CN106929607A - A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit - Google Patents
A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit Download PDFInfo
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Abstract
The invention discloses a kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit.The primer pair is made up of sense primer and anti-sense primer, and sequence is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.Kit includes the primer pair, can also include other detection reagents.The method of the detection pig circular ring virus 3 type virus, be the genomic DNA with testing sample as template, carry out real-time fluorescence quantitative PCR reaction using primer pair or kit, result interpretation is carried out according to melting curve and amplification curve Cq values.Accuracy of the present invention is high, specificity is good, reproducible, easy to operate, can accurately, fast and efficiently carry out identification PCV3, be conducive to popularization and application in clinical practice.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of primer pair, method for detecting the type of pig circular ring virus 3 virus
And kit.
Background technology
Pig circular ring virus (Procine circovirus, PCV) are sub-thread cyclic DNA virus, and full-length genome 1.7kb is left
The right side, belongs to PCV-II section, is one of minimum animal DNA virus.Two kinds of pig circular ring virus are had determined that at present, i.e.,
The type of pig circular ring virus 1 (PCV1) and porcine circovirus 2 type (PCV2).PCV1 does not have pathogenic to pig, but PCV2 can be in clinic
Under the conditions of can infect pig and cause disease.Research finds that pig circular ring virus mainly cause piglet multisystem exhaustion syndrome, pig
It is dermatitis nephrotic syndrome, respiratory diseases in pigs mixing disease, pig breeding dysfunction disease, granulomatous enteritis, acute pulmonary edema, Hypertrophic
Downright bad pneumonia, piglet congenital tremors etc..Recently, a kind of new type of pig circular ring virus 3 is viral reports first in the U.S., the disease
Virus gene group total length 2.0kb, two major proteins of its gene code：Cap and Rep, both are in rightabout in nucleic acid chains,
It can cause the symptoms such as pigskin inflammation nephrotic syndrome, breeding difficulty, heart and multisystem inflammation.
Fluorescence quantitative PCR detection technique has merged that the nucleic acid efficient amplification of round pcr, primer specificity be high, spectral technique
Sensitiveness is high and the quantitative advantage of high precision, and the change of fluorescence signal is obtaining quantitative result during direct detection PCR.SYBR
Green I be it is a kind of can be with a kind of dyestuff of double-stranded DNA specific bond, when template is amplified in system, SYBR can be effective
Be attached in the double-strand of new synthesis, with the carrying out of PCR, with reference to SYBR dyestuffs it is more and more, the fluorescence detected by instrument
Signal is more and more stronger, so as to reach quantitative purpose.The appearance of fluorescent quantitative PCR technique, greatly simplify the mistake of quantitative determination
Journey, as a result judges more true and reliable, substantially increases operating efficiency.
The content of the invention
Primary and foremost purpose of the invention is the shortcoming and deficiency for overcoming prior art, there is provided one kind detection pig circular ring virus 3
The primer pair of type virus.
A further object of the present invention is the reagent for providing the primer pair comprising described detection pig circular ring virus 3 type virus
Box.
Another object of the present invention is to provide a kind of method for detecting the type of pig circular ring virus 3 virus.
The present invention selects specific nucleotide sequence conduct according to reference to all PCV3Rep gene orders in comparison GenBank
Amplification region, devises a pair SYBR Green I fluorescent quantitation RT-PCR primers for detecting the type of pig circular ring virus 3.Set up
Special, sensitive, stable, reliable PCV3SYBR GreenRT-qPCR detecting systems, operation is simple and reliable, is conducive to disease
Timely diagnoses and treatment.
The purpose of the present invention is achieved through the following technical solutions：
A kind of primer pair for detecting the type of pig circular ring virus 3 virus, is made up of, described upstream sense primer and anti-sense primer
The nucleotide sequence of primer such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of described anti-sense primer:2 institutes
Show.
A kind of kit for detecting pig circular ring virus 3 types virus, including described detection pig circular ring virus 3 type is viral draws
Thing pair.
Described kit is also including the one kind in fluorescent dye, positive criteria product, negative control and PCR reaction solutions or extremely
It is few two kinds.
Described fluorescent dye is preferably SYBR Green I；The more preferably RealStar of Beijing Kang Run companies
Green Fast Mixture(2×)。
Described PCR reaction solutions, including archaeal dna polymerase, dNTPs, Mg2+With stabilizer etc..
Described negative control is preferably ddH2O。
The preparation method of described positive criteria product is preferably：DNA is extracted from PCV3 positive pathological material of diseases, by described inspection
The primer pair of survey pig circular ring virus 3 type virus enters performing PCR and obtains amplified production, and the amplified production that will be obtained is connected structure with carrier
Recombinant vector is obtained, described recombinant vector is described positive criteria product.
Described carrier is preferably pMD19-T.
A kind of method for detecting the type of pig circular ring virus 3 virus, comprises the following steps：
(1) DNA is extracted from testing sample；
(2) DNA for being obtained with step (1) as DNA profiling, with the primer of described detection pig circular ring virus 3 type virus
Pair or kit carry out quantitative fluorescent PCR reaction；
(3) after reaction terminates, result interpretation is carried out according to melting curve and amplification curve Cq values：Melting curve is single
Peak, melting temperature is between 80.5~82.5 DEG C, and amplification curve Cq values are judged to the positive between 10.17~34.81；Melt
The non-simple spike of curve, or melting curve is for simple spike but melting temperature is not in the range of 80.5~82.5 DEG C, is judged to feminine gender.
The reaction system of described quantitative fluorescent PCR reaction is preferably the reaction system of 20 μ L：Including RealStar
Green Fast Mixture (2 ×) 10 μ L, 0.8 μ L sense primers, 0.8 μ L anti-sense primers, the μ L of DNA profiling 1.0 (treat test sample
Product), ddH2O 7.4μL。
Described sense primer and the concentration of anti-sense primer are preferably 10 μM.
Described sense primer and the proportioning of anti-sense primer are preferably 1:1.
The reaction condition of described quantitative fluorescent PCR reaction is：95 DEG C of predegeneration 2min；95 DEG C of denaturation 15s, 56.4 DEG C are moved back
Fiery 20s, 72 DEG C of extension 30s, 40 circulations；Reaction is first heated to 95 DEG C of reaction 10s after terminating, 65 DEG C are then down to again, starts
The melting curve that 95 DEG C of detection fluorescence signals draw amplified production is incremented to 0.5 DEG C/s.
Application of the described primer pair or kit in the type field of virus detection of pig circular ring virus 3.
The present invention has the following advantages and effect relative to prior art：
The present invention is designed and screened and obtains a kind of type real-time fluorescence quantitative PCR detection primer pair of pig circular ring virus 3, described to draw
The sense primer of thing pair, the sequence of anti-sense primer such as SEQ ID NO：Shown in 1~2；Using the primer can specific amplification go out pig
The part conserved sequence of the type Rep genes of PCV-II 3, real time fluorescent quantitative can be glimmering by double-stranded DNA during real-time monitoring PCR
The combination situation of photoinitiator dye and pcr amplification product, gathers fluorescence data, and PCV3 is differentiated according to melting curve and Cq values；Utilize
Methods described enter performing PCR amplification after can differentiate PCV3, accuracy is high, specificity is good, reproducible, can accurately, quickly, height
Identification PCV3 is carried out to effect, is conducive to popularization and application in clinical practice.
The present invention to multiple PCV3 virus Rep regional genes sequence (NC031753, KX458235, KY354039,
KX966193, KX898030, KX778720, KT869077 and KY354038) carry out comprehensive study, for research institute obtain it is specific
Target amplification region carries out design of primers, and primer specificity preferably, can be with the Strain of effective detection PCV3.
Brief description of the drawings
Fig. 1 is 1% agarose gel electrophoresis figure of the recombinant plasmid pMD19-T-Rep of embodiment 1, wherein, swimming lane M is
DL2000Marker (units：Bp), swimming lane 1 is recombinant plasmid pMD19-T-Rep.
Fig. 2 is the canonical plotting of the positive criteria product SYBR Green I fluorescent quantitations PCR of embodiment 2.
Fig. 3 is the melting curve figure of the positive criteria product SYBR Green I fluorescent quantitations PCR of embodiment 2.
Fig. 4 be embodiment 3 real-time fluorescence quantitative PCR sensitivity experiment in interpretation of result figure；Wherein, 1~8 template is dense
Degree (copies/ μ L) is followed successively by 1.73 × 109、1.73×108、1.73×107、1.73×106、1.73×105、1.73×104、
1.73×103、1.73×102；NC represents negative control, and template is ddH2O。
Fig. 5 is the specific test amplification analysis chart of the real-time fluorescence quantitative PCR of embodiment 4；Wherein, 1 represent
PCV3 positive controls；2~15 successively respectively represent FMDV, SVDV, SVA, PEDV, PKV, PSV, PBoV, PDCoV, PRV,
PRRSV, CSFV, PCV2, PPV and SIV；NC represents negative control, and template is ddH2O。
When the specific of quantitative fluorescent PCR of Fig. 6 embodiments 4 tries melting curve interpretation of result figure；Wherein, PCV3 is the positive
Control melting curve, remaining be respectively FMDV, SVDV, SVA, PEDV, PKV, PSV, PBoV, PDCoV, PRV, PRRSV, CSFV,
PCV2, PPV, SIV and ddH2O。
Fig. 7 is the specific test ddH of the real-time fluorescence quantitative PCR of embodiment 42The melting curve figure of O (NC) and PCV3.
Fig. 8 be the real-time fluorescence quantitative PCR of embodiment 4 specific test in PKV and PCV3 melting curve figure.
Fig. 9 is the melting curve figure of the specific test SVA and PCV3 of the real-time fluorescence quantitative PCR of embodiment 4.
Figure 10 is the melting curve figure of the specific test PCV2 and PCV3 of the real-time fluorescence quantitative PCR of embodiment 4.
Figure 11 is 1% agarose gel electrophoresis figure of Standard PCR detection experiment in comparative example, wherein, swimming lane M is
Marker, the template concentrations (copies/ μ L) of swimming lane 1~8 are followed successively by 1.73 × 109、1.73×108、1.73×107、1.73×
106、1.73×105、1.73×104、1.73×103、1.73×102, swimming lane 9 is negative control, and template is ddH2O。
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
The preparation of the positive criteria product of embodiment 1
First, the extracting of viral STb gene
Carried out by Invitrogen company's T RIZOL LS Reagent RNA extracts kits operation instructions.In 1.5mL
PCV3 positives pathological material of disease (from Guangdong pig farm) that 250 μ L have been dispensed are added in eppendorf pipes, the positive pathological material of disease is by this reality
Test room new discovery and obtained by extracting organizational form.The μ L TRIZOL of supernatant 750, fully mix, and room temperature places 10min；Plus
Enter the chloroform of 200 μ L, be aggressively shaken 15s, be stored at room temperature 5min；4 DEG C, 12000rpm centrifugations 15min；Supernatant is transferred to new
In 1.5mL eppendorf pipes, 500 μ L isoamyl alcohol are added, fully mixed, room temperature places 10min；4 DEG C, 12000rpm centrifugations
10min；Abandoning supernatant, precipitates the ice-cold μ L of 70% ethanol 1000, gently mixes, and washed once, 4 DEG C, 12000rpm
Centrifugation 10min；Abandoning supernatant, air-dries；The tri-distilled water dissolving DNA processed with 20 μ L DEPC, -80 DEG C of preservations.
2nd, design of primers
According to PCV3 in GenBank Rep regional genes sequence (No. Genbank be NC031753, KX458235,
KY354039, KX966193, KX898030, KX778720, KT869077 and KY354038), design PCR primer：Sense primer
P1：5'-TGAAGTTGCGGAGAAGAT-3'(SEQ ID No.1)；Anti-sense primer P2：5'-CCTGGAGGACCAATAAAA-3'
(SEQ ID No.2), Rep Gene Partial conserved sequences are expanded using the primer, and reaction system is as follows：Premix TaqTM(LA
TaqTMVersion 2.0) enzyme (be purchased from Takara companies, article No. RR900Q) 25 μ L, the μ L of sense primer P1 1, anti-sense primer P2
1 μ L, the μ L of template DNA (DNA that step one finally gives) 1, finally use ddH2O supplies reaction system cumulative volume for 50 μ L.
Amplification program is：(1) 94 DEG C of predegeneration 5min；(2) 94 DEG C of denaturation 1min, 50 DEG C of annealing 30s, 72 DEG C of extension 30s,
Totally 30 circulations；(3) 72 DEG C of extension 10min.
PCR primer is reclaimed (with reference to Omega companiesThe operation instructions of Gel Extraction Kit), enter
Row agarose gel electrophoresis, are accredited as correct target fragment, for follow-up clone.
3rd, the clone of genes of interest
(1) recovery product connection pMD19-T carriers
Reference19-T Vector kits (being purchased from Takara companies, article No. 3271) specification, coupled reaction
System is：By the μ L of PCR primer 4, the μ L of pMD19-T vector 1, the μ L of Solution I 5 that are purified after glue reclaim；Finally connect
The cumulative volume for connecing reaction system is 10 μ L.
In Premix TaqTM(LA TaqTMVersion 2.0) the effect amplification of enzyme obtains the PCR primer of purpose fragment
3 ' ends have " A " base, can be directly connected to pMD19-T, carry out TA clones.
After the mixing of above-mentioned reaction system and being slightly centrifuged, 4 DEG C of connections are overnight.
(2) connection product conversion
The μ L of connection product 5 that will be obtained in step (1) are lightly added to the E.coli DH5 α competent cells (purchase of 50 μ L
From Takara companies, article No. 9057) in, ice bath 30min；Then 42 DEG C of heat shock 90s are quickly transferred to cool down 2min in ice bath；
It is added in 200 μ L LB fluid nutrient mediums, 37 DEG C, 160rpm shaking culture 45min make Escherichia coli recover, resistant gene table
Reach；The competent cell even spread Amp of recovery by more than+/ LB solid medium plates, 37 DEG C of overnight incubations.
(3) the PCR identifications and screening of recon
Three single bacterium colonies of picking, are inoculated in respectively in the solid medium plate of the incubated overnight obtained from step (2)
1mL contains 100 μ g/mL Amp+LB fluid nutrient mediums in, 37 DEG C, 220rpm shaking cultures more than 6h (OD600Value 0.3~
0.4)；Take bacterium solution and enter performing PCR identification.Reaction system is as follows：2 × Taq MasterMix (Dye) enzyme (it is ShiJi Co., Ltd purchased from health,
Article No. CW0682S) 10 μ L, sense primer P1 (SEQ ID No.1) (10 μM) 1 μ L, anti-sense primer P2 (SEQ ID No.2) (10 μ
M) 1 μ L, the μ L of template DNA 1, finally use ddH2O supplies reaction system cumulative volume for 20 μ L.
Amplification program is：(1) 94 DEG C of predegeneration 5min；(2) 94 DEG C of denaturation 1min, 50 DEG C of annealing 30s, 72 DEG C of extension 30s,
Totally 32 circulations；(3) 72 DEG C of extension 10min.
Product is detected with 1% agarose gel electrophoresis.
The positive bacterium solution of 20 μ L identifications is taken with 1：100 ratio is inoculated into 2mL Amp+LB fluid nutrient mediums in, 37 DEG C,
220rpm shakes overnight incubation.
(4) extraction of recombinant plasmid and identification
With reference to Omega companiesThe specification of Plasmid Mini Kit I, to incubated overnight in step (3)
The bacterium solution for obtaining carries out plasmid extraction, and plasmid extraction effect is detected with 1% agarose gel electrophoresis.
Recombinant plasmid extract product is detected with 1% agarose gel electrophoresis, qualification result is shown in that Fig. 1, M are DL2000
Marker (is purchased from Takara companies, article No. 3427Q), and unit is bp.Positive recombinant plasmid is named as pMD19-T-Rep.Through
Identify that positive restructuring pMD19-T-Rep plasmids send Beijing AudioCodes biotechnology Co., Ltd to carry out nucleotide sequence survey
It is fixed.
So as to prepare PCV3 positive criteria products.
The quantitative fluorescent PCR reaction condition optimization of embodiment 2 and standard curve making
Optimized rear quantitative fluorescent PCR reaction system is：RealStar Green Fast Mixture (2 ×) is (purchased from north
Jing Kang moistens Science and Technology Ltd., article No. A301) 10 μ L, 0.8 μ L sense primers (SEQ ID No.1) (10 μM), 0.8 μ L downstreams are drawn
Thing (SEQ ID No.2) (10 μM), DNA profiling 1 μ L, ddH2O 7.4μL.Optimum reaction condition is：95 DEG C of predegeneration 2min；95
DEG C denaturation 15s, 56.4 DEG C annealing 20s, 72 DEG C extension 30s, 40 circulation；Reaction is first heated to 95 DEG C of reaction 10s after terminating, so
It is down to 65 DEG C again afterwards, starts to be incremented to the melting curve that 95 DEG C of detection fluorescence signals draw amplified production with 0.5 DEG C/s.
Determine the OD of PCV3 positive criteria products (pMD19-T-Rep) obtained in embodiment 1260Nm and OD280Nm values and its ratio
Value, calculates plasmid concentration, and is converted into copy number, and 8 gradients (10 are serially diluted into again with distilled water 102~109copies/μ
L).8 pMD19-T-Rep of concentration gradient with gained set up 20 μ L reaction systems, after optimization as DNA profiling respectively
Quantitative fluorescent PCR reaction system, expanded on Bio-Rad quantitative real time PCR Instruments, gained standard curve is shown in Fig. 2, SYBR amplifications
Efficiency (E) is 94.3%, and slope of standard curve is -3.465, and y intercept is 42.338.Result shows, standard curve and Cq values
Between linear relationship closely, r2(coefficient correlation) is up to 0.998.
As shown in figure 3, melting curve is simple spike, melting temperature is 82.5 DEG C, without specificity for the melting curve set up
Peak value is produced.
The sensitivity experiment of the real-time fluorescence quantitative PCR of embodiment 3
It is serially diluted again as template (l.73 × 10 using PCV3 positive criteria products distilled water 10 obtained in embodiment 10
~l.73 × 109Copies/ μ L, totally 10 gradients, template concentrations are according to OD260Nm and OD280Nm values and its ratio, calculate plasmid
Concentration, and it is converted into copy number) carry out real time fluorescent quantitative by the quantitative fluorescent PCR reaction system after optimizing in embodiment 2
PCR, to determine their detection lower limit.Result as shown in figure 4,1~8 template concentrations (copies/ μ L) be followed successively by 1.73 ×
109、1.73×108、1.73×107、1.73×106、1.73×105、1.73×104、1.73×103、1.73×102, ddH2O
Be negative control (NC) template, analysis understands, the detection lower limit of real-time fluorescence quantitative PCR of the invention for 1.73 ×
102copies/μL。
The specific test of the real-time fluorescence quantitative PCR of embodiment 4
It is popular with swine foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), pig Sai Neijia paddy virus (SVA), pig
Diarrhea virus (PEDV), pig storehouse cloth virus (PKV), porcine sapelo virus (PSV), pig bocavirus (PBoV), pig triangle are coronal
Viral (PDCoV), PRV (PRV), PRRS virus (PRRSV), CSFV (CSFV), 2 porcine circovirus
The cDNA or DNA of type (PCV2), pig parvoviral (PPV), swine influenza virus (SIV) and PCV3 be template, wherein PBoV, PRV,
The template of PCV2 and PPV is DNA, and remaining is cDNA.Above Strain is animal science institute of Agricultural University Of South China poultry and grinds
Study carefully room and preserve strain.The reaction system and reaction condition of the optimization that Application Example 2 is set up carry out real-time fluorescence quantitative PCR expansion
Increase, as a result such as Fig. 5, Fig. 6, Fig. 7, shown in Fig. 8, Fig. 9 and Figure 10, PCR amplifications only have PCV3 to be positive, and melting curve is single
Peak, and melting temperature is 82.5 DEG C；And other viral cDNA or DNA are without specific amplification, melting curve is non-simple spike, or
Melting curve is for simple spike but melting temperature is far below 82.5 DEG C.Show that the PCV3 SYBR Green I of present invention foundation are real-time
Fluorescent quantitative PCR detection method has good specificity.
The replica test of the real-time fluorescence quantitative PCR of embodiment 5
The positive criteria product plasmid (i.e. pMD19-T-Rep obtained in embodiment 1) of PCV3 is diluted to 1.9 × 10 respectively9、
1.9×108、1.9×107、1.9×106、1.9×105、1.9×104、1.9×103、1.9×102Copies/ μ L totally 8 gradients,
Successively using it as template, the real time fluorescence quantifying PCR method augmentation detection set up with the present invention carries out the weight and between group in group
Renaturation is checked, and each gradient carries out 3 repetitions and tests, 5 repetitions every time.By this hair of pMD19-T-Rep standard items plasmid pairs
Bright set up PCV3 real time fluorescence quantifying PCR methods carry out Repeatability checking.As shown in table 1, group is interior and between group for result
The coefficient of variation (CV) be respectively less than 2.27%, illustrate the present invention foundation the real-time fluorescence quantitative PCRs of PCV3SYBR Green I detection
Method has good repeatability.
The repeatability analysis of the PCV3 real-time fluorescence quantitative PCRs of table 1 amplification PMD19-T-Rep
The composition of the kit of embodiment 6
It is formulated for detecting the kit of the type of pig circular ring virus 3 according to following composition：Sense primer, anti-sense primer, the positive
Standard items (i.e. pMD19-T-Rep obtained in embodiment 1), RealStar Green Fast Mixture (2 ×), ddH2O。
The reaction system of the kit is：RealStar Green Fast Mixture (2 ×) 10 μ L (are purchased from
Genstar, article No. A301), 0.8 μ L sense primers (10 μM), 0.8 μ L anti-sense primers (10 μM), DNA profiling 1.0 μ L, ddH2O
7.4 μ L, reaction cumulative volume is 20 μ L.Reaction condition is：95 DEG C of predegeneration 2min；95 DEG C of denaturation 15s, 56.4 DEG C of anneal 20s, 72
DEG C extend 30s, 40 circulation；Reaction is first heated to 95 DEG C of reaction 10s after terminating, 65 DEG C are then down to again, starts with 0.5 DEG C/s
It is incremented to the melting curve that 95 DEG C of detection fluorescence signals draw amplified production.
Result judgement method：Melting curve is simple spike, and melting temperature is between 80.5~82.5 DEG C, and amplification curve Cq
Value is judged to positive (Fig. 5~Figure 10) between 10.17~34.81.The non-simple spike of melting curve (Fig. 8), or melting curve is
Simple spike but melting temperature is not in the range of 80.5~82.5 DEG C (Fig. 9, Figure 10), is judged to feminine gender.
The detection experiment of comparative example's Standard PCR
With PCV3 positive criteria products dilution obtained in embodiment 3 as template (l.73 × 102~l.73 × 109copies/μ
L, totally 8 gradients) standard PCR amplification is carried out, reaction system is as follows：2 × Taq MasterMix (Dye) enzyme (is century purchased from health
Company, article No. CW0682S) 10 μ L, sense primer P1 (SEQ ID No.1) (10 μM) 1 μ L, anti-sense primer P2 (SEQ ID
No.2) (10 μM) 1 μ L, the μ L of template DNA 1, finally use ddH2O supplies reaction system cumulative volume for 20 μ L.
Amplification program is：(1) 94 DEG C of predegeneration 5min；(2) 94 DEG C of denaturation 1min, 56.4 DEG C of annealing 30s, 72 DEG C of extensions
30s, totally 32 circulations；(3) 72 DEG C of extension 10min.
Product is detected with 1% agarose gel electrophoresis.
Result is as shown in figure 8, the template concentrations (copies/ μ L) of swimming lane 1~8 are followed successively by 1.73 × 109、1.73×108、
1.73×107、1.73×106、1.73×105、1.73×104、1.73×103、1.73×102, ddH2O is negative control (NC)
Template, it follows that the detection lower limit of Standard PCR is 1.73 × 104Copies/ μ L, the result of comparative example 3 shows, this
(detection lower limit is 1.73 × 10 to the sensitivity of the real-time fluorescence quantitative PCR detection PCV3 of invention2Copies/ μ L) compare Standard PCR
Sensitivity 2 orders of magnitude high, i.e., it is sensitive 100 times.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>A kind of primer pair for detecting the type of pig circular ring virus 3 virus, method and kit
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Sense primer P1
<400> 1
tgaagttgcg gagaagat 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Anti-sense primer P2
<400> 2
cctggaggac caataaaa 18
Claims (10)
1. a kind of primer pair for detecting pig circular ring virus 3 types virus, it is characterised in that：It is made up of sense primer and anti-sense primer,
The nucleotide sequence of described sense primer such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ of described anti-sense primer
ID NO:Shown in 2.
2. a kind of kit for detecting the type of pig circular ring virus 3 virus, including the type of detection pig circular ring virus 3 described in claim 1
The primer pair of virus.
3. the kit of detection pig circular ring virus 3 type virus according to claim 2, it is characterised in that：Described reagent
Box also includes the one kind or at least two in fluorescent dye, positive criteria product, negative control and PCR reaction solutions.
4. the kit of detection pig circular ring virus 3 type virus according to claim 3, it is characterised in that：Described fluorescence
Dyestuff is SYBR Green I.
5. the kit of detection pig circular ring virus 3 type virus according to claim 3, it is characterised in that the described positive
Standard items are prepared via a method which to obtain：DNA is extracted from PCV3 positive pathological material of diseases, by the detection pig described in claim 1
The primer pair of the type of PCV-II 3 virus enters performing PCR and obtains amplified production, and the amplified production that will be obtained is connected structure and obtains with carrier
Recombinant vector, described recombinant vector is described positive criteria product.
6. a kind of method for detecting the type of pig circular ring virus 3 virus, comprises the following steps：
(1) DNA is extracted from testing sample；
(2) DNA for being obtained with step (1) as DNA profiling, with the detection pig circular ring virus 3 type virus described in claim 1
Kit described in primer pair or any one of claim 2~5 carries out quantitative fluorescent PCR reaction；
(3) after reaction terminates, result interpretation is carried out according to melting curve and amplification curve Cq values：Melting curve is simple spike, is melted
Solution temperature is between 80.5~82.5 DEG C, and amplification curve Cq values are judged to the positive between 10.17~34.81；Melting curve
Non- simple spike, or melting curve is for simple spike but melting temperature is not in the range of 80.5~82.5 DEG C, is judged to feminine gender.
7. the method for detection pig circular ring virus 3 type virus according to claim 6, it is characterised in that described fluorescence is determined
The reaction system of amount PCR reactions is the reaction system of 20 μ L：Including RealStar Green Fast Mixture10 μ L, 0.8 μ L
Sense primer, 0.8 μ L anti-sense primers, the μ L of DNA profiling 1.0 (i.e. testing sample), ddH2O 7.4μL。
8. the method for detection pig circular ring virus 3 type virus according to claim 6, it is characterised in that described fluorescence is determined
Amount PCR reaction reaction condition be：95 DEG C of predegeneration 2min；95 DEG C of denaturation 15s, 56.4 DEG C of annealing 20s, 72 DEG C extend 30s, 40
Individual circulation；Reaction is first heated to 95 DEG C of reaction 10s after terminating, 65 DEG C are then down to again, and beginning is incremented to 95 DEG C of inspections with 0.5 DEG C/s
Survey the melting curve that fluorescence signal draws amplified production.
9. application of the primer pair described in claim 1 in the type field of virus detection of pig circular ring virus 3.
10. application of the kit described in claim 3 in the type field of virus detection of pig circular ring virus 3.
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