CN110527746A - For detecting double PCR primer, detection method and the kit of pig Senecan paddy virus Yu 3 type of annulus virus - Google Patents
For detecting double PCR primer, detection method and the kit of pig Senecan paddy virus Yu 3 type of annulus virus Download PDFInfo
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Abstract
The present invention is provided to detect double PCR primer, method and the kit of Senecan paddy virus Yu 3 type of pig annulus virus.The present invention designs double PCR primer according to the highly conserved specific sequence of pig Senecan paddy virus and 3 type of pig annulus virus, and by primer screening and reaction system optimization, the double PCR detection kit of single tube synchronized detection Senecan paddy virus and 3 type of pig annulus virus is established.Kit has the characteristics of fast and convenient, high specificity, high sensitivity, good reliability, batch sample analysis can be carried out simultaneously, primary first-order equation just can antidiastole sample whether there is Senecan paddy virus and 3 type of pig annulus virus infection conditions, strong technical support is provided for the monitoring and prevention and control of pig Senecan paddy virus and 3 type virus epidemic situation of pig annulus, there is good application prospect.
Description
Technical field
The invention belongs to animal epidemic detection technique fields, and in particular to for detecting pig Senecan paddy virus and annulus 3
Double PCR primer, method and the kit of type.
Background technique
Senecan paddy virus A (Seneca virus A, SVA) is Picornaviridae (Picornaviridae)
The Typical Representative of Senecavirus Tobamovirus.Hales etc. after analyzing SVV-001 genetic sequence by inciting somebody to action
Senecavirus is classified as the new Tobamovirus (Hales et al., 2008) of tiny RNA Viraceae, is the single-stranded positive without cyst membrane
RNA.SVV is initially considered as the pollutant in cell culture, thus it is speculated that its pancreatin or fetal calf serum (Reddy for deriving from pig
et al.,2007).By 2007, there is bubble from the hog snout mirror that Canada transports Minn. in a batch, and hoof is coronal
Band such as festers at the similar blister disease symptoms, excludes aftosa, vesicular stomatitis disease and pig blisters through detection, examines eventually by PCR
Survey is determined as SVA positive (Pasma T et al., 2008).Also there is the report of swinery infection SVA in November, 2014, Brazil.
In March, 2015, the pig on China, pig farm, Guangdong walk lamely, and asoscope bubble, inspection blister fluid, bubble skin, foot skin sample occur
Product, testing result show that Schweineseuche, pig blisters cause of disease are negative.Then data is searched, determines that morbidity swinery infects SVA.
Then in October, 2015, November and in January, 2016,2 months, on other pig farms, discovery swinery infects SVA case successively.
Pig circular ring virus (porcine circovirus, PCV) is the small and nothing of one kind of circovirus section Circovirus
Cyst membrane, icosahedral symmetry, ring-type, covalence closed Single-stranded DNA virus.Virion diameter average out to 17nm, with rolling ring side
Formula is replicated, and is presently found the smallest no cyst membrane DNA virus.Brain, Allan etc. think can be according to pig circular ring virus
Pathogenic, antigenic and nucleotide sequence pig circular ring virus can be divided into two kinds of genotype, i.e. 1 type of pig circular ring virus (PCV1)
With porcine circovirus 2 type (PCV2).PCV1 was used as a kind of pollutants identification in 1974 in PK cell culture for the first time, right
Pig is not pathogenic.PCV2 was reported for the first time in 1998, can cause the pig circular ring virus of pig related in the clinical setting
Disease (Porcine circovirus associated diseases, PCVAD) mainly causes piglet multisystem failure comprehensive
Levy (PMWS), pigskin inflammation nephrotic syndrome (PDNS), porcine respiratory syndrome (PRDC), sow breeding difficulty disease, Hypertrophic intestines
Inflammation, gangrenosum acne interstitial pneumonia, piglet congenital tremors etc. show as breathing, uropoiesis, enteron aisle, lymph, angiocarpy, nerve, numerous
The dysfunction for growing system and skin causes weight huge economic loss to global pig-breeding.Recently, a kind of novel
3 type virus of pig circular ring virus be reported for the first time in the U.S., full length viral genome 2.0kb, gene encode two it is main
Albumen: Cap albumen and Rep albumen, the two are in opposite direction in nucleic acid chains, can cause pigskin inflammation nephrotic syndrome, numerous
Grow the symptoms such as obstacle, heart and multisystem inflammation.
Since SVA and PCV3 are neopathies, the common detection method for detecting SVA and PCV3 at present mainly has virus point
From, Serologic detection and PCR detection etc..But virus purification, Serology test are time-consuming and laborious, sensibility is low and it is easy go out
Existing false positive.And the method for PCR detection not can be carried out a variety of Virus Types mainly based on the single detection of a certain Virus Type
Joint PCR detection, and detection sensitivity with it is specific also irregular.There is bubble symptom in June, 2108 acquisition
There is the presence for detecting SVA and PCV3 in pathological material of disease, inhibits since PCV3 infection easily leads to Swinery immunity, prevent and treat swine disease prevention and control
It is more complicated with it is difficult, to influence the productivity effect on pig farm.Therefore PCV3 is detected while detecting SVA, helps to inspire
The new approaches of control and prevention of disease, while being conducive to the prevention and control of swine disease.
Summary of the invention
In view of this, the present invention is provided to detect the double PCR primer of 3 type of pig Senecan paddy virus and annulus, method
And kit, the kit is fast and convenient, high specificity, high sensitivity, good reliability, and testing cost is cheap, to detector
Device and the level requirement of operator be not high, is provided simultaneously with compared with high detection sensitivity and specificity, can accurately identify and examine
The micro latent infection of disconnected SVA and PCV3 out is retrieved economic losses to implement correct therapeutic scheme as early as possible.
For this purpose, first aspect present invention offer is drawn for detecting the double PCR of pig Senecan paddy virus and annulus 3 type virus
Object, the primer draw according to the highly conserved specific sequence design double PCR of pig Senecan paddy virus and annulus 3 type virus
Object, wherein the detection primer of pig Senecan paddy virus, the forward primer and SEQ of the nucleotide sequence as shown in SEQ ID NO:1
The reverse primer of nucleotide sequence shown in ID NO:2 forms, and the detection primer of 3 type of pig annulus virus is detected, by SEQ ID
The reverse primer of nucleotide sequence shown in the forward primer and SEQ ID NO:8 of nucleotide sequence shown in NO:7 forms.
Second aspect of the present invention provides the double PCR detection side for detecting pig Senecan paddy virus and annulus 3 type virus
Method, comprising the following steps:
1) nucleic acid is extracted from measuring samples；
It 2) is amplification template with nucleic acid obtained by step 1), using double PCR primer as described in claim 1, same
Double PCR amplification is carried out in reaction system, obtains amplified production；
3) amplified production obtains testing result through agarose gel electrophoresis.
In an embodiment of the present invention, described for detecting pig Senecan paddy virus and the double PCR of annulus 3 type virus is anti-
Answering system total volume is 50 μ L, comprising:
1) 10 μM of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, nucleotide sequence shown in SEQ ID NO:8
Each 0.5-2.0 μ L；
2)10×PCR Buffer 5.0μL；
3)dNTP Mixture 4.0μL；
4) 1.0 μ L of rTaq archaeal dna polymerase；
5)RNase free ddH20 28.0-34.0μL；
6) the 4.0 μ L of DNA profiling of sample to be tested.
In an embodiment of the present invention, described for detecting pig Senecan paddy virus and the double PCR of annulus 3 type virus is anti-
It answers in system, the additional amount of nucleotide sequence shown in 10 μM of SEQ ID NO:1, SEQ ID NO:2 is 0.7 μ L, i.e., it is reacted
Concentration is 0.14 μM；The additional amount of nucleotide sequence shown in 10 μM of SEQ ID NO:7, SEQ ID NO:8 be 1.4 μ L, i.e., its
Reaction density is 0.28 μM.
In an embodiment of the present invention, described for detecting pig Senecan paddy virus and the double PCR of annulus 3 type virus is anti-
Answering condition includes: to enter circulation after 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, 35
After circulation terminates；72 DEG C extend 10min eventually.
In an embodiment of the present invention, the double PCR for being used to detect pig Senecan paddy virus and annulus 3 type virus
Detection method, detection sensitivity is up to 102Copy/μ L.
Third aspect present invention provides a kind of for detecting the dual of pig Senecan paddy virus and circovirus 3 type virus
PCR detection kit, including detection pig Senecan paddy virus and circovirus 3 type virus as described in the first aspect of the invention
Double PCR primer further includes 10 × PCRbuffer, dNTP, MgCl2, Taq enzyme, positive quality control product, negative quality-control product.
In an embodiment of the present invention, the double PCR for being used to detect pig Senecan paddy virus and annulus 3 type virus
Detection kit, detection sensitivity is up to 102Copy/μ L.
In an embodiment of the present invention, pair for being used to detect pig Senecan paddy virus and circovirus 3 type virus
Weight PCR detection kit can specific detection identification pig Senecan paddy virus and circovirus 3 type virus, cannot detect pig
Epidemic diarrhea virus, swine vesicular disease virus, vesicular stomatitis virus, PRRS virus, porcine circovirus 2 type, hog cholera
One of poison is a variety of.
Fourth aspect present invention provides double PCR primer as described in the first aspect of the invention, such as second aspect of the present invention
The dual PCR detection method or double PCR detection kit as described in the third aspect of the present invention are in pig Senecan paddy disease
Application in poison and 3 type viral diagnosis of circovirus, wherein for the purpose of the detection is not treated by medical diagnosis on disease.
The beneficial effects of the present invention are: kit provided by the invention has fast and convenient, high specificity, sensitivity
High, good reliability, and testing cost is cheap, it is not high to the level requirement of detecting instrument and operator, it is provided simultaneously with higher
Detection sensitivity and specificity accurately antidiastole can go out the micro of pig Senecan paddy virus and circovirus 3 type virus
Latent infection, therefore, suitable for pig Senecan paddy virus and circovirus 3 type virus micro subclinical infection period monitoring and
Early warning, conducive to being applied and promoting in breeding enterprise.
Detailed description of the invention
Fig. 1 is the primer screening electrophoretogram of double PCR provided in an embodiment of the present invention detection, and wherein M is DL2000
DNAmarks, swimming lane 1 be negative control, swimming lane 2 be SVA-1-F/R and PCV3-1-F/R, swimming lane 3 be SVA-2-F/R with
PCV3-2-F/R, swimming lane 4 are SVA-2-F/R and PCV3-1-F/R, and swimming lane 5 is SVA-1-F/R and PCV3-2-F/R, and swimming lane 6 is
SVA-1-F/R and PCV3-3-F/R, swimming lane 7 are SVA-2-F/R and PCV3-3-F/R, and swimming lane 8 is SVA-3-F/R and PCV3-1-
F/R, swimming lane 9 are SVA-3-F/R and PCV3-2-F/R, and swimming lane 10 is SVA-3-F/R and PCV3-3-F/R, and wherein swimming lane 2 is
The amplification efficiency of SVA-1-F/R and PCV3-1-F/R is optimal；
Fig. 2 is the single primer concentration optimization of pig Senecan paddy virus SVA-1-F/R provided in an embodiment of the present invention, wherein M
For DL2000 DNAmarks, swimming lane 1 is negative control, swimming lane 2-10 be respectively 0.10 μM of primer reaction density, 0.14 μM,
0.16 μM, 0.18 μM, 0.20 μM, 0.22 μM, 0.24 μM, 0.26 μM, 0.28 μM of reaction system, as a result when pig Senecan paddy disease
The reaction density of malicious primer SVA-1-F/R is 0.14 μM, and the amplification of the single primer PCR of pig Senecan paddy virus SVA-1-F/R is imitated
Fruit is best；
Fig. 3 is that the primer concentration of double PCR provided in an embodiment of the present invention detection optimizes electrophoretogram, wherein M DL2000
DNA marks, 1 is negative control, and swimming lane 2-9 is respectively that SVA-1-F/R reaction density is 0.14 μM and PCV3-1-F/R reaction
Reaction system when concentration is 0.14 μM, 0.18 μM, 0.20 μM, 0.22 μM, 0.24 μM, 0.28 μM, 0.32 μM, 0.36 μM,
When middle swimming lane 7 is that the reaction density of SVA-1-F/R and PCV3-1-F/R is respectively 0.14 μM, 0.28 μM, double PCR detection knot
Fruit is optimal；
Fig. 4 is that the annealing temperature of double PCR provided in an embodiment of the present invention detection optimizes electrophoretogram, wherein M DL2000
DNA marks, 1 is negative control, and 2-8 is different annealing temperature, respectively 50.0 DEG C, 51.9 DEG C, 53.7 DEG C, 56.1 DEG C,
58.0 DEG C, 59.2 DEG C, 60.0 DEG C, when annealing temperature is 56.1 DEG C as the result is shown, band is brighter, clear, and expanding effect is preferable；
Fig. 5 is the electrophoretogram of the specific test of double PCR provided in an embodiment of the present invention detection, and wherein M is DL2000
DNA marks, swimming lane 1 are negative control, and swimming lane 2 is the electricity of 3 type virus hybrid detection of pig Senecan paddy virus and circovirus
As a result, swimming lane 3 is the electrophoresis result of pig Sai Neijia paddy viral diagnosis, swimming lane 4 is the electrophoresis knot of 3 type viral diagnosis of circovirus for swimming
Fruit, 5,6,7,8,9,10 be respectively Porcine epidemic diarrhea virus, swine vesicular disease virus, vesicular stomatitis virus, pig blue-ear disease disease
Poison, porcine circovirus 2 type, swine fever virus；
Fig. 6 is the electrophoretogram of the sensitivity test of double PCR provided in an embodiment of the present invention detection, and wherein M is DL2000
DNA marks, swimming lane 1 are negative control, and swimming lane 2-9 is respectively 107-100Copy/μ L, as the result is shown the detection spirit of double PCR
Sensitivity is 102Copy/μ L；
Fig. 7 is Senecan paddy virus provided in an embodiment of the present invention and the clinical inspection that 3 type virus double PCR of pig annulus detects
Test the electrophoretogram tested.Wherein M be DL2000 DNA marks, swimming lane 1 be negative control, swimming lane 2 be Senecan paddy virus and
The control of 3 type virus double-positive of pig annulus, swimming lane 3 are Senecan paddy virus positive control, and swimming lane 4 is 3 type virus-positive of annulus
Control, swimming lane 5-14 are clinical detection sample to be tested.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.Test method without specific conditions in embodiment, usually according to normal condition.The present invention is implemented
In example unless otherwise noted, agents useful for same and consumptive material are commercial goods.
Embodiment 1
The preparation of 3 type viral nucleic acid template of 1 Senecan paddy virus and pig annulus
1) clinical sample is taken to be used for the extraction of viral nucleic acid after multigelation 3 times in -80 DEG C.Referring to Trizol method extraction side
Method extracts virus total RNA.
2) by above-mentioned steps 1) resulting virus total RNA reverse transcription reagent box preparation cDNA is extracted, -80 DEG C of preservations are standby
With.Reaction system is as follows:
The first step
1 μ L of random primer
RNA 5.75μL
Ice bath 2min after 70 DEG C of water-bath 10min, obtains RNA primer mixture；
Second step
6.75 μ L of RNA primer mixture
5×MLV buffer 2μL
dNTPs 0.5μL
0.25 μ L of RNase inhibitor
0.5 μ L of MLV reverse transcriptase
30 DEG C of water-bath 10min, 42 DEG C of water-bath 1h, ice bath is cooling, and the cDNA synthesized after the completion of reverse transcription is placed in be used on ice
Subsequent experimental is placed in -20 DEG C of preservations.
2 Senecan paddy viruses and the screening of 3 type virus dual-PCR method specific primer of pig annulus and system optimization
1) 3 type virus double PCR design of primers of Senecan paddy virus and pig annulus
According to the full-length genome sequence of the GenBank pig annulus 3 type virus logged in, carried out using application ClustW software
Multiple alignment, in the universal primer that highly conserved position is guarded with 5.0 software design of Primer.Choose Sai Neijia paddy disease
The conservative gene VP1 gene of poison, the specific primer guarded with 5.0 genetic analysis software design height of Primer, all primers
There is the synthesis of Beijing Rui Bo Biotechnology Co., Ltd, particular sequence is shown in Table 1.
Table 1 is directed to the specific primer of Senecan paddy virus and pig annulus 3 type virus:
2) reaction system and reaction condition of Senecan paddy virus and 3 type virus double PCR of pig annulus
The reaction system of 3 type virus double PCR of Senecan paddy virus and pig annulus is shown in Table 2.
The reaction system of 3 type virus double PCR of 2 Senecan paddy virus of table and pig annulus
Reaction condition: enter circulation, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extensions after 94 DEG C of initial denaturation 5min
30s, 35 circulations, 72 DEG C extend 10min eventually, identify after reaction according to through 1.2% agarose gel electrophoresis.
As a result as shown in Figure 1, SVA-1-F/R and PCV3-1-F/R specific band is brighter, clear, therefore, SVA- is screened
1-F/R and PCV3-1-F/R is that Senecan paddy virus and 3 type virus double PCR of pig annulus identify primer.
3) the primer concentration optimization of 3 type virus double PCR of Senecan paddy virus and pig annulus
To the single primer concentration optimization (μm ol/L) of resulting pig Senecan paddy virus SVA-1-F/R is screened, see Table 3 for details:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
| SVA | 0 | 0.10 | 0.14 | 0.16 | 0.18 | 0.20 | 0.22 | 0.24 | 0.26 | 0.28 |
As a result as shown in Fig. 2, when the reaction density of pig Senecan paddy virus primer SVA-1-F/R is 0.14 μM, in pig plug
The expanding effect for blocking the single primer PCR of paddy virus SVA-1-F/R is best.
Senecan paddy virus and 3 type virus double PCR primer SVA-1-F/R of pig annulus and PCV3-1-F/R do primer concentration
Optimization experiment, wherein the reaction density of pig Senecan paddy virus SVA-1-F/R is 0.14 μM, and primer concentration combination is as shown in table 4.
Table 4SVA-1-F/R and PCV3-1-F/R primer reaction density optimization (μm ol/L)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
| SVA | 0 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 | 0.14 |
| PCV3 | 0 | 0.14 | 0.18 | 0.22 | 0.24 | 0.26 | 0.28 | 0.30 | 0.32 |
It is expanded by above-mentioned PCR reaction condition, after reaction according to conventional agarose gel electroresis appraisal.
As a result as shown in figure 3, combination 6 primer concentration combination double PCR effect it is best, i.e., Senecan paddy virus and pig
When 3 type virus primer concentration of annulus is 0.12 μm of ol/L, band is brighter, clear, determines that each primer concentration of double PCR is 0.2
μm ol/L, 0.28 μm of ol/L are optimal.
4) the annealing temperature optimization of 3 type virus double PCR of Senecan paddy virus and pig annulus
According to the reference value that Primer5.0 when design of primers is provided, double PCR annealing temperature is groped, setting 50
7 gradients (50.0 DEG C, 51.9 DEG C, 53.7 DEG C, 56.1 DEG C, 58.0 DEG C, 59.2 DEG C, 60.0 DEG C) in~60 DEG C respectively with
CDNA template carries out PCR amplification.
As a result as shown in figure 4, band is brighter, clear, and therefore, optimum annealing temperature is when annealing temperature is 56.1 DEG C
56.1℃。
Embodiment 2
The performance verification of kit of the present invention
1) specific test
Using the dual-PCR method after optimization to pig Senecan paddy viral (SVA), 3 type of pig circular ring virus viral (PCV3),
Porcine epidemic diarrhea virus (PEDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), PRRS virus
(PRRSV), porcine circovirus 2 type (PCV2), swine fever virus (CSFV) are expanded, and aqua sterilisa are selected to do negative control, inspection
Test the specificity of institute's method for building up.
As a result as shown in figure 5, the target fragment size of pig Senecan paddy viral (SVA) is 772bp；3 type of pig circular ring virus
The target fragment size of viral (PCV3) is 398bp；And Sai Neijia paddy virus and 3 type of pig circular ring virus viral (PCV3) mixing sense
Dye target fragment size has two, respectively 772bp and 398bp；PEDV, SVDV, VSV, PRRSV, PCV2, CSFV virus are
Have no that specific band expands.
2) double PCR sensitivity tests
With the plasmid (10 of mixing 10 times of doubling dilutions of plasmid of PCV3 and SVA7~100) it is used as template, with established pair
Weight PCR method is expanded, and amplification is as shown in fig. 6, show Senecan paddy virus provided by the invention and pig annulus 3 type disease
The detection sensitivity of malicious double PCR can be to 102Copy/μ L.
3) double PCR repetitive test
With the mixing plasmid 10 of SVA and PPV311~100Copy/μ L be template, using established dual-PCR method into
3 repetitions of row are tested, as a result identical, show that this double PCR has higher repeatability.
4) clinical test
It chooses 10 parts of South China hoof coronary band to fester the equal pathological material of disease similar to blister disease symptoms, with established dual
PCR method is expanded, as a result as shown in fig. 7, part pathological material of disease the case where there are mixed infections in 10 parts of pathological material of diseases of display.
In conclusion provided by the present invention for the double PCR for detecting pig Senecan paddy virus and circovirus 3 type virus
Detection kit has higher detection sensitivity and specificity, while detection kit provided by the invention is to equipment and people
Member's skill requirement is lower, suitable for the monitoring to pig Senecan paddy virus and circovirus 3 type virus micro subclinical infection period
And pig Senecan paddy virus and circovirus 3 can be significantly increased conducive to being applied and promoting in breeding enterprise in early warning
Cause of disease recall rate under the micro infection conditions of type virus accurately antidiastole can go out the cause of disease that pig infects, be conducive to suit the medicine to the illness
Prescribe medicine implements correct therapeutic scheme, has a good application prospect.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although ginseng
The present invention is explained in detail according to preferred embodiment, those skilled in the art should understand that, it can be to of the invention
Technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
Sequence table
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Claims (8)
1. the double PCR primer for detecting pig Senecan paddy virus and annulus 3 type virus, which is characterized in that the primer root
Double PCR primer is designed according to the highly conserved specific sequence of pig Senecan paddy virus and annulus 3 type virus, wherein pig plug
The detection primer of interior card paddy virus, shown in the forward primer and SEQ ID NO:2 of the nucleotide sequence as shown in SEQ ID NO:1
Nucleotide sequence reverse primer composition, detect 3 type of pig annulus virus detection primer, the core as shown in SEQ ID NO:7
The reverse primer of nucleotide sequence shown in the forward primer and SEQ ID NO:8 of nucleotide sequence forms.
2. for detecting the dual PCR detection method of pig Senecan paddy virus and annulus 3 type virus, which is characterized in that including with
Lower step:
1) nucleic acid is extracted from measuring samples；
It 2) is amplification template with nucleic acid obtained by step 1), using double PCR primer as described in claim 1, in same reaction
Double PCR amplification is carried out in system, obtains amplified production；
3) amplified production obtains testing result through agarose gel electrophoresis.
3. the dual PCR detection method of detection pig Senecan paddy virus and annulus 3 type virus as claimed in claim 2, special
Sign is that the reaction system total volume of the double PCR is 50 μ L, comprising:
1) 10 μM of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, nucleotide sequence is each shown in SEQ ID NO:8
0.5-2.0μL；
2)10×PCR Buffer 5.0μL；
3)dNTP Mixture 4.0μL；
4) 1.0 μ L of rTaq archaeal dna polymerase；
5)RNase free ddH20 28.0-34.0μL；
6) the 4.0 μ L of DNA profiling of sample to be tested.
4. it is as claimed in claim 2 for detecting the dual PCR detection method of pig Senecan paddy virus and annulus 3 type virus,
It is characterized in that, described for detecting the reaction condition of the dual PCR detection method of pig Senecan paddy virus and annulus 3 type virus
It include: to enter circulation after 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulations knots
Shu Hou；72 DEG C extend 10min eventually.
5. the double PCR detection kit for detecting pig Senecan paddy virus and circovirus 3 type virus, which is characterized in that
Including for detecting the double PCR primer of pig Senecan paddy virus and circovirus 3 type virus, going back as described in claim 1
Including 10 × PCR buffer, dNTP Mixture, MgCl2, Taq enzyme, positive quality control product, negative quality-control product.
6. the double PCR detection examination as claimed in claim 5 for detecting pig Senecan paddy virus and circovirus 3 type virus
Agent box, which is characterized in that the double PCR detection kit is minimum to pig Senecan paddy virus and circovirus 3 type virus
Detected level can be down to 102Copy/μ L.
7. the double PCR detection examination as claimed in claim 5 for detecting pig Senecan paddy virus and circovirus 3 type virus
Agent box, which is characterized in that the Dual-PCR Kit can specific detection identification pig Senecan paddy virus and 3 type of circovirus
Virus cannot detect Porcine epidemic diarrhea virus, swine vesicular disease virus, vesicular stomatitis virus, PRRS virus, pig circle
One of 2 type of circovirus virus, swine fever virus are a variety of.
8. the double PCR primer as described in claim 1 for detecting pig Senecan paddy virus and circovirus 3 type virus,
The as claimed in claim 2 dual PCR detection method for detecting pig Senecan paddy virus and circovirus 3 type virus, such as
For detecting the double PCR detection kit of pig Senecan paddy virus and circovirus 3 type virus in pig described in claim 5
Application in 3 type viral diagnosis of Senecan paddy virus and circovirus, wherein the detection is not using medical diagnosis on disease treatment as mesh
's.
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