CN109913586A - A method of PCV-2 is detected using PCR-ELISA - Google Patents
A method of PCV-2 is detected using PCR-ELISA Download PDFInfo
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Abstract
The invention discloses a kind of methods using PCR-ELISA detection PCV-2, specifically include: extracting sample DNA, obtain amplification template DNA；Using specific primer pair, PCR reaction, specific amplification template DNA are carried out；Amplified production is subjected to ELISA detection and analysis.The present invention provides a kind of to utilize the method for PCR-ELISA detection PCV-2 for the purpose of non-diagnostic treatment, effectively PCR and ELISA method are combined, PCV-2 is detected, has many advantages, such as that high specificity, high sensitivity and detection method are easy to operate, quick.
Description
Technical field
The present invention relates to molecular Biological Detection fields, more particularly to a kind of side using PCR-ELISA detection PCV-2
Method.
Background technique
Pig annulus (Porcine circo, PC) refers to 2- type circovirus (2-Porcine circo disease
Virus, PCV-2) it is main pathogen, a series of general name of independent or the other pathogenic microorganisms of secondary or mixed infection diseases.
PCV-2 infection can be propagated through a variety of ways, caused different degrees of infection morbidity, be to seriously affect large scale of pig farm
A kind of important virus of industry development and intelligent clean manufacturing, mainly with air, feed, drinking-water, utensil, the excrement etc. in environment
It is propagated as medium.
The detection method of PCV-2 routine is agar diffusion test in the prior art, though this method is easy to operate, and it is time-consuming
Long, sensibility is low；Molecule and amynologic diagnostic method developed in recent years are mainly round pcr and elisa technique, PCR
Detection technique has high sensitivity, but false positive results easily occurs, and result needs to carry out electrophoresis judgement, needs to contact toxic
Property EB dyestuff and have limitation；Fluorescent quantitative PCR technique is also widely recognized because of its sensibility height, high specificity,
But equipment is expensive, at high cost shorter with the fluorescence probe holding time etc. limits the popularization of this method；ELISA method operation letter
It is single, do not need expensive instrument, be suitble to extensive epidemiological survey, but need to prepare antibody with high specificity, and susceptibility compared with
It is low.These methods are not able to satisfy detection demand accurate to PCV-2, economic, stable.
Therefore it provides a kind of easy to operate, high sensitivity, high specificity utilize PCR- for the purpose of non-diagnostic treatment
The problem of method that ELISA detects PCV-2 is those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, there is high specificity, spirit the present invention provides a kind of method using PCR-ELISA detection PCV-2
The advantages that sensitivity is high and detection method is easy to operate, quick.To achieve the goals above, the present invention uses following technical side
Case:
The PCV-2 gene high conserved region sequence according to disclosed in Genbank:JF272498.1 designs a kind of using PCR-
ELISA method detects the specific primer pair of PCV-2, and the nucleotide sequence of the specific primer pair is as follows:
Upstream primer: 5 '-GCGGTGGACATGATGAGATT-3 '；SEQ ID NO.1；
Downstream primer: 5 '-GTTATGGTATGGCGGGAGG-3 '；SEQ ID NO.2；
Its upstream primer 5 ' end utilizes digoxigenin labeled, and the end of downstream primer 5 ' utilizes biotin labeling.
A method of PCV-2 being detected for the purpose of non-diagnostic treatment using PCR-ELISA, comprising the following steps:
The PCR amplification of S1:PCV-2 gene:
S11: extracting sample DNA, obtains amplification template DNA；
S12: using above-mentioned specific primer to progress PCR reaction, the template DNA that specific amplification step S11 is obtained；
S2:ELISA is tested and analyzed:
S21: Streptavidin coated elisa plate is used；
S22: after the amplified production of step S1 is mixed with dilution according to the volume ratio of 1:8-10, it is added to S21
In ELISA Plate obtained, 37 DEG C of warm bath 30min；
S23: after incubation, being sucked out the liquid in ELISA Plate, and rinsing liquid is added and washs to it, gets rid of after washing every time
It is dry, it is repeated 3 times, the anti digoxin antibody that concentration is 0.20 μ l/ml, 35-40 DEG C of incubation 50-70min is added later；
S24: after incubation, being sucked out the liquid in ELISA Plate, and rinsing liquid is added and washs to it, gets rid of after washing every time
It is dry, it is repeated 5 times, color developing agent is added later, be protected from light 5-10min；
S25: terminate liquid being added into ELISA Plate, after detect light absorption value at 450 nm.
Preferably, work as OD450Value is greater than 0.32, can determine that as positive sample；It is negative sample less than 0.25, in 0.25 He
It is suspicious specimen between 0.32.
Preferably, detection method disclosed by the invention can be widely used for feed in piggery enviroment, drinking-water, breeding facility, life
The detection of PCV-2 pathogen in fresh pork and excrement.
The PCR-ELISA rapid detection method of PCV-2 of the invention compared with detection technique compare, this method combines
PCR and ELISA method have stronger specificity and higher sensitivity to PCV-2, and remolding sensitivity agarose gel electrophoresis is high
About 1000 times；Compared with common PCR method, the high special of ELISA antigen-antibody reaction is utilized in PCR-ELISA method
Property, enables the method more specific detection target gene fragment into sample；It is avoided compared with ELISA and prepares the numerous of antibody
It is trivial, improve the sensibility of detection method；Present invention employs Streptavidin-biotin-digoxin-anti digoxin antibody systems
System, improves the sensibility of detection method；From result judgement, the method that this method uses ELISA colour developing avoids leakage of electricity
Swimming process keeps result judgement more direct.It can detecte in environment, as viral in excrement, feed, air, for the prevention of the disease
Certain technical support is provided with propagating.
Preferably, PCR amplification system includes: 1 μ l of template DNA solution, each 1 μ l of upstream and downstream primer, Ex Taq in step S12
0.5 μ l of archaeal dna polymerase, dNTP are 2 μ l, 10x buffer5 μ l, 39.5 μ l of sterile deionized water.
Preferably, PCR amplification condition in step S12 are as follows: 96 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s；54.8 DEG C annealing
30s；72 DEG C of extension 25s；Expand 30 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of pcr amplification product preservations.
Preferably, step S21 method particularly includes: be added the solution of streptavidin of 2.5 μ g/ml into ELISA Plate hole
100-150 μ l, 4 DEG C of coatings overnight, pour out liquid in plate hole, and dry, and 300 μ l rinsing liquids are added and wash plate hole, wash every time
After dry, in triplicate.
Preferably, dilution described in step S22 is PBS buffer solution.
Preferably, rinsing liquid PBST, PBST are the PBS buffer solution of 0.5%Tween20.
Preferably, color developing agent described in step S24 is TMB developing solution.
Preferably, terminate liquid described in step S25 is the sulfuric acid solution of 2M.
It can be seen via above technical scheme that compared with prior art, being examined the present invention provides a kind of using PCR-ELISA
The method for surveying PCV-2 has following technological merit:
1. the method provided by the invention that PCV-2 is quickly detected using PCR-ELISA, combines PCR and ELISA method,
To PCV-2 have it is stronger specificity and higher sensitivity, and it is easy to operate, quick, at low cost, high throughput can be carried out
Sample detection, can be widely used for the quick detection of PCV-2 in pig farm.
2. the method provided by the invention for quickly detecting PCV-2 using PCR-ELISA and traditional PCR-ELISA method phase
Than eliminating the use of probe, label substance markers directly being simplified operating procedure on primer, and also avoid
The error occurred when probe is in conjunction with PCR product effectively prevents the false positive of PCR amplification so that sensitivity further increases
As a result, improving the accuracy of testing result.
3. the method provided by the invention for quickly detecting PCV-2 using PCR-ELISA, PCR cycle number is less, contracts significantly
Short test period.
4. the method provided by the invention for quickly being detected PCV-2 using PCR-ELISA, can be realized in ELISA Plate to sample
The high-throughput of product detects, and only needs common instrument, reagent and consumptive material in detection process, reduces testing cost.
5. present invention employs the methods of ELISA colour developing, high using Streptavidin-biotin-ground from result judgement
Octyl- anti digoxin antibody system, avoids electrophoresis process, keeps result judgement more direct.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is the PCV-2 electrophoretogram of various concentration provided by the invention
Wherein, M Marker；1-17 be respectively positive control, 2 μ g/ml, 1.5 μ g/ml, 0.5 μ g/ml, 0.25 μ g/ml,
0.1μg/ml、0.05μg/ml、0.025μg/ml、0.01μg/ml、0.005μg/ml、0.004μg/ml、0.003μg/ml、
0.002μg/ml、0.001μg/ml、0.0005μg/ml、0.0004μg/ml、0.0003μg/ml、0.0002μg/ml、0.0001μ
g/ml。
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The design of specific primer pair
According to PCV-2 gene high conserved region sequence disclosed in GeneBank JF272498.1, designs and a kind of utilize PCR-
ELISA method detects the specific primer pair of PCV-2, and the nucleotide sequence of specific primer pair is as follows:
Upstream primer: 5 '-GCGGTGGACATGATGAGATT-3 '；SEQ ID NO.1；
Downstream primer: 5 '-GTTATGGTATGGCGGGAGG-3 '；SEQ ID NO.2；
The end of upstream primer 5 ' utilizes digoxigenin labeled, and the end of downstream primer 5 ' utilizes biotin labeling.
Embodiment 2
The method of PCV-2 is detected for the purpose of non-diagnostic treatment
1, the preparation of pcr template
The excrement of acquisition infection PCV-2 illness pig, utilizes kit (TaKaRa MiniBEST Viral RNA/DNA
Extraction Kit Ver.5.0) extract sample DNA, then by purifying, connection, conversion, upgrading grain, be sequenced and etc. obtain mould
Plate DNA.
The genomic DNA of CSFV, PRRSV, PRV, PEDV are extracted respectively.
2, pcr amplification reaction
200 μ l PCR tubules are taken, each 1 μ l, Ex Taq archaeal dna polymerase, 0.5 μ of 1 μ l template DNA solution, upstream and downstream primer is added
L, dNTP be 2 μ l, 10x buffer be 5 μ l, sterile deionized water is 39.5 μ l, total volume is 50 μ l.It is slight to mix, briefly from
The heart carries out PCR amplification.PCR amplification condition are as follows: 96 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s；54.8 DEG C of annealing 30s；72 DEG C are prolonged
25s is stretched, 30 circulations, last 72 DEG C of extensions 10min are expanded.4 DEG C of pcr amplification product preservations.
3, the ELISA of amplified production is tested and analyzed
(1) the solution of streptavidin 100 μ l of 2.5 μ g/ml is added into 96 ELISA Plate holes, is coated with overnight at 4 DEG C；It is sucked out
Liquid in ELISA Plate hole, and dry, 300 μ l PBST (PBS buffer solution of 0.5%Tween20) are added and wash plate hole, after washing
Drying, in triplicate；
(2) the 200 μ l of BSA that mass fraction is 2% is added into ELISA Plate, incubates 15min in 37 DEG C of insulating boxs；It inhales
Liquid in ELISA Plate hole out, and dry, 300 μ lPBST are added and wash plate hole, are dried after washing, in triplicate；
(3) it takes the pcr amplification product of 10 μ l in the PBS buffer solution of 90 μ l, is added after slight mixing by Streptavidin
In the ELISA Plate after BSA coating, 30min is incubated in 37 DEG C of insulating boxs；Liquid in ELISA Plate hole is sucked out and dries, is added
300 μ l PBST wash plate hole, dry after washing, in triplicate；
(4) Antidigoxigenin-detecting that 100 μ l concentration are 0.20 μ l/ml is added into ELISA Plate
Antibody Fab fragments (Germany, Roche), incubates 60min in 37 DEG C of insulating boxs；Liquid in ELISA Plate hole is sucked out
And dry, 300 μ l PBST are added and wash plate hole, dries after washing, so repeats five times；
(5) 50 μ l TMB solution are added into ELISA Plate, are protected from light colour developing 5min；
(6) sulfuric acid solution that the 2M of 50 μ l is added terminates reaction；It is then placed in the extinction detected under its 450nm in microplate reader
Value, testing result is as shown in table 1, determines testing result according to the size of light absorption value, wherein OD450Value is greater than 0.32, can determine that
For positive sample；It is negative sample less than 0.25, is suspicious specimen between 0.25 and 0.32.
Table 1 infects the result in PCV-2 illness swine excrement to PCV-2 specific detection
| Virus Name | OD450Value | As a result |
| PCV-2 | 1.26690 | It is positive |
| CSFV | 0.10120 | It is negative |
| PRRSV | 0.13850 | It is negative |
| PRV | 0.12240 | It is negative |
| PEDV | 0.16010 | It is negative |
As shown in Table 1, the testing result for infecting only PCV-2 in PCV-2 illness swine excrement is positive, remaining is yin
Property, illustrate detection method high specificity provided by the invention, does not there is false positive and false negative result.
Embodiment 3
Sensitivity technique
The present embodiment is by taking live fresh pork as an example: the live fresh pork of acquisition infection PCV-2 illness pig utilizes kit
(TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0) extracts sample DNA, then by purifying,
Connection, conversion, upgrading grain, sequencing and etc. template DNA.
By obtained template DNA be diluted to respectively 2 μ g/ml, 1.5 μ g/ml, 1 μ g/ml, 0.5 μ g/ml, 0.25 μ g/ml,
0.1μg/ml、0.05μg/ml、0.025μg/ml、0.01μg/ml、0.005μg/ml、0.004μg/ml、0.003μg/ml、
0.002μg/ml、0.001μg/ml、0.0005μg/ml、0.0004μg/ml、0.0003μg/ml、0.0002μg/ml、0.0001μ
g/ml.The DNA of various concentration is detected respectively using the method in embodiment 2, as a result such as table 2.
Table 2 infects the result in PCV-2 illness live fresh pork to PCV-2 sensitivity technique
As shown in Table 2, when being detected using PCR-ELISA method of the invention, the detection of positive sample is limited to 0.2ng/ml,
Sensitivity with higher.
At the same time, it to the positive DNA sample for being diluted to various concentration, is detected using PCR gel electrophoresis, as a result such as
Shown in Fig. 1.As shown in Figure 1, when PCR detected through gel electrophoresis, the positive DNA sample band of 0.1 μ g/ml is very faint.Cause
The traditional agarose gel electrophoresis of the remolding sensitivity of this PCR-ELISA rapid detection method disclosed by the invention is about 1000 times high.
Embodiment 4
Sample detecting is carried out to from Xinxiang City, Henan Province periphery pig farm from during 1-5 month in 2017, is utilized respectively this
Method, quantitative fluorescent PCR and the PCR method using PCR-ELISA detection PCV-2 that inventive embodiments 2 are established carry out practical
The detection of sample, every group has detected 56 parts of swine excrements respectively, and testing result is as shown in table 3.
Comparison of the table 3 using different detection methods to PCV-2 testing result
| Detection method | Quantitative fluorescent PCR | PCR | PCR-ELISA |
| Positive findings (part) | 50 | 43 | 50 |
The result shows that detecting 50 parts of PCV-2 positive samples, fluorescence quantifying PCR method detection using the method for embodiment 2
50 parts of PCV-2 positive samples out, PCR method detect 43 parts of positive samples, illustrate PCR-ELISA method in the detection of actual sample
In have very high accuracy, can be used for the detection of batch samples in pig farm.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
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<120>a kind of method using PCR-ELISA detection PCV-2
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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gttatggtat ggcgggagg 19
Claims (9)
1. a kind of specific primer pair using PCR-ELISA method detection PCV-2, which is characterized in that the specific primer
Pair nucleotide sequence it is as follows:
Upstream primer: 5 '-GCGGTGGACATGATGAGATT-3 '；SEQ ID NO.1；
Downstream primer: 5 '-GTTATGGTATGGCGGGAGG-3 '；SEQ ID NO.2；
The end of upstream primer 5 ' utilizes digoxigenin labeled, and the end of downstream primer 5 ' utilizes biotin labeling.
2. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA, which is characterized in that including following
Step:
The PCR amplification of S1:PCV-2 gene:
S11: extracting sample DNA, obtains amplification template DNA；
S12: using specific primer described in claim 1 to progress PCR reaction, the mould that specific amplification step S11 is obtained
Plate DNA；
S2:ELISA is tested and analyzed:
S21: Streptavidin coated elisa plate is used；
S22: after the amplified production that step S1 is obtained is mixed with dilution according to the volume ratio of 1:8-10, it is added to step
In ELISA Plate made from S21,37 DEG C of warm bath 30min；
S23: after incubation, being sucked out the liquid in ELISA Plate, and rinsing liquid is added and washs to it, dries after washing every time,
In triplicate, the anti digoxin antibody that concentration is 0.20 μ l/ml, 35-40 DEG C of incubation 50-70min are added later；
S24: after incubation, being sucked out the liquid in ELISA Plate, and rinsing liquid is added and washs to it, dries after washing every time,
It is repeated 5 times, color developing agent is added later, be protected from light 5-10min；
S25: terminate liquid being added into ELISA Plate, after detect light absorption value at 450 nm.
3. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, PCR amplification system includes: 1 μ l of template DNA solution, each 1 μ l of upstream and downstream primer, Ex Taq DNA in step S12
0.5 μ l of polymerase, dNTP are 2 μ l, 10x buffer5 μ l, 39.5 μ l of sterile deionized water.
4. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 3,
It is characterized in that, PCR amplification condition in step S12 are as follows: 96 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s；54.8 DEG C of annealing 30s；72
DEG C extend 25s；Expand 30 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of pcr amplification product preservations.
5. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, step S21 method particularly includes: the solution of streptavidin 100- of 2.5 μ g/ml is added into ELISA Plate hole
150 μ l, 4 DEG C of coatings overnight, pour out liquid in plate hole, and dry, 300 μ l rinsing liquids are added and wash plate hole, get rid of after washing every time
It is dry, in triplicate.
6. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, dilution described in step S22 is PBS buffer solution.
7. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, the rinsing liquid is PBST, the PBST is the PBS buffer solution of 0.5%Tween20.
8. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, color developing agent described in step S24 is TMB developing solution.
9. a kind of method for detecting PCV-2 for the purpose of non-diagnostic treatment using PCR-ELISA according to claim 2,
It is characterized in that, terminate liquid described in step S25 is the sulfuric acid solution of 2M.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030096377A1 (en) * | 2001-06-28 | 2003-05-22 | Virginia Tech Intellectual Properties, Inc. | Differential PCR-RFLP assay for detecting and distinguishing between nonpathogenic PCV-1 and pathogenic PCV-2 |
| CN102495208A (en) * | 2011-10-24 | 2012-06-13 | 山东省农业科学院畜牧兽医研究所 | Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method |
| CN103472225A (en) * | 2013-09-06 | 2013-12-25 | 青岛易邦生物工程有限公司 | Fluorescence immunoassay kit used for detecting porcine circovirus type 2 |
| CN104745703A (en) * | 2015-03-31 | 2015-07-01 | 大连工业大学 | Vbrio parahaemolyticus PCR-ELISA detection kit and detection method |
| CN106086238A (en) * | 2016-07-01 | 2016-11-09 | 江苏农牧科技职业学院 | The PCR detection method of porcine circovirus 2 type, the primer and detection kit |
| CN109439775A (en) * | 2018-08-29 | 2019-03-08 | 铜仁职业技术学院 | A kind of multi-PCR detection method of porcine pathogen |
-
2019
- 2019-03-25 CN CN201910228559.2A patent/CN109913586A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030096377A1 (en) * | 2001-06-28 | 2003-05-22 | Virginia Tech Intellectual Properties, Inc. | Differential PCR-RFLP assay for detecting and distinguishing between nonpathogenic PCV-1 and pathogenic PCV-2 |
| CN102495208A (en) * | 2011-10-24 | 2012-06-13 | 山东省农业科学院畜牧兽医研究所 | Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method |
| CN103472225A (en) * | 2013-09-06 | 2013-12-25 | 青岛易邦生物工程有限公司 | Fluorescence immunoassay kit used for detecting porcine circovirus type 2 |
| CN104745703A (en) * | 2015-03-31 | 2015-07-01 | 大连工业大学 | Vbrio parahaemolyticus PCR-ELISA detection kit and detection method |
| CN106086238A (en) * | 2016-07-01 | 2016-11-09 | 江苏农牧科技职业学院 | The PCR detection method of porcine circovirus 2 type, the primer and detection kit |
| CN109439775A (en) * | 2018-08-29 | 2019-03-08 | 铜仁职业技术学院 | A kind of multi-PCR detection method of porcine pathogen |
Non-Patent Citations (3)
| Title |
|---|
| 杨少华等: "禽流感病毒RT-PCR ELISA诊断方法的建立", 《中国农学通报》 * |
| 王宪文等: "猪圆环病毒 II型 PCR 检测方法的研究", 《畜牧兽医科学》 * |
| 陈晴等: "PCR-ELISA在疫病病原分子诊断中的应用", 《中国家禽》 * |
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Application publication date: 20190621 |