carry out agarose gel electrophoresis qualification detection amplification.
3, use and carry out second with the ReversePCR primer of biotin (Biotin) mark and ForwardPCR primer and take turns expansionIncrease, template is the PCR product of first round amplification. In 50 μ L reaction systems, the concentration of second primer the best is 50nmol/L.PCR course of reaction is as follows:
First at 94 DEG C, carry out preheating 10 minutes; At 94 DEG C, keep carrying out for 45 seconds keeping annealing for 45 seconds at sex change, 64 DEG C,At 72 DEG C, keep increasing and extending for 90 seconds, cycle annealing process 30 times; Finally at 72 DEG C, keep 10 minutes.
The PCR product of getting 2 μ L carries out 2% agarose gel electrophoresis detection, electrophoresis 20 minutes under 12V/cm electric potential gradient, afterUnder uviol lamp, check expanding effect. Fig. 1 is the HA1 genetic fragment RT-PCR product electrophoretogram to H1N1virus,Wherein M is index zone, 1～8 HA genetic fragment RT-PCR product that is H1N1virus. In figure, can see and depositingAt a band that single signal is stronger, and there is no remaining primer, there is no primer dimer or other non-specific bands yetExist.
PCR second primer with sequence shown in the first primer of sequence shown in H1F and Forward primer is that two forwards drawThing. PCR first primer with sequence shown in H1R is two with PCR second primer with sequence shown in Reverse primerBar reverse primer plays mark effect with biotin labeled primer in detection.
Pyrophosphoric acid order-checking
1. detect reagent preparation
Binding buffer liquid: by 10mmol/LTris-HCl, 2mol/LNaCl, 1mmol/LEDTA and 0.1%Tween20 go fromThe water-soluble solution of son, with 1mol/LHCl tune pH to 7.6.
Sex change buffer solution (denatureationbuffer): 0.5mol/LNaOH.
Annealing buffer: 20mmol/LTris-Acetate, 2mmol/LMgAc2 solution are mixed, use 4mol/L acetic acid to adjust pH extremely7.6。
Lavation buffer solution (washingbuffer): use 4mol/L acetic acid that 10mmol/LTris-Acetate solution is adjusted to pH to 7.6.70% ethanol: add high purity water 20mL in 70mL absolute ethyl alcohol, be settled to 100mL after fully mixing.
2. detecting instrument and matched reagent
(1) the full-automatic pyrophosphoric acid sequenator of PyroMarkQ96ID, the strand of German QIAGEN company are prepared tool stand (VacuumPrepworkstation), vacuum sample translator (Vacuumpreptool), vavuum pump, vortex oscillator, PCR instrument.
(2) German QIAGEN company produces pyrosequencing kit (PSQ96MASQAUpgradeKit) and the U.S.The coated magnetic bead of streptavidin that GEHealthcare company produces (StreptavidinSepharoseHighPerformance,Cat.No.17-5113-01）。
3. pyrophosphoric acid order-checking detecting step
(1) PCR product is fixed: in PCR plate, put into the pcr amplification product that has of 40 μ L, then add respectively the knot of 37 μ LClose buffer solution and the coated magnetic bead of 3 μ L streptavidin. PCR plate is placed on to normal temperature on vortex oscillator and vibrates 20 minutes, make PCRProduct is fixed on magnetic bead.
(2) purifying of single-stranded template: open vavuum pump, vacuum sample translator is moved on in PCR plate, capture in conjunction with PCR productMagnetic bead, check whether most of magnetic bead has all been attracted on vacuum sample translator. Again vacuum sample translator is put into 70%In ethanol, slight vibration 5 seconds, then moves on in sex change buffer solution and aspirates 5 seconds, makes DNA sex change to obtain the single stranded DNA of purifyingTemplate, finally moves on in lavation buffer solution and cleans 5～10 seconds, washs loose single stranded DNA, thereby obtains as order-checking mouldThe single stranded DNA of plate.
(3) primer hybridization: first add annealing buffer 50 μ L in the pyrophosphoric acid order-checking each hole of microwell plate, then add and there is order shown in table 1Row sequencing primer. The concentration of sequencing primer is 20nmol/L. Again vacuum sample translator is moved into pyrophosphoric acid order-checking from PCR plate micro-Orifice plate, closes vavuum pump, by mixed to the single stranded DNA template that purifying is good and sequence order-checking thing primer, and 2 points of sex change at 80 DEG CClock, is then cooled to room temperature, makes primer and template annealing hybridization.
The sequence of sequencing primer: 5-GTTACTTTGGCCGTT-3
Pyrophosphoric acid order-checking: utilize PyroMarkQ96ID sequenator and kit by German QIAGEN company, successively in agent binAdd enzyme, substrate and 4 kinds of dNTPs, select SQA detecting pattern, alkali with A, G, C, T sequential loop application of sample 16 timesBase addition sequence carries out sequencing reaction. By CCD optical system, each application of sample afterproduct is detected, to obtain special inspectionSurvey peak, can read DNA sequence dna information accurately according to the peak value figure obtaining.
Fig. 2 is the HA gene cracking site pyrophosphoric acid sequencing result that adopts SQA pattern H1N1virus, detects the order drawingClassify aggaatgttccgtctattcaatctagaggcctatttggg as. By the HA gene cracking of itself and H1N1virus type strainSite sequence is compared, and can show whether cracking site gene order suddenlys change.
Claims (1)
1. the hemagglutinin that utilizes based on pyrophosphoric acid order-checking detects the primer of H1N1virus pathogenicity, and its feature existsIn the PCR primer comprising for H1N1 virus hemagglutinin gene being carried out to RT-PCR amplification, and pcr amplification is producedThing carries out the primer of pyrophosphoric acid order-checking;
Wherein, PCR primer comprises
First round amplification PCR primer sequence is as follows:
H1F:ACGTGTTACCCAGGAGATTTC
H1R:TCTTTACCYACTRCTGTGAA
Second to take turns amplification PCR primer sequence as follows:
Reverse primer 5`-GGTTCCCACGATATTTGTGGT-3
Forward primer 5 '-AAAAGGTCCACCAACTTGAGAAAT-3-
The sequence of sequencing primer is as follows: 5-GTTACTTTGGCCGTT-3
。
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| CN107881224A (en) * | 2017-11-22 | 2018-04-06 | 凯杰（苏州）转化医学研究有限公司 | A kind of kit and its detection method of detection HER3 mutation |
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| CN109266784B (en) * | 2018-07-27 | 2022-04-08 | 广东海洋大学 | Closed DNA fluorescent biosensor and application thereof in detection of influenza A H1N1 virus |
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