CN106065419A - The quick real-time fluorescence quantitative PCR detection primer differentiating duck circovirus genotype and detection method thereof - Google Patents
The quick real-time fluorescence quantitative PCR detection primer differentiating duck circovirus genotype and detection method thereof Download PDFInfo
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Abstract
The present invention relates to real-time fluorescence quantitative PCR detection primer and the detection method thereof of a kind of quick discriminating duck circovirus genotype, wherein the sequence of primer is: upstream primer P1:5 ' CAGATCCCCGGGCACGAGA 3 '；Downstream primer P2:5 ' CCTCACCTTCAGGGATTC 3 ', described detection method is: utilize described primer to set up based on SYBR Green I real time fluorescence quantifying PCR method, the difference of the gene 1 type duck circovirus of primer amplification and gene 2 type duck circovirus gene-specific fragments Region Nucleotide G/C content causes the difference of melting curve temperature, gene 1 type duck circovirus and gene 2 type duck circovirus are directly infected and detect by the difference according to melting curve Tm value, the present invention only needs one group of primer just can distinguish different duck circovirus genotype infection, technology is provided to ensure for duck culturing industry healthy aquaculture.
Description
Technical field
The present invention relates to zoonosis quick diagnosis technical field, be specifically related to a kind of quick discriminating duck circovirus base
Real-time fluorescence quantitative PCR detection primer and detection method thereof because of type.
Background technology
Duck circovirus (duck circovirus, DuCV) is one duck group neopathy in recent years poison, each kind duck
Infect, report by Hattermann etc. early than 2003, domestic by Fu Guanghua etc. report the earliest (DuCV-MH25 strain,
GenBank EF451157).Clinical symptoms show as skin and lymphatic system damage, make animal body subject to other cause of diseases
Concurrent and scabies secondary infection；Also Symptoms is had to be dysplasia and feather is in disorder；And can the immune system of infected duck, cause disease
The immunosupress of duck, provisions duck industry causes great economic loss.
Duck circovirus is the animal virus without cyst membrane, icosahedral symmetry, and its genome is sub-thread cyclic DNA, for
One of little pathogenicity virus.The genome of duck circovirus is about 2.0kb, and its genome is annular, comprises 6
The ORF:V1 (49～927) of more than 200bp, V2 (1370～1585), V3 (1661～1918), C1 (1932～1159), C2
(1741～1379), C3 (400～164), be distributed in normal chain and the minus strand that is complementary to.Wherein ORF V1 and ORF C1 is duck
2 main code districts (ORF) of PCV-II genome.
According to duck circovirus genome sequence feature, duck circovirus can be subdivided into two genotype: gene 1 type duck is round
Circovirus virus (Duck Circovirus Type 1, DuCV-1) and gene 2 type duck circovirus (Duck Circovirus Type
2,DuCV-2).Currently, seen there is the report of two kinds of genotype duck circovirus infected duck groups.Although antidiastole gene 1 type and
The dual-PCR method of gene 2 type duck circovirus has been shown in and studies have reported that (Li Z, Wang X, Zhang R, et
al.Evidence of possible vertical transmission of duck circovirus.Vet
Microbiol.2014,174 (1-2): 229-232.), the method is for gene 1 type and gene 2 type duck circovirus difference
District separately designs the primer upstream and downstream primer of a group-specific (gene 1 type and the gene 2 type duck circovirus respectively design).But,
Yet there are no gene 1 type and the real-time fluorescence quantitative PCR (real-time of gene 2 type duck circovirus antidiastole
Quantitative PCR, qPCR) report of method, especially only need one group of primer can be to gene 1 type and gene 2 type duck circle
Kit that circovirus virus differentiates and the research report of method, it is blank that the present invention can fill up association area.
Content of the invention
It is an object of the invention to provide the real-time fluorescence quantitative PCR detection of a kind of quick discriminating duck circovirus genotype
Primer and detection method thereof.This primer has very strong specific, it is only necessary to the primer for qPCR of a group-specific can have
Effect is distinguished different duck circovirus genotype (gene 1 type and gene 2 type) and is infected, and provides technology to protect for duck culturing industry healthy aquaculture
Card.
The purpose of the present invention is achieved through the following technical solutions:
The primer of one group of real-time fluorescence quantitative PCR detection quickly differentiating duck circovirus genotype, the sequence of described primer
Arrange as follows:
Upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 '；
Downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 '.
Described primer needs all can expand DuCV-1 and DuCV-2 simultaneously, and in this amplification region DuCV-1 and
There is the G/C content difference (Tm value difference is different) of gene in DuCV-2.
As shown in Figure 1: the present invention is when carrying out upstream primer P1, downstream primer P2 design, according to according to duck circovirus
The 151-253 potential difference allogene district design of gene 1 type and gene 2 type, the duck circovirus gene 1 type sequence in this region is consistent,
Duck circovirus gene 2 type sequence is consistent, but the sequence between duck circovirus gene 1 type and gene 2 type is inconsistent, exists
G/C content difference, based on the primer of this sequence-specific characteristic Design.As shown in Figure 2: during the primer Standard PCR of the present invention without
Method is distinguished, and duck circovirus gene 1 type and gene 2 type clip size are 103bp, it is impossible to making a distinction, in fig. 2, M is
DL500 DNA Marker；1 is DuCV-1；2 is DuCV-2, and 3 is negative control.
Inventor also designs based on duck circovirus gene 1 type of Fig. 3 and the 532-633 potential difference allogene district of gene 2 type
Upstream primer P3 and downstream primer P4, G/C content is consistent, it is impossible to make a distinction DuCV-1 and DuCV-2, wherein upstream primer P3
As follows with downstream primer P4 sequence:
Upstream primer P3:5 '-TTGAATTTCCCGCCGAAAACAAGT-3 '；
Downstream primer P4:5 '-CCAACCATAAAAGTCGTCCATGA-3 '；
Utilize upstream primer P3 and downstream primer P4 simultaneously to gene 1 type duck circovirus and gene 2 type duck circovirus
Carrying out based on the detection of SYBR Green I real-time fluorescence quantitative PCR, as shown in Figure 4, Tm value is 82.3 DEG C to the melting curve obtaining
Left and right, it is impossible to the different duck circovirus genotype of effective differentiation.
The real-time fluorescence quantitative PCR detection method of a kind of quick discriminating duck circovirus genotype, utilizes upstream primer P1:
5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 ' set up based on SYBR
Green I real time fluorescence quantifying PCR method, the gene 1 type duck circovirus of described primer amplification and gene 2 type duck circovirus
The difference of gene-specific fragments Region Nucleotide G/C content causes the difference of melting curve temperature, according to the difference of melting curve Tm value
Different directly infection gene 1 type duck circovirus and gene 2 type duck circovirus is detected.
The described quick real-time fluorescence quantitative PCR detection method differentiating duck circovirus genotype, it includes following step
Rapid:
1) DNA of gene 1 type duck circovirus and gene 2 type duck circovirus is extracted；
2) utilize upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-
Gene 1 type duck circovirus and gene 2 type duck circovirus are carried out based on SYBR by CCTCACCTTCAGGGATTC-3 ' simultaneously
Green I real-time fluorescence quantitative PCR detects, and reaction makes melting curve after completing；
3) difference according to melting curve specific peak Tm value is directly to gene 1 type duck circovirus and gene 2 type duck circle
Circovirus virus infection type situation directly judges.
The application of the described quick real-time fluorescence quantitative PCR detection method differentiating duck circovirus genotype, in detection
Application in terms of gene 1 type duck circovirus and gene 2 type duck circovirus infection conditions.
For prior art, it is an advantage of the current invention that: authentication method of the present invention is simple, quick and accuracy rate height:
The undergrowth duck pathological material of disease to 14 parts of clinical acquisitions for the present invention detects, and carries out based on SYBR after extracting genomic DNA
Green I real-time fluorescence quantitative PCR detects, and from amplification curve it can be seen that there is the positive pathological material of disease 5 parts of DuCV, positive rate is 35.71%
(5/14)；Analyzing from melting curve Tm value, in the positive pathological material of disease 5 parts of DuCV, DuCV-1 type infects 4 parts, and DuCV-2 type infects 1
Part, it is not detected by 14 parts of samples and there is coinfection phenomenon；The present invention only needs upstream primer P1 and this group of downstream primer P2
Differentiating gene 1 type and gene 2 type duck circovirus, without empty complicated loaded down with trivial details condition optimizing process, the present invention is quickly
It is quick accurate to differentiate in duck circovirus genotype detection, can fill up domestic and international association area research blank.
Brief description
Fig. 1 is that duck circovirus (gene 1 type and gene 2 type) is used for primer P1 and P2 genetic analysis figure.
Fig. 2 is the gel electrophoresis figure after utilizing primer of the present invention to carry out Standard PCR.
Fig. 3 is that duck circovirus (gene 1 type and gene 2 type) is used for primer P3 and P4 genetic analysis figure.
Fig. 4 is the melting curve utilizing primer P3 and P4 to carry out gained after real-time fluorescence quantitative PCR detection is reacted.
Fig. 5 is the melting curve that DuCV-1 is detected by real-time fluorescence quantitative PCR detection primer of the present invention.
Fig. 6 is the melting curve that DuCV-2 is detected by real-time fluorescence quantitative PCR detection primer of the present invention.
Fig. 7 is the melting curve that DuCV-1 and DuCV-2 is detected by real-time fluorescence quantitative PCR detection primer of the present invention.
Detailed description of the invention
Below in conjunction with Figure of description and embodiment, present invention is described in detail:
A kind of primer of the real-time fluorescence quantitative PCR detection of quick discriminating duck circovirus genotype, the sequence of described primer
Arrange as follows:
Upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 '；
Downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 '.
The real-time fluorescence quantitative PCR detection method of a kind of quick discriminating duck circovirus genotype, utilizes upstream primer P1:
5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 ' set up based on SYBR
Green I real time fluorescence quantifying PCR method, the gene 1 type duck circovirus of described primer amplification and gene 2 type duck circovirus
The difference of gene-specific fragments Region Nucleotide G/C content causes the difference of melting curve temperature, according to the difference of melting curve Tm value
Different directly infection gene 1 type duck circovirus and gene 2 type duck circovirus is detected.
The described quick real-time fluorescence quantitative PCR detection method differentiating duck circovirus genotype, it includes following step
Rapid:
1) DNA of gene 1 type duck circovirus and gene 2 type duck circovirus is extracted；
2) utilize upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-
Gene 1 type duck circovirus and gene 2 type duck circovirus are carried out based on SYBR by CCTCACCTTCAGGGATTC-3 ' simultaneously
Green I real-time fluorescence quantitative PCR detects, and reaction makes melting curve after completing；
3) difference according to melting curve specific peak Tm value is directly to gene 1 type duck circovirus and gene 2 type duck circle
Circovirus virus infection conditions carries out visualizing quantitatively detection.
Wherein, step 2) described in based on SYBR Green I real-time fluorescence quantitative PCR detection reaction system be: at 20 μ
Addition following component in L system:
Step 2) described in based on the reaction condition that the detection of SYBR Green I real-time fluorescence quantitative PCR is arranged be: 95 DEG C are pre-
Denaturation 2min；Carry out 94 DEG C of 20s, 56 DEG C of 10s, 68 DEG C of 10s subsequently, after carrying out 40 circulations, make the melting curve of qPCR.
Specific experiment operation is as follows:
1 materials and methods:
1.1 experiment reagents and consumptive material: real time fluorescent quantitative eight comb (Axygen) is purchased from Corning company；Real-time fluorescence
Quantitative reagent SYBR Premix Ex Taq II (Tli RNaseH Plus) is purchased from precious bioengineering (Dalian) Co., Ltd；Glue
Reclaim kit and the little extraction reagent kit of plasmid is purchased from Omega company.
1.2 experiment strains:
Aquatic bird parvovirus (MDPV and GPV), duck plague virus (DPV), Egg Drop syndrome virus (EDSV), gene 1 type
(DuCV-1) (representative strains DuCV-1-PT07, GenBank EU499310) and gene 2 type (DuCV-2) (representative strains DuCV-2-
MH25, GenBank EF451157), E. coli isolated from ducks (Escherichia coli), Riemerlla anatipestifer (Riemerella
Aliatipestifer) separate by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute with Pasteurella (Pasteurella)
Identify and preserve.
1.3 methods:
1.3.1 the design of primers of real-time fluorescence quantitative PCR:
Utilizing Oligo7 to carry out design of primers to it, need when this is to design of primers all can be to DuCV-1 and DuCV-2 simultaneously
Expanding, and there is the G/C content difference of gene in this amplification region DuCV-1 and DuCV-2, described primer is:
Upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 '；
Downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 '.
Expected amplified fragments size is 103bp.
1.3.2 reaction system and the optimization of reaction condition based on the detection of SYBR Green I real-time fluorescence quantitative PCR:
The real time fluorescent quantitative reagent SYBR Premix Ex of precious bioengineering (Dalian) Co., Ltd that the present invention uses
Taq II (Tli RNaseH Plus), real-time fluorescence quantitative PCR reactant liquor uses the reaction system configuration that kit is recommended, real
When quantitative fluorescent PCR reaction system be 20 μ L, concrete configuration method is shown in Table 1.So that the highest fluorescent value (△ Rn), minimum real to occur
When fluorescence threshold (cycle threshold, Ct value) and melting curve analysis in occur without non-specific peak be index, to reaction
Condition (annealing temperature) is optimized, and the reaction condition of optimization is: 95 DEG C of denaturations 2min；Carry out subsequently 94 DEG C of 20s, 56 DEG C
10s, 68 DEG C of 10s, make the melting curve of qPCR after carrying out 40 circulations.
The configuration of table 1 real-time fluorescence quantitative PCR reactant liquor
In the optimization carrying out reaction system and the reaction condition detecting based on SYBR Green I real-time fluorescence quantitative PCR,
Primer concentration is a key factor affecting fluorescent quantitative PCR, if primer concentration is too low, reaction can be made incomplete；If
Primer concentration is too high, then the possibility of generation mispairing and generation nonspecific products can be greatly increased；Therefore inventor passes through
0.8 μ L is used, to ensure reaction completely, it is to avoid produce nonspecific products after optimization.
1.3.3DuCV-1 the extraction with DuCV-2DNA: extract the DNA of DuCV-1 and DuCV-2 in conventional manner.
1.3.4 the preparation of standard items
With DuCV-1 and DuCV-2DNA that extract as template, to stride across real time fluorescent quantitative primer P1 and P2 amplification region
Design primer (P5:5 '-AGCTGCCCAAGTGTTTAATCCCT-3 ' and P6:5 '-CAATGGCGAAGAGCGGCAACTACT-3 ')
Carry out to DuCV-1 and DuCV-2 expanding that (this primer 2 kind genotype duck circovirus all can effectively expand, but cannot be distinguished by gene
Type), amplified production, after agarose gel electrophoresis, utilizes DNA gel to reclaim kit to meeting the special of purpose clip size
Property amplified band reclaim, be connected to after purification on pGEM-T Easy carrier, construction recombination plasmid, and convert to DH5 α sense
By state cell, picking positive colony carries out Zengjing Granule, illustrates that extracting DNA carries out order-checking mirror by plasmid extraction kit
Fixed.PGEM-DuCV1 and pGEM-DuCV-2 can be respectively designated as plasmid positive standard items after identifying correctly through order-checking.
1.3.5 melting curve Tm value is analyzed
1.3.5.1 the acquisition of different genotype duck circovirus melting curve
With ultraviolet specrophotometer measure gene 1 type (DuCV-1) (representative strains DuCV-1-PT07, No. GenBank
And the 260nm of gene 2 type (DuCV-2) (representative strains DuCV-2-MH25, GenBank EF451157) nucleic acid DNA EU499310)
Place's absorbance value (A260), calculates DNA copy number, carries out continuously standard items pGEM-DuCV1 and pGEM-DuCV-2 respectively dilute
As the standard items of qPCR after releasing, set up the real time fluorescent quantitative of different genotype duck circovirus (DuCV-1 and DuCV-2)
PCR method, after reaction completes, utilizes real-time fluorescence quantitative PCR software to observe melting curve, it is thus achieved that corresponding Tm value.
1.3.5.2DuCV-1 the melting curve Tm value of real-time fluorescence qPCR is analyzed
Carrying out based on the detection of SYBR Green I real-time fluorescence quantitative PCR by the condition optimizing, reaction can expand after terminating
The melting curve of product is as shown in Figure 5: from such as the melting curve analysis of Fig. 5. occur single at Tm=(88.80 ± 0.1) DEG C
The specific peak of one, without primer dimer and nonspecific products.To aquatic bird encountered pathogenic (MDPV and GPV, DPV, EDSV,
Escherichia coli, Riemerella aliatipestifer, Pasteurella) detect, observe and have or not appearance
Positive amplification signal.
1.3.5.3DuCV-2 the melting curve Tm value of real-time fluorescence qPCR is analyzed
Carrying out based on the detection of SYBR Green I real-time fluorescence quantitative PCR by the condition optimizing, reaction can expand after terminating
The melting curve of product is as shown in Figure 6: from Fig. 6 melting curve analysis: single Tm=(90.08 ± 0.13) DEG C of appearance
Specific peak, without primer dimer and nonspecific products.To aquatic bird encountered pathogenic (MDPV and GPV, DPV, EDSV,
Escherichia coli, Riemerella aliatipestifer, Pasteurella) detect, observe and have or not appearance
Positive amplification signal.
1.3.6 the Tm value of double real time fluorescent quantitative qPCR is analyzed
With standard items pGEM-DuCV1 and pGEM-DuCV-2 as template, carry out based on SYBR by the condition optimizing
Green I real-time fluorescence quantitative PCR detects, and reaction can obtain the melting curve of amplified production as shown in Figure 7 after terminating: can from Fig. 7
Know: only gene 1 type (DuCV-1) (representative strains DuCV-1-PT07, GenBank EU499310) and gene 2 type (DuCV-2) (generation
Table strain DuCV-2-MH25, GenBank EF451157) visible specific positive amplified signal.From fig.7, it can be seen that work as Tm=
It when (88.80 ± 0.1) DEG C, is judged to DuCV-1 and infects the positive；It when Tm=(90.08 ± 0.13) DEG C, is judged to DuCV-2 and infects sun
Property.
1.3.7 clinical practice
Utilize the real-time fluorescence quantitative PCR detection primer of a kind of quick discriminating duck circovirus genotype that the present invention provides
And the undergrowth duck pathological material of disease that detection method is to 14 parts of clinical acquisitions detects, from amplification curve it can be seen that there is DuCV sun
5 parts of venereal disease material, positive rate is 35.71% (5/14)；Analyze from melting curve Tm value, DuCV-in the positive pathological material of disease 5 parts of DuCV
1 type infects 4 parts, and DuCV-2 type infects 1 part, is not detected by DuCV-1 and DuCV-2 coinfection positive in 14 parts of clinical samples.This
The method that invention is passed through i.e. can determine that the duck circovirus gene type of infection with one group of real-time fluorescence quantitative PCR primer and carries out
Accurate quantitative analysis.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>the quick real-time fluorescence quantitative PCR detection primer differentiating duck circovirus genotype and detection method thereof
<160> 2
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
cagatccccg ggcacgaga 19
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
cctcaccttc agggattc 18
Claims (6)
1. the primer of the real-time fluorescence quantitative PCR detection of a quick discriminating duck circovirus genotype, it is characterised in that: described
The sequence of primer is as follows:
Upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 '；
Downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 '.
2. the real-time fluorescence quantitative PCR detection method of a quick discriminating duck circovirus genotype, it is characterised in that: in utilization
Trip primer P1:5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-CCTCACCTTCAGGGATTC-3 ' sets up base
In SYBR Green I real time fluorescence quantifying PCR method, the gene 1 type duck circovirus of described primer amplification and gene 2 type duck
The difference of PCV-II gene-specific fragments Region Nucleotide G/C content causes the difference of melting curve temperature, according to melting curve
Gene 1 type duck circovirus and gene 2 type duck circovirus are directly infected and detect by the difference of Tm value.
3. the real-time fluorescence quantitative PCR detection method of quick discriminating duck circovirus genotype according to claim 2, its
It is characterised by: it comprises the following steps:
1) DNA of gene 1 type duck circovirus and gene 2 type duck circovirus is extracted；
2) utilize upstream primer P1:5 '-CAGATCCCCGGGCACGAGA-3 ' and downstream primer P2:5 '-
Gene 1 type duck circovirus and gene 2 type duck circovirus are carried out based on SYBR by CCTCACCTTCAGGGATTC-3 ' simultaneously
Green I real-time fluorescence quantitative PCR detects, and reaction makes melting curve after completing；
3) difference according to melting curve specific peak Tm value is directly sick to gene 1 type duck circovirus and gene 2 type duck annulus
Poison infection conditions carries out visualization and differentiates to judge.
4. the real-time fluorescence quantitative PCR detection method of quick discriminating duck circovirus genotype according to claim 3, its
Be characterised by: step 2) described in based on SYBR Green I real-time fluorescence quantitative PCR detection reaction system be: at 20 μ L bodies
Addition following component in system:
5. the real-time fluorescence quantitative PCR detection method of quick discriminating duck circovirus genotype according to claim 3, its
Be characterised by: step 2) described in based on SYBR Green I real-time fluorescence quantitative PCR detection arrange reaction condition be: 95 DEG C
Denaturation 2min；Carrying out 94 DEG C of 20s, 56 DEG C of 10s, 68 DEG C of 10s subsequently, the melting making qPCR after carrying out 40 circulations is bent
Line.
6. quickly differentiate the primer of the real-time fluorescence quantitative PCR detection of duck circovirus genotype according to claim 1
Application, it is characterised in that: the application in terms of detection gene 1 type duck circovirus and gene 2 type duck circovirus infection conditions.
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| CN111826465A (en) * | 2020-07-30 | 2020-10-27 | 福建省农业科学院畜牧兽医研究所 | Primers and probe for real-time fluorescent quantitative PCR detection of duck circovirus type 1 |
| CN111910018A (en) * | 2020-07-30 | 2020-11-10 | 福建省农业科学院畜牧兽医研究所 | Primers and probe for real-time fluorescent quantitative PCR detection of duck circovirus type 2 |
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