CN107338332A - The type SYBR Green I real-time fluorescence quantitative PCRs detection primers pair of pig circular ring virus 3 and kit - Google Patents
The type SYBR Green I real-time fluorescence quantitative PCRs detection primers pair of pig circular ring virus 3 and kit Download PDFInfo
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Abstract
The invention discloses a kind of type SYBR Green I real-time fluorescence quantitative PCRs detection primers pair of pig circular ring virus 3 and kit, the primer pair includes SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:The reverse primer of nucleotide sequence shown in 2.The primer pair is used for the ORF2 Gene Partial fragments for expanding the type of pig circular ring virus 3, and the target gene clip size amplified is 131bp.The present invention also provides the SYBR Green I real-time fluorescence quantitative PCR detection kits of the type of pig circular ring virus 3 and the application method of the kit.Have the characteristics that high specificity, susceptibility are high, reproducible, quick using detection of the primer pair of the present invention to the type of pig circular ring virus 3, the pathogeny detection available for the type of pig circular ring virus 3.
Description
Technical field
The invention belongs to biological technical field, it is related to the detection method of the type of pig circular ring virus 3, and in particular to pig circular ring virus
The SYBR Green I real-time fluorescence quantitative PCRs detection primers pair and kit of 3 types.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) is known to carry out self in mammalian cell
The minimum virus of duplication, belong to PCV-II section (Circoviridae) Circovirus (Circovirus) member, it is
One kind is without cyst membrane, single stranded circle DNA virus, and main infection Monocytes/Macrophages system cell, wherein porcine alveolar macrophage is it
Main target cell.According to PCV antigenic, pathogenic and nucleotide sequence difference, PCV is divided into PCV-1, PCV-2
With tri- kinds of genotype of PCV-3, wherein PCV-1 no pathogenicities, but be widely present in pig body and pig cell lines；PCV-2, which has, to be caused
Characteristic of disease, a variety of diseases of pig, including pmws, pigskin inflammation and nephrotic syndrome and breeding can be caused
The diseases such as obstacle, and be in break out trend in China's most area；PCV-3 is pathogenic not to be understood also, only in aborted fetus, has lung
Detected in scorching pig.
At present, commonly used Standard PCR detection method detection PCV-3, but the sensitivity of regular-PCR detection is relatively low,
And the drawbacks such as viral level can not be quantified, and clinically pig morbidity with viral level into certain positive correlation.Cause
This, establishes the type SYBR Green I real-time fluorescence quantitative PCRs detection primers pair of pig circular ring virus 3 and kit is very necessary.This
Invention designs primer according to the ORF2 genes of the type of pig circular ring virus 3, available for establish efficiently, accurately and rapidly molecular biology
Diagnostic techniques.
The content of the invention
It is an object of the present invention to provide the type SYBR of pig circular ring virus 3 of a kind of hypersensitivity, specificity and accuracy
Green I real-time fluorescence quantitative PCRs detection primers pair and kit.
The SYBR Green I real-time fluorescences that one of technical scheme is to provide a kind of type of pig circular ring virus 3 are determined
PCR detection primers pair are measured, including：SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:Core shown in 2
The reverse primer of nucleotide sequence.
In an embodiment of the present invention, the annealing temperature of the primer pair is 58 DEG C.
Primer pair provided by the invention is used for the ORF2 Gene Partial fragments for expanding the type of pig circular ring virus 3, the mesh amplified
Genetic fragment size be 131bp.
The SYBR Green I real-time fluorescences that the two of technical scheme are to provide a kind of type of pig circular ring virus 3 are determined
PCR detection kit is measured, it includes：SEQ ID NO:The forward primer of nucleotide sequence shown in 1；With SEQ ID NO:Shown in 2
The reverse primer of nucleotide sequence.
As the technical scheme of optimization, the SEQ ID NO:The concentration of the forward primer of nucleotide sequence shown in 1 is 10 μ
mol/L；The SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 2 is 10 μm of ol/L.
As the technical scheme of optimization, kit provided by the invention also include SYBR Premix Ex Taq premixed liquids,
ddH2O, negative quality-control product and positive quality control product.
Preferably, described negative quality-control product is ddH2O；The positive quality control product is the positive matter of the type of pig circular ring virus 3
Grain.
The three of technical scheme are to provide the application method of the kit, comprise the following steps：To treat test sample
The DNA of product is template, in SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:Nucleotides sequence shown in 2
In the presence of the reverse primer of row, detected by SYBR Green I real-time fluorescence quantitative PCRs, pig annulus is judged according to testing result
The presence situation and content of viral 3 types.
In an embodiment of the present invention, the SEQ ID NO:The concentration of the forward primer of nucleotide sequence shown in 1 is 10 μ
mol/L；The SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 2 is 10 μm of ol/L.
In embodiments of the present invention, the reaction system of the PCR detections is 20 μ L, and the reaction system includes：
(1)10μmol/L SEQ ID NO:The μ L of forward primer 1 of nucleotide sequence shown in 1；With 10 μm of ol/L SEQ ID
NO:The μ L of reverse primer 1 of nucleotide sequence shown in 2；
(2) the μ L of DNA profiling 2 of testing sample；
(3) the μ L of SYBR Premix Ex Taq premixed liquids 10；
(4)ddH2O 6μL。
In embodiments of the present invention, the amplification program of the SYBR Green I real-time fluorescence quantitative PCRs detection is：
(1) 95 DEG C of pre-degeneration 90s；
(2) 94 DEG C of denaturation 5s, 58 DEG C of annealing 30s, 40 circulate；
(3) 95 DEG C of denaturation 15s, 58 DEG C of extension 1min；95 DEG C of denaturation 30s, 58 DEG C of extension 15s.
The beneficial effects of the invention are as follows：The type SYBR Green I real time fluorescent quantitatives of pig circular ring virus 3 provided by the invention
PCR detection primers pair and kit, the primer pair include SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ
ID NO:The reverse primer of nucleotide sequence shown in 2.The primer pair is used for the ORF2 Gene Partials for expanding the type of pig circular ring virus 3
Fragment, the target gene clip size amplified are 131bp.The present invention also provides the SYBR Green I of the type of pig circular ring virus 3
The application method of real-time fluorescence quantitative PCR detection kit and the kit.Using primer pair of the present invention to pig annulus
The detection specificity of viral 3 types is good, and discord pig correlated virus reacts, and the only type of pig circular ring virus 3 is the positive, detection it is sensitive
Degree is high, is 1.0 × 101Copy/μ L.Etiological available for the type of pig circular ring virus 3.
Brief description of the drawings
Fig. 1 is the type fluorescent quantitative PCR standard curve of pig circular ring virus 3；Wherein, 1-6 corresponds to the sample of various concentrations
PCR primer；The concentration of sample is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0×105、1.0×
104、1.0×103Copy/μ L；
Fig. 2 is the type quantitative fluorescent PCR standard curve of pig circular ring virus 3；
Fig. 3 is the type fluorescent quantitative PCR melting curve of pig circular ring virus 3；
Fig. 4 is the type quantitative fluorescent PCR sensitivity tests result of pig circular ring virus 3；Wherein, 1-9 corresponds to the sample of various concentrations
The PCR primer of product；The concentration of sample is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0×105、1.0×
104、1.0×103、1.0×102、1.0×101、1.0×100Copy/μ L；10 be negative control；
Fig. 5 is the type quantitative fluorescent PCR specific test result of pig circular ring virus 3；Wherein, 1-10 corresponds to different virus sample
PCR primer, viral sample be followed successively by from left to right PCV-3, PCV-1, PCV-2, PRV, CSFV, PRRSV, PEDV, PDCoV,
TGEV and PAstV.
Embodiment
It is to preferably further understand the present invention to provide following embodiments, it is not limited to the optimal embodiment party
Formula, most preferred embodiment are not construed as limiting to present disclosure and protection domain, and anyone is under the enlightenment of the present invention or incites somebody to action
Method and production as the present invention and any and present invention that the features of other prior arts are combined and drawn are same or like
Product, it is within the scope of the present invention.
Experiment material source involved in the present invention is as follows, and other unaccounted materials are common commercially available product, can be led to
Market purchase is crossed to obtain：
1st, bacterial strain and plasmid
Escherichia coli Top10 competent cells are purchased from Beijing Tiangeng biochemical technology Co., Ltd.
PCV-1 (type of pig circular ring virus 1), PCV-2 (porcine circovirus 2 type), PRV (PRV), CSFV (swine fevers
Virus), PRRSV (porcine reproductive and respiratory syndrome virus), PEDV (Porcine epidemic diarrhea virus), PDCoV (the coronal diseases of pig fourth type
Poison), TGEV (transmissible gastro-enteritis virus), PAstV (pig astrovirus) separate from each pig farm clinical sample in Jiangxi and obtain
Obtain and preserved by this laboratory.
2nd, reagent
Yeast extract, tryptone, ampicillin are purchased from Beijing Suo Laibao Science and Technology Ltd；
Viral RNA/DNA extraction kit (centrifugation column type), SYBR Premix Ex Taq premixed liquids, DL2000 DNA
Marker, pMD18-T carrier purchase Takara (Dalian) company；
The small extraction reagent kit of plasmid is purchased from Beijing Tiangeng biochemical technology Co., Ltd；
Glue reclaim kit is purchased from OMEGA companies.
3rd, equipment
SorvallST16R high speed freezing centrifuges, Legend Micro 21R high speed freezing centrifuges and Nano
Drop2000 is purchased from Thermo companies of the U.S.；
Applied Biosystems 7500PCR instrument is purchased from Applied biosystems；
Agarose horizontal electrophoresis tank is purchased from the bio tech ltd of Beijing 61；
Gel imaging system is purchased from Shanghai Peiqing Science Co., Ltd.
Embodiment 1：The SYBR Green I real-time fluorescence quantitative PCR detection methods of the type of pig circular ring virus 3 are established
1st, design of primers and synthesis
, should according to the ORF2 conservative regions of PCV-3 (KX966193) the PCV3-US/SD2016 strains logged in GenBank
With Primer Premier 5.0 Software for Design, a pair of specific primers, primer is by Shanghai Sheng Gong biotechnologies service company
Synthesis, the sequence of the primer pair of gained is as shown in table 1 (wherein, " F " represents forward primer, and " R " represents reverse primer)：
Table 1：Primer information
2nd, viral DNA extracts
According to the MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 Viral extractions of TaKaRa companies
Operational manual extraction viral RNA/DNA of kit, operating procedure are as follows：
(1) after mill grinding pathological material of disease, centrifuging and taking supernatant；
(2) supernatant, 200 μ L Buffer VGB, 20 μ L Proteinase Ks and 1 μ L Carrier RNA are mixed after taking 200 μ L centrifugations
It is even, in 56 DEG C of water-bath 10min；
(3) liquid after cracking adds 200 μ L absolute ethyl alcohols and mixed, and is transferred to Spin Columns, adds step by step after centrifugation
Enter Buffer RWA/RWB, centrifuge.
(4) 30 μ L are added without RNAase ddH after centrifuging2O dissolving DNAs/RNA, -80 DEG C save backup.
3rd, PCR is expanded
DNA by previous step extraction is the ORF2 Gene Partial sequences PCR amplifications that template carries out the type of pig circular ring virus 3, is pressed
Following reaction systems and response procedures are expanded：
The reaction system of the pig circular ring virus 3 type ORF2 Gene Partial sequences PCR amplifications is 25 μ L, including：
(1)10μmol/L SEQ ID NO:The μ L of forward primer 1 of nucleotide sequence shown in 1；With 10 μm of ol/L SEQ ID
NO:The μ L of reverse primer 1 of nucleotide sequence shown in 2；
(2) the μ L of DNA profiling 2.5 of testing sample；
(3) the μ L of Premix Taq premixed liquids 12.5；
(4)ddH2O 8μL。
The PCR detection response procedures be：
(1) 94 DEG C of pre-degeneration 10min；
(2) 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 36 circulate；
(3) 72 DEG C re-extend 7min.
The agarose gel electrophoresis of PCR primer 2% observes electrophoresis result.Amplification PCR primer corresponds to stripe size and is about
131bp, it is consistent with the length of the type ORF2 genetic fragments of pig circular ring virus 3.
4th, purpose product glue reclaim
The previous step PCR target gene for expanding to obtain is entered according to the operating instruction of the glue reclaim kit of Omega companies
Row glue reclaim purifies.
5th, target gene is cloned
The purified target gene of previous step is connected with pMD 18-T carriers, 2h or 4 DEG C of connection of 16 DEG C of connections overnight, connects
Junctor system is as follows：
6th, the conversion of connection product
Connection product obtained in the previous step is followed the steps below to the conversion of connection product：
(1) Escherichia coli Top10 competent cells are taken out from -80 DEG C of refrigerators, is immediately placed on ice or frozen water mixes
Thawed in thing.
(2) the Escherichia coli Top10 competent cells for taking 5 μ L connection products and 25 μ L to thaw are placed in 1.5mL centrifuge tubes
It is gently mixed mixing, ice bath 30min.And hold water-bath successfully in advance, temperature is adjusted to 42 DEG C.
(3) 42 DEG C of water-bath heat stress 60s, and centrifuge tube is transferred quickly to place 5min in trash ice.
(4) the LB fluid nutrient mediums of 900 μ L preheatings, 180rpm shaken cultivations about 90min are added.
(5) 8000rpm normal temperature centrifugation 5min, abandons 800 μ L of supernatant, and thalline is resuspended and takes 50 μ L and 150 μ L bacterium solutions difference uniform
It is coated on the LB solid mediums added with ampicillin, 37 DEG C of incubators are inverted culture 12-16h after just putting culture 10min.
7th, the picking of bacterium colony and PCR identifications
White, moistening, smooth single bacterium colony add 300 μ into 1.5mL EP pipes in the LB culture mediums of five coatings of picking
L LB fluid nutrient medium culture 4h, using the bacterium solution as DNA profiling, enter performing PCR detection according to following reaction system and response procedures：
The reaction system of PCR detections is 25 μ L, including：
(1)10μmol/L SEQ ID NO:The μ L of forward primer 1 of nucleotide sequence shown in 1；With 10 μm of ol/L SEQ ID
NO:The μ L of reverse primer 1 of nucleotide sequence shown in 2；
(2) the μ L of bacterium solution 2.5；
(3) the μ L of Premix Taq premixed liquids 12.5；
(4)ddH2O 8μL。
The PCR detection response procedures be：
(1) 94 DEG C of pre-degeneration 10min；
(2) 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 36 circulate；
(3) 72 DEG C of overall elongation 7min.
The agarose gel electrophoresis of gained PCR primer 2% is observed.The bacterium colony that picking 2 is accredited as positive colony carries out next
Step experiment.
8th, the determination and analysis of sequence of PCV-3 ORF2 genes
2 bacterium colonies for being accredited as positive colony that previous step obtains are delivered into Shanghai Sheng Gong biotechnologies service company
It is sequenced, sequencing primer M13, and carries out sequence alignment analysis.Sequencing result is compared through blast and analyzed, of the present invention
PCR method amplification gene order and PCV-3 strains homology more than 98%, illustrate that primer pair provided by the invention expands
The target gene of increasing is PCV-3 types.
9th, the drafting of standard curve
Plasmid is extracted after the bacterium colony for being accredited as positive restructuring bacterium is expanded into culture, the plasmid of extraction is calculated into copy number
(calculation formula of copy number is：Copies/ml=(6.02 × 1023) × (g/ml)/(DNA length × 660)), do 10 times
Gradient dilution, with 1.0 × 108、1.0×107、1.0×106、1.0×105、1.0×104、1.0×103Carried out for standard form
PCR is expanded, and each gradient is done repeats (it is to ensure result accuracy to do the purpose repeated three times) three times, and the type of pig circular ring virus 3 is glimmering
Fluorescent Quantitative PCR amplification standard curve is shown in Fig. 1, and 0.056598 is fluorescence threshold in Fig. 1, and 1-6 corresponds to the PCR of the sample of various concentrations
Product；The concentration of sample is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0×105、1.0×104、1.0
×103Copy/μ L；Interpretation of result is carried out to amplification, standard curve is drawn, sees Fig. 2.
Gradually heating after reaction terminates, often heats once, reads first order fluorescence signal.Using temperature as abscissa, fluorescence signal
Change turn to ordinate, draw the type fluorescent quantitative PCR melting curve of pig circular ring virus 3 and see Fig. 3, Tm values are 84.5 DEG C, molten
Solution curve is unimodal, illustrates that the product specificities of amplification are good.
Embodiment 2：The SYBR Green I real-time fluorescence quantitative PCR optimum determinations of the type of pig circular ring virus 3
Using 48 DEG C, 50 DEG C, 52 DEG C, 54 DEG C of thermograde optimizes to reaction, by optimizing reaction condition, confirms
Following reaction system and amplification program are optimum condition：
Optimum response program：20 μ L reaction system, including：
(1)10μmol/L SEQ ID NO:The μ L of forward primer 1 of nucleotide sequence shown in 1；With 10 μm of ol/L SEQ ID
NO:The μ L of reverse primer 1 of nucleotide sequence shown in 2；
(2) the μ L of testing sample DNA profiling 2；
(3) the μ L of SYBR Premix Ex Taq premixed liquids 10；
(4)ddH2O 6μL。
Optimal amplification program is：
(1) 95 DEG C of pre-degeneration 90s；
(2) 94 DEG C of denaturation 5s, 58 DEG C of annealing 30s, 40 circulate；
(3) 95 DEG C of denaturation 15s, 58 DEG C of extension 1min；95 DEG C of denaturation 30s, 58 DEG C of extension 15s.
Embodiment 3：It is prepared by the kit of the type SYBR Green I real-time fluorescence quantitative PCRs of pig circular ring virus 3 detection
The type kit for detecting nucleic acid of pig circular ring virus 3 provided by the invention, including：SYBR Premix Ex Taq are premixed
Liquid, ddH2O、SEQ ID NO:Forward primer, the SEQ ID NO of nucleotide sequence shown in 1:Nucleotide sequence shown in 2 it is reverse
Primer, negative quality-control product (ddH2O), positive quality control product (standard plasmid of preparation).
Reagent used is mainly purchased from precious bioengineering (Dalian) Co., Ltd (Takara) in this kit preparation process,
Remaining is purchased from TIANGEN Biotech (Beijing) Co., Ltd., Beijing Chemical Plant.The preparation process of the kit is as follows：
1st, prepared by PCR reaction solutions
(1) design of primers and synthesis
The PCV-3 ORF2 gene orders delivered according to GenBank, carry out sequence alignment with the softwares of MEGA 6.0 and find out
Conserved sequence.Pig circular ring virus 3 is detected with the Software for Design of Primer Premier 5.0 according to the type conserved sequence of pig circular ring virus 3
Type PCR primer.Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd, and primer includes：SEQ ID NO:Nucleotides sequence shown in 1
The forward primer of row, and SEQ ID NO:The reverse primer of nucleotide sequence shown in 2.
(2) dissolving of primer
Take synthetic primer (SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:Shown in 2
The reverse primer of nucleotide sequence) dry powder pipe, 12,000rpm centrifugation 1min, ddH is added according to primer dissolving specification2O will be done
Powder dissolves, and it is respectively 10 μM to make two kinds of primer concentrations, and vibration mixes centrifugation, standby；
(3) PCR reaction solutions are prepared：
PCR reaction solutions are prepared in following ratios：SEQ ID NO:The forward primer (10 μm of ol/L) of nucleotide sequence shown in 1
1 μ L, SEQ ID NO:μ L, SYBR the Premix Ex Taq premixed liquids of reverse primer (10 μm of ol/L) 1 of nucleotide sequence shown in 2
10 μ L, ddH2O 6μL。
2nd, prepared by negative quality-control product
The effect of negative quality-control product be for prevent because of pollution and caused by false positive.The present invention uses ddH2O is as cloudy
Property quality-control product, is monitored to whole experiment process.
3rd, prepared by positive quality control product
The effect of positive quality control product is that whether monitoring reagent fails and whether performance declines.The present invention is using PCV-3 types
Positive recombinant plasmid is as positive quality control product.According to molecule clone technology conventional in the art, positive recombinant plasmid
Structure and extracting method are as follows：
First with SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:Nucleotides sequence shown in 2
The reverse primer of row enters performing PCR amplification to the genomic DNA of extraction；Then reclaimed using omega Gel Extraction Kit
PCR primer is as purpose fragment；Using the pMD18-T Cloning Vector kits of the precious biology in Dalian, by above-mentioned purpose piece
In section insertion pMD18-T carriers, construction recombination plasmid, by converting and screening, obtain containing the weight inserted with above-mentioned purpose fragment
The positive colony bacterium of group plasmid；The positive colony bacterium extracts culture bacterium after expanding and cultivating, with Tiangeng plasmid extraction kit
Plasmid in liquid is simultaneously quantified with ultraviolet specrophotometer.
4th, quality control standard：
Negative quality-control product：It is negative；
Positive quality control product：It is positive.
Conditions above should meet that otherwise, this time experiment is considered as invalid, and total Test should re-start simultaneously.
5th, analysis judges：
SYBR Green I real-time fluorescence quantitative PCRs detection fluorescence is strong and weak, have amplification curve for the positive, do not expand song
Line for feminine gender.And the viral nucleic acid content in sample can be calculated according to the ct values of sample and the standard curve of drafting.
Embodiment 4：The type SYBR Green I real-time fluorescence quantitative PCR detection method specific tests of pig circular ring virus 3
Using the type PCR detection method of pig circular ring virus 3 established of the present invention respectively with PCV-3, PCV-1, PCV-2, PRV,
CSFV, PRRSV, PEDV, PDCoV, TGEV, PAstV nucleic acid are that template enters performing PCR amplification, as a result see Fig. 5,1-10 is corresponding in figure
The PCR primer of different virus sample, viral sample be followed successively by from left to right PCV-3, PCV-1, PCV-2, PRV, CSFV, PRRSV,
PEDV, PDCoV, TGEV and PAstV.
As a result illustrate：The SYBR Green I real-time fluorescence quantitative PCRs of the type of pig circular ring virus 3 provided using the application are examined
Survey method is the moon to the testing result of PCV-1, PCV-2, PRV, CSFV, PRRSV, PEDV, PDCoV, TGEV, PAstV virus
Property, only PCV-3 PCR testing results are positive, and show that the specificity of the PCR detection method is good.
Embodiment 5：The type SYBR Green I real-time fluorescence quantitative PCR detection method sensitivity tests of pig circular ring virus 3
Plasmid is extracted after the positive restructuring bacterium obtained in embodiment 1 is expanded into culture, extracting method is：By 200 μ L PCR
It is accredited as the positive and correct bacterium solution of sequencing result to be inoculated in LB fluid nutrient mediums of the 20mL added with ampicillin, cultivates 8h
Afterwards, 12000rpm centrifuges 2min, abandons supernatant；Add 2mL PBS to be resuspended, 12000rpm centrifugation 2min, abandon after supernatant with reference to plasmid
Small extraction reagent kit specification (Beijing Tiangeng) extracts plasmid, is surveyed after extraction by ultramicrospectrophotometer Nanodrop 2000
Surely the concentration of the recombinant plasmid extracted, plasmid concentration is converted into further according to recombinant plasmid molecular mass and Avgadro constant
Unit volume molecule copy number, and by recombinant plasmid with 1.0 × 108Copy/μ L are 10 times of gradient dilutions of progress after original concentration,
Respectively with 1.0 × 108、1.0×107、1.0×106、1.0×105、1.0×104、1.0×103、1.0×102、1.0×101Copy
Shellfish/μ L are that template carries out real-time fluorescence quantitative PCR amplification to examine the type SYBR Green I of pig circular ring virus 3 provided by the invention
The sensitivity of real-time fluorescence quantitative PCR detection method, testing result are shown in Fig. 4, and in figure, 1-9 corresponds to the PCR productions of various concentrations sample
Thing；The concentration of sample is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0×105、1.0×104、1.0×
103、1.0×102、1.0×101、1.0×100Copy/μ L；10：Negative control.
As a result show：The SYBR Green I real-time fluorescence quantitative PCRs detection side of the type of pig circular ring virus 3 provided by the invention
The detection limit of method is 1.0 × 101Copy/μ L, show that established PCR method sensitiveness is good.
Embodiment 6：The type SYBR Green I real-time fluorescence quantitative PCR detection method replica tests of pig circular ring virus 3
Using the SYBR Green I real-time fluorescence quantitative PCR detection methods of the type of pig circular ring virus 3 of the present invention, with
The PCV-3 type positive plasmids DNA of various concentrations is template, amplification three times (interval five days) is carried out, to determine the weight of testing result
Renaturation, it the results are shown in Table 2.
Table 2：The type quantitative fluorescent PCR replica test result of pig circular ring virus 3
As a result illustrate：Examined using the SYBR Green I real-time fluorescence quantitative PCRs of the type of pig circular ring virus 3 of the present invention
Survey method is measured with the PCV-3 type positive plasmids of various concentrations in different time points, batch in and batch between replica test knot
Fruit statistical analysis shows that its coefficient of variation scope is respectively less than 0.5%, shows that method repeatability of the present invention is good.
Embodiment 7：The type SYBR Green I real-time fluorescence quantitative PCR detection methods of pig circular ring virus 3 are to clinical sample
Detection
Utilize the SYBR Green I real-time fluorescence quantitative PCR detection methods pair of the type of pig circular ring virus 3 of the present invention
Pick up from 32, Jiangxi, 256 parts of pig farm sample (lung, lymph node, spleen) to be detected, to verify the practicality of this method.As a result 5
Part detection sample is positive, and the positive rate of detection is 1.95%.
As a result show：SYBR Green I real-time fluorescence quantitative PCR detection methods of the present invention are in clinical practice
With feasibility.
SEQUENCE LISTING
<110>Agricultural University Of Jiangxi
<120>The type SYBR Green I real-time fluorescence quantitative PCRs detection primers pair of pig circular ring virus 3 and kit
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
agaggctttg tcctgggtga g 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
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Claims (10)
1. the SYBR Green I real-time fluorescence quantitative PCR detection primers pair of the type of pig circular ring virus 3, including：SEQ ID NO:1 institute
Show the forward primer of nucleotide sequence, and SEQ ID NO:The reverse primer of nucleotide sequence shown in 2.
2. primer pair according to claim 1, it is characterised in that the annealing temperature of the primer pair is 58 DEG C.
3. primer pair according to claim 1, it is characterised in that the primer pair is used to expand the type of pig circular ring virus 3
ORF2 Gene Partial fragments, the target gene clip size amplified are 131bp.
4. the SYBR Green I real-time fluorescence quantitative PCR detection kits of the type of pig circular ring virus 3, it includes：SEQ ID NO:1
The forward primer of shown nucleotide sequence；With SEQ ID NO:The reverse primer of nucleotide sequence shown in 2.
5. kit according to claim 4, it is characterised in that the SEQ ID NO:Nucleotide sequence shown in 1 is just
It is 10 μm of ol/L to the concentration of primer；The SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 2 is 10 μm of ol/
L。
6. kit according to claim 4, it is characterised in that also including SYBR Premix Ex Taq premixed liquids,
ddH2O, negative quality-control product and positive quality control product；Preferably, described negative quality-control product is ddH2O；The positive quality control product is pig
The positive plasmid of the type of PCV-II 3.
7. the application method of the kit according to claim any one of 4-6, it is characterised in that comprise the following steps：With
The DNA of testing sample is template, in SEQ ID NO:The forward primer of nucleotide sequence shown in 1, and SEQ ID NO:Core shown in 2
In the presence of the reverse primer of nucleotide sequence, detect by SYBR Green I real-time fluorescence quantitative PCRs, judged according to testing result
The presence situation and content of the type of pig circular ring virus 3.
8. application method according to claim 7, it is characterised in that the SEQ ID NO:Nucleotide sequence shown in 1
The concentration of forward primer is 10 μm of ol/L；The SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 2 is 10 μ
mol/L。
9. application method according to claim 7, it is characterised in that the SYBR Green I real-time fluorescence quantitative PCRs
The reaction system of detection is 20 μ L, and the reaction system includes：
(1)10μmol/L SEQ ID NO:The μ L of forward primer 1 of nucleotide sequence shown in 1；With 10 μm of ol/L SEQ ID NO:2
The μ L of reverse primer 1 of shown nucleotide sequence；
(2) the μ L of DNA profiling 2 of testing sample；
(3) the μ L of SYBR Premix Ex Taq premixed liquids 10；
(4)ddH2O 6μL。
10. application method according to claim 7, it is characterised in that the SYBR Green I real-time fluorescence quantitative PCRs
The amplification program of detection is：
(1) 95 DEG C of pre-degeneration 90s；
(2) 94 DEG C of denaturation 5s, 58 DEG C of annealing 30s, 40 circulate；
(3) 95 DEG C of denaturation 15s, 58 DEG C of extension 1min；95 DEG C of denaturation 30s, 58 DEG C of extension 15s.
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| CN107653348A (en) * | 2017-11-30 | 2018-02-02 | 山东新希望六和集团有限公司 | For detecting the primer and probe of the type of pig circular ring virus 3, PCR kit for fluorescence quantitative and method, application |
| CN107828916A (en) * | 2017-11-30 | 2018-03-23 | 山东新希望六和集团有限公司 | For detecting primer, PCR kit and the application of the type of pig circular ring virus 3 |
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| CN116064939A (en) * | 2022-08-25 | 2023-05-05 | 广东方道基因生物科技有限公司 | Multiplex fluorescent quantitative special primer group and kit for simultaneously detecting PCV3, PRV and PPV |
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