CN104789700A - DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method - Google Patents
DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method Download PDFInfo
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Abstract
The invention discloses a DHAV (duck hepatitis A virus) typing detection method based on a fluorescent quantitative PCR (polymerase chain reaction) melting curve method. The DHAV typing detection method comprises steps as follows: virus RNA (ribonucleic acid) extraction and cDNA (complementary deoxyribonucleic acid) synthesis; preparation of positive standard forms; establishment of the DHAV typing detection method based on the fluorescent quantitative PCR melting curve method; method validation adopting a specificity test, a sensitivity test and a reproducibility test. According to the DHAV typing detection method, all that is required is to add a pair of primers, A and C gene types are detected by single tube, the method is simple and feasible, and time consumption is low; the whole amplification and detection process adopts closed tube operation, so that pollution of PCR amplification products is avoided, and false positive results are reduced; amplified fragments are short, higher annealing temperature is adopted, and amplification specificity is guaranteed. The fast DHAV typing detection method is established, great convenience is provided for correct diagnosis and control of the DHAV, and economic benefits of agricultural production are achieved.
Description
Technical field
The present invention relates to a kind of DHAV based on quantitative fluorescent PCR melting curve method (A type duck hepatitis virus) somatotype detection method.
Background technology
A type duck hepatitis virus (Duck hepatitis A virus, DHAV) is a kind of main pathogen causing Duck Hepatitis Virus.Mainly there is acute necrotizing hepatitis for pathological characters with the duckling of 1 ~ 21 age in days in it, morbidity rapidly and mortality ratio is high and have the infectivity of height, at present in worldwide distribution.DHAV can be divided into DHAV-A according to genetic evolution distance, and DHAV-B, DHAV-C be totally three kinds of genotype, wherein advantage strain mainly DHAV-A and DHAV-C of China.Due to for DHAV specific antibody be difficult to pass to duckling by laying ducks, therefore current main dependence prevents and treats the generation of this disease to duckling injection attenuated vaccine or specific yolk antibody.But great many of experiments confirms, lacks cross-protection ability between the DHAV of different genotype, namely use for the attenuated vaccine of DHAV-A or the infection of yolk antibody to control DHAV-C inoperative, vice versa.Due to the clinical symptom that shows after two kinds of genotypic virus infection ducklings and pathological change closely similar, only cannot distinguish with observing pathological change; And the common popular phenomenon of current DHAV-A and DHAV-C is on the rise, bring great difficulty to the correct Diagnosis and treatment of this disease.Therefore in the urgent need to setting up a kind of feasible method, which kind of genotype the DHAV that the ill duckling of energy rapid detection infects belongs to, and instructs yolk antibody and the attenuated vaccine of clinical proper use of homotype with this.
At present, the gene fragment using inverse transcription polymerase chain reaction (RT-PCR) to detect DHAV is its main method for quick, but the method can not be carried out quantitatively virus.Also have report to use two pairs of primers in order to detect and to distinguish DHAV-A and DHAV-C, but the simplicity of the sensitivity of the method and operation is short of all to some extent simultaneously, and needs to carry out nucleic acid electrophoresis and just can complete thus add the consuming time of detection.Fluorescence quantitative RT-RCR technology, because having the advantages such as specificity, accuracy, totally-enclosed reaction, has become the important means of rapid detection pathogenic micro-organism.Detection RRT-PCR being applied to DHAV has been reported.The RRT-PCR technology that Miao etc. establish Taq man probe method detects the DHAV-A in duck embryo and chick embryo allantoic liquid respectively.According to 3D gene conserved regions sequences Design hydrolysis probes and the primer of DHAV-A, the geneome RNA of the DHAV-A of about 10 copies can be detected.Huang etc. establish the RRT-PCR technology of SYBR Green method in order to detect the DHAV-C in clinical tissue.According to the 2C gene conserved regions sequences Design pair of primers of DHAV-C, the geneome RNA of the DHAV-C of about 3000 copies can be detected.But the report at present not yet about using a kind of RRT-PCR to carry out differential diagnosis different genotype DHAV.Because SYBR Green fluorescence dye is because combining with all DNA double chains, selectivity be there is no to template, and its low price therefore be more suitable for the detection of multiple object product.Solubility curve analytical method be the amplification of SYBR Green method after PCR primer from certain temperature, slowly rise to 95 DEG C, amplified production double-stranded DNA raises sex change gradually with temperature and to unwind generation strand.The fluorescence dye SYBR Green be embedded in dehybridization procedure in double-strand can be released, the change of instrument automatic detection reaction inner fluorescent tube signal, finally draw out the temperature variant solubility curve of amplified production fluorescent signal, and obtain the solubility curve peak figure of corresponding PCR primer.Therefore different size, the PCR primer of different genes composition, type figure is different at its solubility curve peak.
The present invention, by than the DHAV of relatively large different genotype, in the obvious 5 ' UTR region of its G+C content difference, designs pair of primers and can to increase DHAV-A and DHAV-C simultaneously; And by the solubility curve that compares amplified production come easy, differentiate to detect the virogene type in sample rapidly.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of DHAV based on quantitative fluorescent PCR melting curve method (A type duck hepatitis virus) somatotype detection method.Detection method only adds pair of primers in a PCR reaction system, and single tube detects A, C genotype, and method is simple, consuming time few; Whole amplification and testing process are stopped pipe operation, avoid the pollution of pcr amplification product, decrease false positive results; This detection method amplified fragments is short, adopts higher annealing temperature, ensure that the specificity of amplification.After quantitative real time PCR Instrument amplification after melting curve step i.e., rapidly discriminating DHAV genotype easy by Tm value.The present invention establishes a kind of method of rapid detection somatotype duck hepatitis A virus (HAV), provides a great convenience, for agriculture production brings economic benefit to the correct Diagnosis and treatment of this disease.
The present invention is achieved by the following technical solutions:
First aspect, the invention provides a kind of A type duck hepatitis virus somatotype detection primer based on quantitative fluorescent PCR melting curve method, described primer is as follows:
DHAV-F:5'GTTGTGAAACGGATTACCGGTAGT 3'，
DHAV-R:5'ACTCGACCAGCCGCGACCCTAT 3'。
Second aspect, the invention provides a kind of A type duck hepatitis virus somatotype detection positive plasmid based on quantitative fluorescent PCR melting curve method, described plasmid comprises pMD-19T-DHAV-A and pMD-19T-DHAV-C.
The third aspect, the invention provides a kind of A type duck hepatitis virus somatotype detection positive plasmid standard substance based on quantitative fluorescent PCR melting curve method;
Described in described positive plasmid standard substance, the concentration of plasmid is 1 × 10
11copies/ μ L; Can dilute for concentration gradient in use procedure.
Fourth aspect, the invention provides a kind of A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method, described detection method comprises:
The extraction of step one, viral RNA and cDNA synthesis: extract A type duck hepatitis virus total serum IgE from attacking malicious duck tissue, reverse transcription obtains cDNA;
The preparation of step 2, positive criteria template: with described cDNA for template, with described DHAV-F and DHAV-R for primer, carries out pcr amplification to the goal gene of A type duck hepatitis virus; Described positive recombinant plasmid is built with described pcr amplification product;
Step 3, foundation based on the A type duck hepatitis virus genotyping detection method of quantitative fluorescent PCR melting curve method: with described positive recombinant plasmid for template, optimize described PCR reaction conditions, drawing standard curve, melting curve;
Step 4, method validation: the real-time fluorescence quantitative PCR detection method that step 3 is set up is verified as follows:
Specific test: adopt fluorescent quantitative PCR detection method described in step 3, according to described typical curve and melting curve, specific detection carried out to A type duck hepatitis virus and non-A type duck hepatitis fowl source virus;
Sensitivity test: dilute described positive plasmid standard substance, set up regular-PCR control group simultaneously, increase respectively, obtains minimum template and detects copy number;
Replica test: in respectively described positive plasmid standard substance being done batch and batch between repeat, establish negative control simultaneously, by the calculating to the TM value of A type duck hepatitis virus and the analysis covariation coefficient of melting curve, verify the repeatability of described A type duck hepatitis virus genotyping detection method.
Preferably, in step one, described duck comprises Beijing duck, and described tissue comprises liver.
Preferably, in step 3, described PCR reaction conditions comprises primer concentration, annealing temperature.
Preferably, described primer concentration scope is 0.04 ~ 1.04umol/L; Described annealing temperature is 54 ~ 62 DEG C.
Preferably, described primer concentration is 0.24umol/L; Described annealing temperature is 55 DEG C.
Preferably, in the specific test of step 4, described A type duck hepatitis virus comprises DHAV-A, DHAV-C; Described non-A type duck hepatitis fowl source virus comprises duck reovirus, Avian pneumo-encephalitis virus, avian influenza virus, avian infectious bronchitis virus, goose parvovirus, duck plague, duck egg drop syndrome virus, duck tembusu virus etc.
Preferably, in the sensitivity test of step 4, described dilution comprises according to 10 times of gradient dilutions; It is 100 copies/μ L that this operation is implemented to show that minimum template that fluorescent quantitative PCR detection method detects detects copy number.
Preferably, in the replica test of described step 4, repeat in described batch and between criticizing specifically to refer to do in 3 batches to repeat between repetition and 3 batches, positive recombinant plasmid concentration adopts respectively: 1 × 10
7copies/ μ L, 1 × 10
5copies/ μ L, 1 × 10
4copies/ μ L.
Preferably, described positive plasmid concentration is 1 × 10
7copies/ μ L, 1 × 10
5copies/ μ L.
In sum, the present invention successfully establishes A type duck hepatitis virus somatotype detection method based on quantitative fluorescent PCR melting curve method by adopting pair of primers, after quantitative real time PCR Instrument amplification after melting curve step i.e., rapidly discriminating DHAV genotype easy by Tm value.
Compared with prior art, the present invention has following beneficial effect:
1, be only added with pair of primers in a PCR reaction system in this detection method, single tube detects A, C genotype, and method is simple, consuming time less;
2, this detection method is without the need to using Taq Man probe, reduces testing cost, reduces the appearance of false negative result;
3, whole amplification and testing process are stopped pipe operation, do not need to uncap to draw amplified production and carry out electrophoresis, avoid the pollution of pcr amplification product, decrease false positive results;
4, this detection method amplified fragments is short, adopts higher annealing temperature, also can ensure the specificity increased;
5, SYBR Green is a kind of asymmetric nitrile fluorescein, can non-specifically be embedded in the ditch in DNA double spirane structure, the number of the size representation DNA duplex molecule of fluorescence intensity, therefore tentatively can judge the number of DNA double chain molecule from the power of fluorescence intensity;
6, when two DNA molecular renaturation in conjunction with time fluorescence the strongest, reduce with temperature rising fluorescence intensity and occur a more constant gradient, when temperature reaches Tm value, fluorescence intensity sharply declines; Melting curve figure can be obtained, the Tm value of the temperature representative double chain DNA molecule at its crest place through software analysis; Namely easy by Tm value, differentiate DHAV genotype rapidly;
7, the size of Tm value depends on G/C content in the length of amplification of DNA fragments and sequence thereof, therefore the different corresponding Tm value of amplified production is also different, namely DHAV genotype is differentiated by Tm value after melting curve method is analyzed, reduce and get rid of the interference of non-specific amplification product and primer dimer, ensure that the specificity of this detection method.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the structure of recombinant plasmid;
Wherein, A:pMD-19T-DHAV-A C:pMD-19T-DHAV-C;
Fig. 2 is the amplification curve, the canonical plotting that detect DHAV-A;
Wherein, A is the amplification curve diagram of DHAV-A;
1 ~ 7:pMD-19T-DHAV-C 1 × 10
9, 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3the series standard template of copies/ μ L;
B is the canonical plotting of DHAV-A;
Fig. 3 is the amplification curve, the canonical plotting that detect DHAV-C;
Wherein, A is the amplification curve diagram of DHAV-C,
1 ~ 7:pMD-19T-DHAV-C 1 × 10
9, 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3the series standard template of copies/ μ L;
B is the canonical plotting of DHAV-C;
Fig. 4 is DHAV-A, DHAV-C melting curve figure;
Wherein A:DHAV-A, C:DHAV-C;
Fig. 5 is the specific amplification curve of fluorescence quantitative RT-RCR;
Wherein 1:DHAV-A 2:DHAV-C 3-9:DRV, NDV, AIV, IBV, GPV, DEV, DTMUV;
Fig. 6 is regular-PCR amplification electrophorogram;
Wherein A: plasmid pMD-19T-DHAV-A, C: plasmid pMD-19T-DHAV-C
1 ~ 8:1 × 10
9, 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the series standard template of copies/ μ L;
Fig. 7 is duck viral hepatitis tissue distribution figure;
Wherein A is DHAV-A tissue distribution figure; B is DHAV-C tissue distribution figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
1.1 strain
DHAV-A ZJV strain, DHAV-C LS strain (KP233203), DRV TH11 strain, Avian pneumo-encephalitis virus (NDV), H7 avian influenza virus (AIV), avian infectious bronchitis virus (IBV), goose parvovirus (GPV), duck plague virus (DEV), duck tembusu virus (DTMUV) provide by this laboratory.
1.2 reagent and instrument
Trizol is purchased from invitrogen company; Taq enzyme, RNA enzyme inhibitors, dNTP, DNA Marker, pMD-19T carrier, 2 × Fast SYBR Green I Mixture etc. are all purchased from the precious biological company limited in Dalian; Fowl source ThermoScript II (AMV), T4DNA ligase enzyme are purchased from Promega company; Glue reclaims test kit, and bacillus coli DH 5 alpha is purchased from Tian Gen company; Plasmid extraction kit is purchased from QIAGen company; Taq enzyme, RNA enzyme inhibitors, dNTP, key instrument are Mastercycler ep realplex 4 quantitative real time PCR Instrument (Eppendorf company).
1.3 primer
The genomic sequence of MegAlign software to 2 pairs of strains that 84 strain DHAV strains in US National Bioinformatics Institute NCBI gene pool Genbank and this laboratory are separated in Lasergene is used to carry out homology analysis, design and synthesize a pair Auele Specific Primer at DHAV-A DHAV-C 5' non-coding region, primer sequence is as follows:
DHAV-F:5'GTTGTGAAACGGATTACCGGTAGT 3'，
DHAV-R:5'ACTCGACCAGCCGCGACCCTAT 3'，
Expanding fragment length is 203bp.
The extraction of 1.4 viral RNAs and cDNA synthesis
The virus total RNA in malicious Beijing Duck Liver tissue is attacked according to Trizol method specification sheets extraction DHAV-A ZJV strain, DHAV-C LS strain.Prepare reaction solution according to following table 1 and carry out reverse transcription, reaction conditions is 37 DEG C and hatches 2h, and hatch 10min for 75 DEG C ,-20 DEG C save backup, for subsequent experimental.
Table 1RT reaction system
| Reaction reagent | Cumulative volume | |
| Template ribonucleic acid solution | 10uL | |
| 5×M-MLV Buffer | 5.0uL | |
| dNTPs(10.0mmol/μL) | 2.0uL | |
| Primer R(10pmol/μL) | 95 DEG C of l0min, on ice 5min | 2.0uL |
| RNase Inhibitor | 1.0uL | |
| M-MLV Reverse Transcriptase | 1.0uL | |
| DEPC H 2O | up to 25uL |
The preparation of 1.5 positive criteria templates
With above-mentioned product cDNA for template, DHAV-F and DHAV-R is that primer carries out pcr amplification to the goal gene of DHAV-A ZJV strain, DHAV-C LS strain respectively.PCR reaction system is as shown in table 2.
Table 2PCR reaction system
| Reaction reagent | Cumulative volume |
| CDNA solution | 2uL |
| 10×LA Taq Buffer | 5.0uL |
| TaKaRa LA Taq | 1.0uL |
| dNTPs(10.0mmol/μL) | 2.0uL |
| DHAV-F(10umol/μL) | 1.0uL |
| DHAV-R(10umol/μL) | 1.0uL |
| DEPC H20 | up to 50uL |
Pcr amplification condition is as follows:
After reaction terminates, get 10 μ L PCR primer electrophoresis detection amplification on 1% sepharose.Reclaim purifying object fragment respectively, be connected with pMD-19T carrier, be converted into DH5 α E bacterial classification, extraction plasmid carries out enzyme and cuts qualification and sequencing, positive recombinant plasmid called after pMD-19T-DHAV-A, pMD-19T-DHAV-C, measure concentration, according to formula: copy number (Copies/uL)=(plasmid concentration × 6.02 × 10
23)/(DNA molecular quality), calculate the copy number of plasmid standard.As calculated two kinds of recombinant plasmid dna molecule number are adjusted to 1 × 10
10copies/ μ L.Respectively two kinds of standard substance plasmids are done 10 times of gradient dilutions, obtain 1 × 10
9, 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the series standard template of copies/ μ L.
The foundation of 1.6 SYBR Green real-time fluorescence quantitative PCR detection methods
1.6.1 the foundation of SYBR Green quantitative fluorescent PCR reaction system and optimization
Respectively with two kinds of plasmid standards for template, reaction system is 25 μ L, is optimized respectively and draws melting curve to primer concentration and reaction conditions.
2.4.3.1 the optimization of fluorescence quantitative RT-PCR primer concentration
Get respectively and be diluted to 1 × 10
7copies/ μ L positive plasmid standard substance pMD-19T-DHAV-A and pMD-19T-DHAV-C is template.PCR reaction (annealing temperature is 55 DEG C) is carried out, to obtain best primer concentration during virus amplification respectively with primer volume 0.1 μ L, the 0.2 μ L of 10umol/L, 0.4 μ L, 0.6 μ L, 0.8 μ L, 1.0 μ L, 1.2 μ L, 1.4 μ L, 1.6 μ L, 1.8 μ L, 2.0 μ L.
2.4.3.2 the optimization of fluorescence quantitative RT-RCR annealing temperature
Get respectively and be diluted to 1 × 10
7copies/ μ L positive plasmid standard substance pMD-19T-DHAV-A and pMD-19T-DHAV-C is template, for annealing temperature, pcr amplification (primer concentration is for 0.24uM) is carried out to virus, to determine optimum annealing temperature with 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C respectively.
1.6.2 the foundation of typical curve
Get the DNA molecular number 1 × 10 of pMD-19T-DHAV-A, pMD-19T-DHAV-C recombinant plasmid respectively
9copies/ μ L ~ 1 × 10
3copies/ μ L is totally 7 concentration, carries out real-time fluorescence quantitative RT-PCR reaction as standard substance.Adopt the condition optimized to carry out SYBR Green I real-time fluorescence quantitative PCR, obtain respective Ct value, with Ct value for X-coordinate, with the logarithm of initial template concentration for ordinate zou, production standard curve.
1.7 specific test
The method that application is set up carries out specific test to DHAV-A, DHAV-C and DRV, NDV, AIV, IBV, GPV, DEV, DTMUV strain.
1.8 sensitivity test
Two kinds of positive plasmid standard substance pMD-19T-DHAV-A and pMD-19T-DHAV-C are done 10 times of gradient dilutions, sets up regular-PCR control group simultaneously.Increase, detect copy number with the minimum template that this obtains the DNA of fluorescence quantitative RT-RCR reaction.
1.9 replica test
Do in 3 batches two kinds of positive plasmid standard substance pMD-19T-DHAV-A and pMD-19T-DHAV-C respectively and repeat and repeat between 3 batches, plasmid concentration adopts respectively: 1 × 10
7copies/ μ L, 1 × 10
5copies/ μ L, 1 × 10
4copies/ μ L, establishes negative control simultaneously, by the calculating to the TM value of virus and the analysis covariation coefficient of melt curve analysis, and the repeatability of checking DHAV-A and DHAV-C fluorescent quantitative PCR detection method.
The detection of 1.10 laboratory infection samples
Choose the heart of the 3 age in days ducklings that 36h gathers after DHAV-A ZJV strain, DHAV-C LS strain experimental infection, liver, spleen, lung, kidney, brain, pancreas and totally 21 parts, jejunum internal organs respectively, use establishment method to detect respectively.
2 results
The preparation of 2.1 positive criteria product
RT-PCR amplification obtains DHAV-A, DHAV-C goal gene fragment, be cloned into construction recombination plasmid in pMD-19T carrier, PCR qualification is carried out to it, obtain the specific fragment that 1 size is about 203bp, conform to (Fig. 1) with expection object clip size, show that goal gene fragment has been cloned in pMD-19T carrier.Positive recombinant plasmid pMD-19T-DHAV-A concentration is 239ng/ μ L, and pMD-19T-DHAV-C concentration is 223ng/ μ L, and pMD-19T-DHAV-A, pMD-19T-DHAV-C recombinant plasmid dna molecule number regulates and is 1. × 10 as calculated
10copies/ μ L.
The determination of 2.2SYBR Green real-time fluorescence quantitative PCR reaction conditions
3.1.1.1 the determination of best primer concentration and annealing temperature
Show that when annealing temperature is 55 DEG C, Ct value is minimum, fluorescence intensity is maximum when primer final concentration is 0.24uM by test-results, negative control unstressed configuration signal produces (see table 3 and table 4).
The determination of the best primer concentration of table 3
The determination of table 4 optimum annealing temperature
3.1.3 the drafting of DHAV-A, DHAV-C real-time fluorescence quantitative RT-PCR typical curve is detected
Adopt the condition (see table 4) optimized, carry out real-time fluorescence quantitative RT-PCR reaction, obtain respective Ct value, respectively with recombinant plasmid pMD-19T-DHAV-A and pMD-19T-DHAV-C 1.0 × 10
2~ 1.0 × 10
9the logarithm of copies/ μ L copy number is X-axis, and cycle index (Ct) is Y-axis, Criterion curve.
Table 5PCR reaction system
| Reaction reagent | Cumulative volume |
| CDNA solution | 2uL |
| 2×SYBR Green | 12.5uL |
| DHAV-F(10umol/μL) | 0.6uL |
| DHAV-R(10umol/μL) | 0.6uL |
| 50×ROX Reference Dye | 0.5uL |
| DEPC H 20 | up to 25uL |
Result display (see Fig. 2-A, 2-B and 3-A, 3-B): two kinds of recombinant plasmid standard substance each extent of dilution amplification curves are parallel, the Ct value difference evenly different dilution standard substance Ct value of reflection and copy number is good linear relationship.
A relation conefficient is R
2=0.999, amplification efficiency reaches 100%, and logarithm and the Ct value linear relationship of starting copy number are Y=-3.331X+42.12.
C relation conefficient is R
2=0.997, amplification efficiency reaches 98%, and logarithm and the Ct value linear relationship of starting copy number are Y=-3.382X+42.87.
The melting curve of 2.3 SYBR Green real-time fluorescence quantitative PCRs
With StepOne Plus software, result is analyzed, melting curve figure can be obtained through software analysis, the Tm value (melting temperature) of the temperature representative double chain DNA molecule at its crest place.Tm value according to amplified production can judge its genotype.Result shows, and the unimodal of narrow and point all to appear in pMD-19T-DHAV-A and pMD-19T-DHAV-C recombinant plasmid, and fusing point peak does not appear in negative control.PMD-19T-DHAV-A melting temperature (Tm) is (85.8 ± 0.5) DEG C pMD-19T-DHAV-C melting temperature (Tm) is (83.8 ± 0.5) DEG C (Fig. 4), shows that the reaction of this SYBR Green real-time fluorescence quantitative PCR is for specific amplification.
2.4 the specific test of fluorescence quantitative RT-RCR
PCR melting curve shows, the melting peak of DHAV-A with DHAV-C virus is concentrated consistent, occur without assorted peak and small peak, explanation amplified production is single, without non-specific amplification, and the detected result of the strains such as DRV, NDV, AIV, IBV, GPV, DEV, DTMUV is feminine gender (fluorescent signal does not arrive and detects credible threshold value) (Fig. 5), illustrate that present method has good specificity.
2.5 sensitivity test results
Detect the Ct value >0 of sample in real-time fluorescence quantitative RT-PCR 35 circulation, then result is judged to be the positive.By the method set up to pMD-19T-DHAV-A and pMD-19T-DHAV-C recombinant plasmid 1 × 10
9copies/ μ L ~ 1 × 10
2copies/ μ L detects.Result shows that the method is 1.0 × 10 to two kinds of recombinant plasmid limit of identification
3copies/ μ L, negative control unstressed configuration amplification (see Fig. 2 and Fig. 3).And the limit of detection of regular-PCR is 1.0 × 10
4copies/ μ L (see Fig. 6).The method that this test is set up has higher sensitivity, and sensitivity is 1.0 × 10
3copies/ μ L, sensitivity is 10 times of regular-PCR.
2.6 repeated detected results
In pMD-19T-DHAV-A group, the variation coefficient is all less than 1.5%, and between-group variation coefficient is all less than 1%, and repeatability is good; In pMD-19T-DHAV-C group, the variation coefficient is all less than 1.5%, and between-group variation coefficient is all less than 1.5%, and repeatability is good, (see table 6-A, 6-B).
Replica test result in table 6-A quantitative fluorescent PCR group, between group
Replica test result in table 6-B quantitative fluorescent PCR group, between group
2.7 sample detection results
Choose the heart of the 3 age in days ducklings that 36h gathers after DHAV-A ZJV strain, DHAV-C LS strain experimental infection, liver, spleen, lung, kidney, brain, pancreas and totally 21 parts, jejunum internal organs (see Fig. 7-A and 7-B) respectively, result shows, A type A type duck hepatitis virus and C type A type duck hepatitis virus all in liver titre the highest, and A type A type duck hepatitis virus titre levels is overall higher than C type A type duck hepatitis virus.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1., based on an A type duck hepatitis virus somatotype detection primer for quantitative fluorescent PCR melting curve method, it is characterized in that, described primer is as follows:
DHAV-F:5'GTTGTGAAACGGATTACCGGTAGT 3'，
DHAV-R:5'ACTCGACCAGCCGCGACCCTAT 3'。
2. a positive recombinant plasmid is used in the A type duck hepatitis virus somatotype detection based on quantitative fluorescent PCR melting curve method, and described positive recombinant plasmid comprises pMD-19T-DHAV-A and pMD-19T-DHAV-C.
3. the A type duck hepatitis virus somatotype based on quantitative fluorescent PCR melting curve method according to claim 2 detects with positive recombinant plasmid standard substance.
4., based on an A type duck hepatitis virus genotyping detection method for quantitative fluorescent PCR melting curve method, it is characterized in that, described detection method comprises:
The extraction of step one, viral RNA and cDNA synthesis: extract A type duck hepatitis virus total serum IgE from attacking malicious duck tissue, reverse transcription obtains cDNA;
The preparation of step 2, positive criteria template: with described cDNA for template, with DHAV-F and DHAV-R described in claim 1 for primer, carries out pcr amplification to the goal gene of A type duck hepatitis virus; Described positive recombinant plasmid according to claim 2 is built with described pcr amplification product;
Step 3, foundation based on the A type duck hepatitis virus genotyping detection method of quantitative fluorescent PCR melting curve method: with described positive recombinant plasmid for template, optimize described PCR reaction conditions, drawing standard curve, melting curve, set up the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method;
Step 4, method validation: the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method that step 3 is set up is verified as follows:
Specific test: based on the A type duck hepatitis virus genotyping detection method of quantitative fluorescent PCR melting curve method, according to described typical curve and melting curve described in employing step 3, specific detection is carried out to A type duck hepatitis virus and non-A type duck hepatitis fowl source virus;
Sensitivity test: dilute described positive recombinant plasmid standard substance, set up regular-PCR control group simultaneously, increase respectively, obtain minimum template and detect copy number;
Replica test: in respectively described positive regroup plasmid standard being done batch and batch between repeat, establish negative control simultaneously, by the calculating to the TM value of A type duck hepatitis virus and the analysis covariation coefficient of melting curve, verify the repeatability of described A type duck hepatitis virus genotyping detection method.
5. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 4, it is characterized in that, in step one, described duck comprises Beijing duck, and described tissue comprises liver.
6. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 4, is characterized in that, in step 3, described PCR reaction conditions comprises primer concentration, annealing temperature.
7. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 6, it is characterized in that, described primer concentration scope is 0.04 ~ 1.04umol/L; Described annealing temperature is 54 ~ 62 DEG C.
8. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 4, is characterized in that, in the specific test of step 4, described A type duck hepatitis virus comprises DHAV-A, DHAV-C; Described non-A type duck hepatitis fowl source virus comprises duck reovirus, Avian pneumo-encephalitis virus, avian influenza virus, avian infectious bronchitis virus, goose parvovirus, duck plague, duck egg drop syndrome virus or duck tembusu virus.
9. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 4, is characterized in that, in the sensitivity test of step 4, described dilution comprises according to 10 times of gradient dilutions.
10. the A type duck hepatitis virus genotyping detection method based on quantitative fluorescent PCR melting curve method according to claim 4, it is characterized in that, in the replica test of described step 4, repeat in described batch and between criticizing specifically to refer to do in 3 batches to repeat between repetition and 3 batches, positive recombinant plasmid concentration adopts respectively: 1 × 10
7copies/ μ L, 1 × 10
5copies/ μ L, 1 × 10
4copies/ μ L.
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