CN103305638A - Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus - Google Patents
Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus Download PDFInfo
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Abstract
The invention discloses a dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, a kit and a detection method for type 1 and type 2 porcine circovirus. The dual real-time fluorescence PCR detection primer pair for type 1 and type 2 porcine circovirus is characterized in that the nucleotide sequence of the type 1 specific porcine circovirus is as shown in SEQ ID NO: 9 and SEQ ID NO: 11. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 14. The nucleotide sequence of the type 2 specific porcine circovirus is as shown in SEQ ID NO: 2 and SEQ ID NO: 6. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 7. The kit provided by the invention has the characteristics of rapidness, simpleness and convenience, strong specificity, high sensitivity and good reliability, and can be used for analyzing samples in batches at the same time, and differentiating whether the sample is type 1 porcine circovirus infection or type 2 porcine circovirus infection or not by reaction at one time, or common infection of type 1 and type 2 porcine circovirus infection, thereby, providing a powerful technical support for monitoring and preventing porcine circovirus epidemic situation. The kit has good application prospect.
Description
Technical field
The invention belongs to biological technical field, relate to dual real-time fluorescence PCR and detect, be specifically related to the porcine circovirus types 1 and 2 dual real-time fluorescence PCR and detect primer, probe, test kit and detection method.
Background technology
Pig circular ring virus (porcine circovirus, PCV) belongs to PCV-II section, Circovirus.This virus is a kind of little and symmetrical, covalence closed without cyst membrane, icosahedron, ring-type, Single-stranded DNA virus, and virus particle diameter average out to 17nm copies to roll the ring mode, is the animal virus of the minimum found at present.Brain, Allan etc. think and can PCV can be divided into PCV1 and two kinds of genotype of PCV2 according to pathogenic, antigenicity and the nucleotide sequence of PCV.It is generally acknowledged at present the PCV1 no pathogenicity, comprise that PCV-PK15 and other are popular in swinery but the strain isolated of no pathogenicity experimental results show that it extensively is present in pig primary cell line and the swinery; PCV2 has pathogenic, with the pandemic pmws in countries in the world (Post Weaning Multisystemic Wasting Syndrome in recent years, PMWS) closely related, this disease causes quite serious financial loss for global pig industry mainly take progressive emaciation and multisystem pathology damage as feature.PCV1 and PCV2 nucleotide homology are 68%.Between each PCV2 strain of GenBank login all there be more than 95% sequence homology.Therefore PCV1 and PCV2 have certain sibship, but larger variation has also occured.The nucleotide homology rate of PCV1 and PCV2ORF2 gene is lower, is 65%～67%.Before In the view of most scholars, although PCV1 extensively be present in do not have in the pig body pathogenic, thereby to the research of PCV also always take PCV2 as main.Yet the PCV1 that Krakowka etc. go down to posterity with cell in vitro, PCV2 and PPV are independent and the newborn piglet of simultaneous inoculation 1 age in days, clinical symptom does not all appear in the piglet of all single virus infectiones as a result, but wherein the PPV viremia in 2 weeks appears reaching in the piglet of inoculation PCV1 and a PPV, and the colon loop is with granulomatous poradenolymphitis; Rosell etc. carry out immunofluorescence experiment to the piglet that infects PCV1 and find have PCV1 antigen to exist at Lymphoid tissue, lung and enteron aisle, therefore, can not get rid of PCV1 to immune damaging action.The infection rate of PCV1 also is very high in the simultaneously production practice, all might have PCV1 in pig body and the pig source sexual cell.
The present detection method commonly used that detects PCV mainly contains virus separation, serology detection and PCR detection etc.But these ordinary methods waste time and energy, susceptibility is low and false positive easily occurs.At present in the PCV detection method, the real-time fluorescence PCR detection method mainly for PCV2 of foundation, or PCV1 real-time fluorescence PCR detection method, detection PCV2 or PCV1 that can only be single can not carry out dual real-time fluorescence PCR to PCV2 and PCV1 and detect.
Therefore, set up that a kind of fast and convenient, high specificity, susceptibility are high, the PCV1 of good reliability and detection method that PCV2 differentiates be significant.
Summary of the invention
According to demand and the deficiency in above-mentioned field, the invention provides the porcine circovirus types 1 and 2 dual real-time fluorescence PCR and detect primer, probe, test kit and detection method.Test kit of the present invention only needs one-time detection can judge just whether sample contains PCV1 or contain PCV2, perhaps both infect jointly, sensitivity, and convenient, high specificity, susceptibility are high, good reliability, can carry out simultaneously large batch of sample analysis, good application prospect is arranged.
The present invention is achieved through the following technical solutions:
The nucleotide sequence that this paper is listed all has 5 ' end to write to 3 ' extreme direction.
One aspect of the invention relates to two kinds of primer pairs (comprising four primers), wherein pig circular ring virus 1 type primer pair comprises the nucleotide sequence shown in SEQ ID NO:9 and SEQ ID NO:11, and the porcine circovirus 2 type primer pair comprises the nucleotide sequence shown in SEQ ID NO:2 and the SEQ ID NO:6.Another aspect of the present invention relates to two kinds of probes, and pig circular ring virus 1 type probe comprises the nucleotide sequence shown in the SEQ ID NO:14, and the porcine circovirus 2 type probe comprises the nucleotide sequence shown in the SEQ ID NO:7.
The invention still further relates to a kind of Oligonucleolide primers for detecting simultaneously PCV1 and PCV2 to the composition of probe, comprise above-mentioned primer pair and probe.
In addition, the present invention also provides the test kit that comprises above-mentioned composition.
Two kinds of probes of mentioned reagent box use respectively the fluorescein-labelled of different sense channels, and described fluorescein is selected from a kind of among FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or the CY5.
Further, the invention still further relates to the purposes that test kit detects PCV1 and PCV2 at the same time, probe wherein is fluorescein-labelled with any one, and two kinds of probe marks must be the fluorescein that uses different sense channels, all can be selected from the FAM(Fluoresceincarboxylic acid), VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5.
A kind of dual real-time fluorescence PCR method of utilizing above-mentioned two kinds of primer pairs and two kinds of probes to detect simultaneously PCV1 and PCV2 comprises:
The Auele Specific Primer that can differentiate PCV1 and PCV2 and probe sequence for PCV ORF2 gene design.With the probe of fluorescein-labeled PCV1, and the probe that passes through the another kind of fluorescein-labeled PCV2 of different sense channels detections; Probe wherein is fluorescein-labelled with any one, and two kinds of probe marks must be the fluorescein that detects by different sense channels, all can be selected from FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5.Will be for detection of primer and the probe sequence of PCV1 or PCV2, or be used for differentiating and primer and the probe sequence of PCV1 and PCV2 place same reaction tubes, use the fluorescent PCR amplification instrument to carry out real-time fluorescence PCR and react.
Distinguishing feature of the present invention is: fully use the efficient amplification of round pcr, the good specificity of Nucleotide hybridization technique and the quick susceptibility of detection technique of fluorescence, same sample is carried out the discriminating detection that the one-time detection operation can be finished PCV1 and PCV2, can determine that sample is that PCV1 infects, or the PCV2 infection, or both infect jointly.Have that fast and convenient, high specificity, susceptibility are high, good reliability, reduction testing cost, the advantage such as detection efficiency that improves.
Multiple experiment is namely carried out quantitative experiment by a plurality of targets that increase in a reaction tubes, in a reaction tubes, the amplification of any one target can both affect the amplification of other targets, so carry out careful experimental design and the optimization of reaction conditions, rather than in same reaction tubes, all primers and template simply mixed.Multiple fluorescence PCR is tested all targets that ubiquitous problem is concentration notable difference in the amplified sample, when in same reaction tubes, increasing, when being higher than another kind of gene if there is a kind of gene amplification efficient, wherein a kind of amplification of gene may be suppressed, and finally causes the amplification efficiency of two kinds of genes in the sample inconsistent.The present invention is by the design of primer and probe, and the optimization of reaction conditions makes the amplification efficiency of two kinds of genes in the sample consistent, and the sensitivity of reacting with each substance is consistent.
Distinguishing feature of the present invention is: fully use the efficient amplification of round pcr, the good specificity of Nucleotide hybridization technique and the quick susceptibility of detection technique of fluorescence, same sample is carried out the discriminating detection that the one-time detection operation can be finished PCV1 and PCV2, can determine that sample is that PCV1 infects, or the PCV2 infection, or both infect jointly.Have that fast and convenient, high specificity, susceptibility are high, good reliability, reduction testing cost, the advantage such as detection efficiency that improves.
Description of drawings
Fig. 1 is PCV1 specificity result schematic diagram
Fig. 2 is PCV2 specificity result schematic diagram
Embodiment:
It is in order further to understand better the present invention that following embodiment is provided; be not limited to described preferred forms; content of the present invention and protection domain are not construed as limiting; anyone makes up with the feature of other prior aries under enlightenment of the present invention or with the present invention and the identical or akin method of any and the present invention and the product that draw, all drops within protection scope of the present invention.
Fluorescein is introduced:
Corresponding 1 passage of general instrument FAM, VIC, HEX, corresponding 2 passages of JOE, corresponding 3 passages of NED, TAMRA, corresponding 4 passages of CY3, ROX, corresponding 5 passages of CY5.
FAM: Fluoresceincarboxylic acid, Carboxyfluorescein, the a kind of of fluorescein derivative, extensively be present in the fluorescent labeling reagent box, also be applicable to the 488nm spectral line of Argon-ion Laser, Abs/Em=492/518nm(pH=9.0), have the universal feature of fluorescein derivative, stable in water.
VIC: green fluorescent protein, Greenfluorescent protein, GFP is a kind of luminescent protein that comes from marine organisms multitube Medusa (Aequoria Victoria).
HEX: the chlordene fluorescein, Hexachloro fluorescein is a kind of of fluorescein derivative.Be applicable to Argon-ion Laser excitation light source, Abs/Em=535/556nm.
JOE: carboxyl-4', 5'-two chloro-2', 7'-dimethoxy fluorescein, Carboxy-4', 5'-dichloro-2', the JOE fluorescence quantum yield is high, a little less than the pH susceptibility, is suitable for labelled protein.
TAMRA: full name carboxyl tetramethyl-rhodamine, namely Carboxytetramethylrhodamine is the rhodamine fluorescein derivative, TAMRA is that a few can be used for labelled protein.
ROX:5-and 6-carboxyl-X-rhodamine, correction dye.
Cy3 or Cy5, Modification is new fluorescence molecule, has preferably light stability, highly water-soluble and high fluorescence efficiency.Their excitation spectrum and Emission Spectrum Peals are respectively 548/562nm and 646/664nm.The molecular structure of Cy3 and Cy5 and molecular weight are all closely similar, but spectrum between the two is very open, and therefore, Cy3 and Cy5 often are used in a lot of double-colored experiments, as are widely used in gene chip and protein chip field.
NED:2 '-chloro-5 '-fluoro-7 ', 8 '-benzene-Isosorbide-5-Nitrae-two chloro-6-Fluoresceincarboxylic acid.
Corresponding sense channel was the present invention's corresponding sense channel of used FTC-3000 instrument in experimentation when above fluorescein detected, those skilled in the art are in repeating process of the present invention, the corresponding passage of the different possible fluoresceins of instrument is different, two or more fluoresceins corresponding sense channel when using the first instrument is different simultaneously, and corresponding sense channel might be identical when using the second instrument.Aim of the present invention is, no matter which kind of instrument to come repetition the present invention with, as long as when using the same instrument, the fluorescein that is used for the probe of mark porcine circovirus types 1 and 2 just can be realized the present invention at different sense channels.
The source of experiment material of the present invention:
Biomaterial:
The source of the used biological reagent of the present invention and specification:
Pig circular ring virus 1 type (PCV1), porcine circovirus 2 type (PCV2), Pestivirus suis (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese B encephalitis virus (JEV), PRV (Pseudorabies virus) (PRV) and pig parvoviral (PPV) are so kind as to give by China Animal Disease Control And Prevention Center.
Applicant's statement, above biomaterial all has preservation in the applicant laboratory, can provide to the public in 20 years to be used for proof test from the applying date.
The source of reagent of the present invention and specification:
RealMasterMix(Probe) test kit is available from sky root biochemical technology company limited.
PCV1 and PCV2TaqMan probe and primer are synthetic by Jikang Biotechnology Co Ltd, Shanghai.
Biospin tissue gene group DNA extraction test kit is available from Hangzhou BIOER Technology Co., Ltd.
Normal experiment method among the following embodiment, referring to " molecular cloning experiment guide " third edition (Beijing: Science Press, 2002) that Sambrook etc. writes, the use of instrument is with reference to the instrumentation specification sheets.
Embodiment 1. porcine circovirus types 1 and 2 dual real-time fluorescence PCR method Auele Specific Primers and probe screening
(1) primer of pig circular ring virus and probe design
The Auele Specific Primer of porcine circovirus PCV 1 and PCV2 refers to: the oligonucleotide chain about 20 bases of length, the specificity nucleotide fragments of pig circular ring virus ORF2 gene high conservative;
The specific probe of porcine circovirus PCV 1 and PCV2 refers to: length is 13 to 30 oligonucleotide chains between the base, the fluorescence excitation groups such as its 5 ' end flag F AM or HEX, the non-luminous quenching group of 3 ' end mark self.
According to the ORF2 gene specific high conservative zone that obtains, utilized Primer Express3.0 software design for a plurality of real-time fluorescence PCR amplimers and the TaqMan probe of porcine circovirus PCV 1 and PCV2, the concrete sequence of primer pair and probe sees Table 1.
Table 1 is used for primer and the probe of porcine circovirus types 1 and 2 dual real-time fluorescence screening
| Title | Sequence (5 '-3 ') | SEQ?ID?NO: |
| PCV2-F1 | ATCCACTCCCCTGTCACCCT | 1 |
| PCV2-F2 | CCCTGTCACCCTGGGTGAT | 2 |
| PCV2-F3 | ATCCCCTGTCACCCTGGGT | 3 |
| PCV2-R1 | CCGCTCTGTGCCCTTTGA | 4 |
| PCV2-R2 | TCGTGCCCTTTGAATACTACAGAATA | 5 |
| PCV2-R3 | CCTGTGCCCTTTGAATACTACAGA | 6 |
| PCV2-P | FAM-TTAAGGTTGAATTCTGGCCCTGCTCCC-BHQ1 | 7 |
| PCV1-F1 | TGTGGCGGGAGGAGTAGTTAAT | 8 |
| PCV1-F2 | CGGGAGGAGTAGTTAATATAGGGGT | 9 |
| PCV1-F3 | TGTGGCGGGAGGAGTAGTTAATATA | 10 |
| PCV1-R1 | ACTGTTGTTATCTTGGATGCCAACT | 11 |
| PCV1-R2 | CCACTGTTGTTATCTTGGATGCC | 12 |
| PCV1-R3 | AGGGTCCACTGTTGTTATCTTGGA | 13 |
| PCV1-P | HEX-TAACCCCCTCCACCAACTTGGCCTA- |
14 |
(2) reaction system of porcine circovirus types 1 and 2 dual real-time fluorescence PCR method and condition
The reaction system of porcine circovirus types 1 and 2 dual real-time fluorescence PCR method sees Table 2, and reaction conditions sees Table 3.
Table 2 porcine circovirus types 1 and 2 dual real-time fluorescence PCR method reaction system
| Reaction reagent | Final concentration |
| Reaction buffer (contains MgCl 2, dNTP and TaqE) | 1× |
| 10pmoL/ μ L primer pair | 400nM |
| 10pmoL/ μ L probe | 200nM |
| The ROX dyestuff | 1× |
| Template | 2μL |
| Moisturizing extremely | 25μL |
The reaction conditions of table 3 porcine circovirus types 1 and 2 dual real-time fluorescence PCR method
(3) result judges
1. interpretation of result condition is set
2. read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control amplification curve, to show that the result is as the criterion.
3. the result describes and identifies
The amplification situation of contrast different primers pair and probe combinations is followed the specificity principle, susceptibility principle and the highest amplification principle, and selected specificity is good, and susceptibility is high, porcine circovirus types 1 and 2 detection usefulness primer pair and the probe best of breed of good reliability.(4) shaker test method and result
The optimization screening method of primer and probe is as follows:
Select arbitrarily pair of primers and a probe to make up and (such as primer pair PCV1-F1/PCV1-R1, add probe PCV1-P; Primer pair PCV1-F2/PCV1-R2 adds probe PCV1-P; Primer pair PCV2-F1/PCV2-R1 adds probe PCV2-P; Etc.), can obtain simultaneously minimum Ct value and the highest amplification efficiency as screening criteria, select best primer and probe combinations.
By repeatedly repeat, simultaneous test, selected porcine circovirus types 1 and 2 detects with the best of breed of primer pair with probe, combined sorting as a result figure slightly, specifically sequence sees Table 4
Table 4 porcine circovirus types 1 and 2 detects the best of breed sequence with primer pair and probe
The optimization of embodiment 2. pig circular ring virus 1 type real-time fluorescence PCR detection method
(1) primer and the probe design of pig circular ring virus 1 type real time fluorescent PCR method
The primer of pig circular ring virus 1 type real time fluorescent PCR method and probe design are referring to embodiment 1, and concrete sequence sees Table 4.
(2) reaction system and the condition of pig circular ring virus 1 type real time fluorescent PCR method
The optimization principles of pig circular ring virus 1 type real time fluorescent PCR method is: make same sample obtain maximum amplification efficiency and minimum Ct value by optimizing following each condition.
Determining of a, best fluorescent primer concentration: fluorescent primer concentration is screened between the 800nM at 300nM.
Determining of b, best concentration and probe concentration: concentration and probe concentration is screened between the 500nM at 100nM.
Through optimizing, the reaction system of pig circular ring virus 1 type real time fluorescent PCR method sees Table 5, and reaction conditions sees Table 6.
Table 5 pig circular ring virus 1 type real-time fluorescence PCR detection system
Table 6 pig circular ring virus 1 type real-time fluorescence PCR detects amplification condition
(3) result judges
1. interpretation of result condition is set
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, to show that the result is as the criterion.
2. quality control standard
Negative control is without the Ct value and without the specific amplification curve;
The Ct value of positive control answers≤30, and the specific amplification curve occurs.Otherwise it is invalid that this time experiment is considered as.
3. the result describes and judges
Negative sample represents in the sample without pig circular ring virus 1 type without the Ct value and without the specific amplification curve.
Positive sample Ct value≤30, and the specific amplification curve appears, in the expression sample pig circular ring virus 1 type is arranged.
Effective principle: sample Ct value need to be carried out revision test between 30＜Ct＜37 time.Ct＜37 o'clock sample is positive as a result for revision test, otherwise negative.
(4) pattern detection test and result
Test is increased to Pestivirus suis (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese B encephalitis virus (JEV), PRV (Pseudorabies virus) (PRV) and pig parvoviral (PPV), and the result only has pig circular ring virus 1 type (PCV1) to obtain specific amplified fluorescence curve.
The optimization of embodiment 3. porcine circovirus 2 type real-time fluorescence PCR detection methods
(1) primer of porcine circovirus 2 type real time fluorescent PCR method and probe design
The primer of porcine circovirus 2 type real time fluorescent PCR method and probe design are referring to embodiment 1, and concrete sequence sees Table 4.
(2) reaction system of porcine circovirus 2 type real time fluorescent PCR method and condition
The optimization principles of porcine circovirus 2 type real time fluorescent PCR method is: make same sample obtain maximum amplification efficiency and minimum Ct value by optimizing following each condition.
Determining of a, best fluorescent primer concentration: fluorescent primer concentration is screened between the 800nM at 300nM.
Determining of b, best concentration and probe concentration: concentration and probe concentration is screened between the 500nM at 100nM.
Through optimizing, the reaction system of porcine circovirus 2 type real time fluorescent PCR method sees Table 7, and reaction conditions sees Table 8.
Table 7 porcine circovirus 2 type real-time fluorescence PCR detection system
Table 8 porcine circovirus 2 type real-time fluorescence PCR detects amplification condition
(3) result judges
4. interpretation of result condition is set
Read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, to show that the result is as the criterion.
5. quality control standard
Negative control is without the Ct value and without the specific amplification curve;
The Ct value of positive control answers≤30, and the specific amplification curve occurs.Otherwise it is invalid that this time experiment is considered as.
6. the result describes and judges
Negative sample represents in the sample without porcine circovirus 2 type without the Ct value and without the specific amplification curve.
Positive sample Ct value≤30, and the specific amplification curve appears, in the expression sample porcine circovirus 2 type is arranged.
Effective principle: sample Ct value need to be carried out revision test between 30＜Ct＜37 time.Ct＜37 o'clock sample is positive as a result for revision test, otherwise negative.
(4) pattern detection test and result
Test is increased to Pestivirus suis (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese B encephalitis virus (JEV), PRV (Pseudorabies virus) (PRV) and pig parvoviral (PPV), and the result only has porcine circovirus 2 type (PCV2) to obtain specific amplified fluorescence curve.
1, PCR reaction solution: each reaction comprises that reaction buffer (contains MgCl
2, dNTP and TaqE) 12.5 μ L, minute be filled to 1mL serum tube, 750 μ L/ pipe ,-20 ℃ of preservations;
2, fluorescent probe: each reaction comprises that 10 μ M probe PCV10.6 μ L, 10 μ M probe PCV20.6 μ L and ROX dyestuff 0.5 μ L form, and totally 1.7 μ L divide to be filled to the brown serum tube of 0.5mL, 100 μ L/ pipe ,-20 ℃ of preservations;
3, primer: each reaction comprises composition 10 μ M PCV1 primer pairs 1.8 μ L, 10 μ M PCV2 primer pairs, 1.8 μ L, and totally 3.6 μ L divide to be filled to the 0.5mL serum tube, 220 μ L/ pipe ,-20 ℃ of preservations;
4, aseptic nuclease free water: divide to be filled to the 0.5mL serum tube 600 μ L/ pipe ,-20 ℃ of preservations;
5, PCV1 positive control: add the PCV1 virus liquid of lysate, Ct value＜30,600 μ L/ pipe ,-20 ℃ of preservations.
6, PCV2 positive control: add the PCV2 virus liquid of lysate, Ct value＜30,600 μ L/ pipe ,-20 ℃ of preservations.
7, negative control: sterilization deionized water, 600 μ L/ pipe ,-20 ℃ of preservations.
8, Digestive system: the 11ml/ bottle, divide to be filled in the transparent white lid Plastic Bottle of white room temperature preservation.
9, DNA is in conjunction with liquid: the 16mL/ bottle minute is filled in the transparent white lid Plastic Bottle of white room temperature preservation.
10, DNA washings: the 52mL/ bottle, divide to be filled in the transparent white lid Plastic Bottle of white room temperature preservation.
11, DNA elutriant: the 4mL/ bottle, divide to be filled in the transparent white lid Plastic Bottle of white room temperature preservation.
12, DNA adsorption column and collection tube: 50 covers/bag, room temperature preservation.
The using method of embodiment 5. porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit
1 sample preparation
1.1 sample collecting: the pig that dies of illness or slaughter, get pathology section and the healthy section intersection tissues such as lung, lymphoglandula; Live hog to be checked is got blood 5mL with syringe.2～8 ℃ of preservations send the laboratory to detect.(require the censorship pathological material of disease fresh, forbid multigelation.）
1.2 sample preparation: every duplicate samples is processed respectively.
1.2.1 tissue sample is processed: organize respectively for every part and take by weighing approximately 1g of sample from three different positions, shred with operating scissors and to get 0.02g behind the mixing and in mill, grind, adding 1.5mL physiological saline continues to grind, after homogenate, go in the 1.5mL sterilization centrifuge tube, the centrifugal 2min of 8000rpm gets supernatant liquor 100 μ L in 1.5mL sterilization centrifuge tube, adds 200 μ L Digestive systems and 20 μ L Proteinase Ks again, behind the vibration mixing, put in 56 ℃ of water-baths digestion 1 hour.
1.2.2 whole blood sample is processed: behind blood clotting, get serum 100 μ L, put in the 1.5mL sterilization centrifuge tube.
1.2.3 positive control is processed: get positive control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
1.2.4 negative control is processed: get negative control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
2 operation stepss
2.1 the extraction of viral DNA
2.1.1 the taking-up sample hose, be down to room temperature after, add 300 μ L DNA in conjunction with liquid (Biospin tissue gene group DNA extraction test kit), put upside down mixing, whole liquid are moved in the adsorption columns, room temperature leaves standstill 3min, the centrifugal 30s of 10000rpm.
2.1.2 discard liquid in the collection tube, add 500 μ L DNA washingss, the centrifugal 30s of 10000rpm.
2.1.3 repeating step 2.1.2.
2.1.4 discard liquid in the collection tube, the centrifugal 30s of 10000rpm is to remove residual DNA washings.
2.1.5 adsorption column is put into new 1.5mL centrifuge tube, add the DNA elutriant 50 μ L of 56 ℃ of preheatings to post central authorities, room temperature is placed 2min, the centrifugal 30s of 10000rpm, liquid is template DNA in the centrifuge tube.
The operation of 3 real-time fluorescence PCRs
3.2.1 reaction system preparation
If test sample, negative control and positive control summation are N, then reaction system is formulated as follows:
Behind the abundant mixing of reaction system with above preparation, each 23 μ L in each reaction tubes of packing.
3.2.2 amplification
Get respectively the viral DNA 2 μ L that dissolved among the 2.1.5, add in the respective reactor, mixing also performs mark, and wherein PCV-2 fluorescence report group is FAM, and PCV-1 is Hex, and quenching group is None, carries out following reaction at the fluorescent PCR instrument: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 35s, 40 circulations, the second step of each circulation is collected respectively fluorescent signal.
4 results judge
4.1 the interpretation of result condition is set
The threshold setting principle: threshold line is set in the vertex just above the negative control amplification curve.
4.2 the result describes and judges
Positive control Ct value≤30 also specific amplification curve occurs, and negative control is without the Ct value and without specific amplification curve, and experimental result is set up; If test sample FAM fluorescent signal Ct value≤30 and specific amplification curve to occur be that PCV-2 is positive; If test sample Hex fluorescent signal Ct value≤30 and specific amplification curve to occur be that PCV-1 is positive; Test sample 30＜Ct＜37 also specific amplification curve occurs, carry out the result after need again trying again and judge, and are identical such as two times result, then are judged to be the positive; Test sample Ct value 〉=37 o'clock surpasses present method detection sensitivity scope, is judged to be feminine gender.
The compliance test result of embodiment 6. test kits of the present invention
1. specific test
Adopt method provided by the invention to prepare 20120809,20120811, the porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit of 20,120,816 3 lot numbers is respectively to porcine reproductive and respiratory syndrome virus (PRRSV), Japanese B encephalitis virus (JEV), PRV (Pseudorabies virus) (PRV), Pestivirus suis (CSFV), pig parvoviral (PPV), the negative pig lungs of PK-15 cell and pig circular ring virus, lymphoglandula, cardiac muscle carries out specific test, PCV1 detected result negative (Fig. 1), PCV, 2 detected results negative (Fig. 2), above this test kit of as a result sufficient proof has good specificity.
2. sensitivity test
To the PCV1 cell toxicant, extract according to embodiment 5DNA extracting method, the DNA that obtains is carried out 10
-1To 10
-5Gradient dilution detects its sensitivity, the results are shown in Table 7.
To the PCV2 cell toxicant, extract according to embodiment 5DNA extracting method, the DNA that obtains is carried out 10
-1To 10
-6Gradient dilution detects its sensitivity, the results are shown in Table 9.
Table 9 porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit sensitivity result
The logarithm of Ct value and template copy number is linear, and the Ct value between adjacent two template concentrations differs approximately 3.3, differs 10 times with the template concentrations of calculating in theory, and the Ct value differs 3.3 and conforms to.
3. replica test
Use the porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit of 20120809,20120811,20,120,816 3 lot numbers, according to the using method of embodiment 5 DNA of pig annulus 1 type and 10 times of serial dilutions of 2 type cell culture fluids is carried out respectively duplicate detection 3 times, with determine this method batch in repeatability; 3 batches of test kits detect same dilute sample, with determine this method batch between repeatability.The results are shown in Table 10.
Three batches of test kit replica tests of table 10 result
Detected result shows: variation within batch coefficient and interassay coefficient of variation are respectively 0.39%～2.50% and 0.65%～2.00%, all less than 5%, illustrate that 20120809,20120811,20120816 these 3 batches of reagent have between good batch repeatable.
4. stability test
Use the porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit of 20120809,20120811,20,120,816 3 lot numbers, to 3 samples, carry out per month one-time detection according to the using method of embodiment 5, do 3 repetitions for every batch, continue 6 months, the stability of detection kit.The result shows: 20120809, the porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit of 20120811,20,120,816 3 lot numbers, Ct value in 6 months changes little, illustrates that porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection kit had good stability in 6 months.
Claims (9)
1. a porcine circovirus types 1 and 2 dual real-time fluorescence PCR detects primer pair, it is characterized in that, specificity pig circular ring virus 1 type primer pair comprises the nucleotide sequence shown in SEQ ID NO:9 and SEQ ID NO:11;
Specificity porcine circovirus 2 type primer pair comprises the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:6.
2. porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection probes, it is characterized in that, described pig circular ring virus 1 type probe comprises the nucleotide sequence shown in the SEQ ID NO:14, and described porcine circovirus 2 type probe comprises the nucleotide sequence shown in the SEQ ID NO:7.
3. the composition that comprises primer pair claimed in claim 1 and probe claimed in claim 2.
4. a test kit is characterized in that, comprises the described composition of claim 3.
5. test kit according to claim 4 is characterized in that, two kinds of probes use respectively the fluorescein-labelled of different sense channels, and described fluorescein is selected from a kind of among FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or the CY5.
6. claim 4 or 5 described test kits detect the purposes of porcine circovirus types 1 and 2 simultaneously.
7. a porcine circovirus types 1 and 2 dual real-time fluorescence PCR detection method is characterized in that, right to use requires 1 described primer pair and probe claimed in claim 2.
8. method according to claim 7 is characterized in that, comprises the steps:
(1) right to use requires 1 described primer pair and probe claimed in claim 2;
(2) use respectively the probe of the fluorescein-labelled porcine circovirus types 1 and 2 of different sense channels;
(3) will for detection of or differentiate and primer and the probe of pig circular ring virus 1 type or 2 types to place same reaction tubes, use the fluorescent PCR amplification instrument to carry out the real-time fluorescence PCR reaction.
9. method according to claim 8 is characterized in that, two kinds of probes are used respectively the fluorescein-labelled of different sense channels, and described fluorescein is selected from a kind of among FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or the CY5.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274256A (en) * | 2015-10-30 | 2016-01-27 | 中国农业科学院兰州兽医研究所 | Taqman Real-time PCR kit for detecting porcine circovirus (PCV1) and using method of kit |
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| CN106148564A (en) * | 2015-03-12 | 2016-11-23 | 四川出入境检验检疫局检验检疫技术中心 | A kind of method utilizing digital pcr detection circovurus type 2 viral level |
| CN108456747A (en) * | 2018-05-14 | 2018-08-28 | 湖北省农业科学院畜牧兽医研究所 | A kind of multiple PCR detection kit differentiating pig circular ring virus |
| CN109593883A (en) * | 2017-09-30 | 2019-04-09 | 洛阳普莱柯万泰生物技术有限公司 | Kit of the pig circular ring virus multiple real time fluorescence PCR detection primer to, probe and preparation |
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| CN111676247A (en) * | 2020-06-30 | 2020-09-18 | 扬州大学 | Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate |
| CN111961761A (en) * | 2020-09-03 | 2020-11-20 | 扬州大学 | Primer probe group and kit for detecting different genotypes of porcine circovirus |
| CN113025755A (en) * | 2021-03-30 | 2021-06-25 | 广东省农业科学院动物卫生研究所 | Reagent for detecting porcine circovirus type 2e and application thereof |
| CN115478118A (en) * | 2022-06-28 | 2022-12-16 | 四川农业大学 | Taqman multiplex fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277455A (en) * | 2011-08-22 | 2011-12-14 | 浙江省农业科学院 | Primer, probe and assay kit used for detecting porcine circovirus, porcine pseudorabies virus and porcine parvovirus |
| CN102676698A (en) * | 2012-05-17 | 2012-09-19 | 贵州省畜牧兽医研究所 | PRV, PCV-2, PPV multiple PCR (Polymerase Chain Reaction) detection kit |
-
2013
- 2013-07-02 CN CN201310275996.2A patent/CN103305638B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277455A (en) * | 2011-08-22 | 2011-12-14 | 浙江省农业科学院 | Primer, probe and assay kit used for detecting porcine circovirus, porcine pseudorabies virus and porcine parvovirus |
| CN102676698A (en) * | 2012-05-17 | 2012-09-19 | 贵州省畜牧兽医研究所 | PRV, PCV-2, PPV multiple PCR (Polymerase Chain Reaction) detection kit |
Non-Patent Citations (1)
| Title |
|---|
| 王东方 等: "猪圆环病毒1、2型多重PCR诊断方法建立及应用", 《中国畜牧兽医学会家畜传染病学分会第七届全国会员代表大会暨第十三次学术研讨会论文集（上册）》, 31 December 2009 (2009-12-31), pages 518 - 521 * |
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