CN107686864A - Porcine epidemic diarrhea virus TaqMan fluorescent quantitation RT PCR kits and its detection method - Google Patents
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Abstract
The invention discloses a kind of Porcine epidemic diarrhea virus TaqMan fluorescent quantitation RT PCR kits and its detection method, according to the N gene orders of Porcine epidemic diarrhea virus, design and synthesize special primer and TanMan probes, pass through the optimization to fluorescent quantitation reaction condition, detection PEDV TaqMan real time fluorescent quantitative RT PCR methods are established, this method is 108‑102There is good linear relationship, sensitivity minimum detectable 10 in the range of copies/ μ L2Copies/ μ L, its sensitiveness is high, can specific quick detection Porcine epidemic diarrhea virus；In batch and batch between repeat the coefficient of variation and be respectively less than 1%, it is reproducible.185 parts of swine excrement samples of collection in 2,015 2017 years are detected using the present invention, its positive rate is 38.4%, and carries out check experiment with the detection of conventional RT PCR methods, and its positive rate is only 26.3%.
Description
Technical field
The invention belongs to animal virology and technical field of molecular biology, more particularly to Porcine epidemic diarrhea virus
TaqMan fluorescence quantitative RT-PCR kits and its detection method.
Background technology
Porcine epidemic diarrhea virus (PEDV) is a kind of common virus for causing pig acute infectious intestinal disease, easy infection, is sent out
Sick rate is high, and has certain seasonality, and its clinical symptoms and transmissible gastro-enteritis virus (TGEV), pig Delta are coronal
Viral (PDCoV), pig ridge viral (PKV), porcine reproductive and respiratory syndrome virus (PRRSV) and foot and mouth disease virus (FMDV) compared with
To be similar.
The existing many kinds of methods of detection for Porcine epidemic diarrhea virus both at home and abroad, including ELISA, RT-PCR and fluorescence
Quantitative PCR etc., but different method sensitiveness has differences.In the above-mentioned methods, it is real compared to other method, TaqMan probe
When fluorescent quantitative RT-PCR method there is higher Sensitivity and Specificity.At present, TaqMan probe real time fluorescent quantitative is passed through
RT-PCR method detection PEDV research is less, and existing detection method detection sensitivity is poor, and positive rate is low, and repeatability is not
It is good, it is impossible to perform well in the diagnosis and epidemiology survey of PEDV clinical samples.
The content of the invention
It is an object of the invention to provide a kind of Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kits and
Its detection method, it is intended to solve method its poor sensitivity of existing detection Porcine epidemic diarrhea virus, positive rate is low, weight
Renaturation is bad, it is impossible to the problem of performing well in the diagnosis and epidemiology survey of Porcine epidemic diarrhea virus clinical sample.
In order to achieve the above object, the technical scheme is that：
First purpose of the present invention is to provide a kind of Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-RCR reagents
Box, described RT-PCR kit include primer and probe；
The sequence of the primer is：
Forward primer：5'-ACTACCTCGGAACAGGACCTCA-3'；
Reverse primer：5'-AGACGCCTTTCTGACACCCA-3'；
The sequence of the probe is：
5'-FAM-CGCCGACCTCCGCTATAGGACTCGT-BHQ1-3'。
Further, the concentration of the primer is 10 μM, and the concentration of probe is 5 μM.
Second object of the present invention is to provide a kind of Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-RCRs inspection
Survey method, the Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-RCRs described in the detection method usage right requirement 1 or 2
Kit, detection method include following steps：
(1) RNA in testing sample is extracted；
(2) RNA standard items are prepared；
(3) TaqMan fluorescence quantitative RT-RCRs amplified reaction：
Primer, the probe used during amplification be respectively：
Forward primer：5'-ACTACCTCGGAACAGGACCTCA-3'；
Reverse primer：5'-AGACGCCTTTCTGACACCCA-3'；
Probe：5'-FAM-CGCCGACCTCCGCTATAGGACTCGT-BHQ1-3'；
Amplification reaction system：2 × RT-PCR buffer, 10 μ L, E × Taq HS 0.4 μ L, the μ of reverse transcriptase mixture 0.4
L, the μ L of forward primer 0.4, the μ L of reverse primer 0.4, probe 0.6 μ L, no μ L of RNase water 5.8；
The μ L of RNA 2 in step (1) and the μ L of negative control 2 are taken to be separately added into the amplification reaction system that total amount is 20 μ L,
The PCR pipe for having configured reaction system is preheated into 5min in 42 DEG C, 95 DEG C of pre-degeneration 10s, 95 DEG C of denaturation 5s, anneals for 57 DEG C and extends
20s, denaturation and annealing process extension totally 40 circulations；
(4) result detects：Software is carried by quantitative real time PCR Instrument and reads corresponding Ct values, judges that testing sample is positive
It is or negative.
Further, the criterion of the testing sample positive or negative is：
(1) it is positive：Detect sample Ct values and be less than or equal to 35.0, and curve has obvious Exponential growth stage；
(2) it is suspicious：Detect sample Ct values and be more than 35.0, and the sample of typical amplification curve occur, carry out repeating experiment；
(3) it is negative：It can't detect sample Ct values and without obvious amplification curve.
Beneficial effects of the present invention are：Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-RCRs examination provided by the invention
Agent box and its detection method, special primer and TanMan probes are designed and synthesized, by the excellent of fluorescent quantitation reaction condition
Change, establish detection PEDV TaqMan real-time fluorescent quantitative RT-PCR methods, the detection PEDV that this method can be special, and right
Transmissible gastro-enteritis virus (TGEV), pig Delta coronavirus (PDCoV), pig ridge viral (PKV), pig breeding are comprehensive with breathing
Simulator sickness virus (PRRSV) and the testing result of foot and mouth disease virus (FMDV) are feminine gender, i.e., the TaqMan that the present invention establishes is visited
Pin real-time fluorescent quantitative RT-PCR method high specificity；The present invention is 108-102There is well linear in the range of copies/ μ L
Relation, sensitivity minimum detectable 102Copies/ μ L, its sensitiveness is high, and repeating the coefficient of variation in batch and between criticizing is respectively less than
1%, it is reproducible.The 185 parts of swine excrement samples gathered using the present invention to 2015-2017 detect, its positive detection
Rate is 38.4%, and carries out check experiment with the detection of conventional RT-PCR method, and its positive rate is only 26.3%.
Brief description of the drawings
Fig. 1 is PEDV N gene PCRs amplified production figure provided in an embodiment of the present invention, and in figure, M- marks, 1- feminine genders are right
According to 2-PCR products.
Fig. 2 is the canonical plotting of PEDV TaqMan real-time fluorescence quantitative PCRs provided in an embodiment of the present invention.
Fig. 3 is PEDV TaqMan real-time fluorescence quantitative PCR dynamic curve diagrams provided in an embodiment of the present invention, in figure, 1-
6 represent that RNA template concentrations are 2.4 × 10 respectively9copies/μL、2.4×108copies/μL、2.4×107copies/μL、
2.4×106copies/μL、2.4×105copies/μL、2.4×104copies/μL。
Fig. 4 is the sensitivity tests result figure of PEDV TaqMan real-time fluorescence quantitative PCRs provided in an embodiment of the present invention,
In figure, 1-8 represents that PEDV RNA standard concentrations are 2.4 × 10 respectively8copies/μL、2.4×107copies/μL、2.4×
106copies/μL、2.4×105copies/μL、2.4×104copies/μL、2.4×103copies/μL、2.4×
102copies/μL、2.4×101copies/μL。
Fig. 5 is the specific test result figure of TaqMan real-time fluorescence quantitative PCRs provided in an embodiment of the present invention, in figure：
1-PEDV, 2-PRRSV, 3-FMDV, 4-PKV, 5-TGEV, 6-PDCoV.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Below in conjunction with the accompanying drawings and specific embodiment is further described to the application principle of the present invention, in following embodiments
Experimental method, it is conventional method unless otherwise specified；Test material used in following embodiments, unless otherwise specified,
To be commercially available.
Embodiment：A kind of Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kits and its detection method.
1st, primer and probe design
According to Porcine epidemic diarrhea virus (PEDV) N gene DNA Star software sequences comparison results, probe primer is utilized
Design software Primer Express5.0 design two pairs of primers and two probes, refer to table 1.
The PEDV primers of table 1 and TaqMan probe sequence Tab.1 The sequence of PEDV primers and
probes
Note：Using PEDV AH2012 strain N gene orders as template
2nd, PEDV RNA extraction
PEDV RNA extraction is carried out according to RNeasy Mini kit (50) QIAGen operation book explanations.Concrete operations：Take
350 μ L virus liquids are added in 1.5ml centrifuge tubes, add isometric RLT and 3.5 μ L beta -mercaptoethanol, in stand 5 on ice~
10min, 700 μ L absolute ethyl alcohol is then rapidly added, slowly mixed, take out mini centrifugal column, add 700 μ L mixture,
12000rpm/min centrifuges 15s, outwells collecting pipe liquid, is repeated once.700 μ L RW1 is added, stands 1min on ice,
12000rpm/min centrifuges 15s.New collecting pipe is replaced, and adds 500 μ L RPE, 12000rpm/min centrifugation 15s, is discarded
Liquid in collecting pipe, add 500 μ L RPE, 12000rpm/min centrifugations 2min.Mini centrifugal column is placed in no RNase
In 1.5ml centrifuge tubes, the centrifuging 1min without RNase water, standing 1min, 12000rpm/min, take out centrifugal column of 30 μ L is added, will
It is standby that the RNA of extraction is stored in -20 DEG C of refrigerators.
3rd, the preparation of RNA standard items
With the amplification that RT-PCR is carried out comprising the specific primer including fluorescent quantitation purpose fragment.Expanded through PCR pure
Change obtain the product of 630bp sizes, PEDV N gene PCRs amplified productions as shown in figure 1, amplified production DNA with 1.5% agar
Sugared detected through gel electrophoresis simultaneously observes result, and amplification is consistent with the purpose fragment designed.By positive PCR primer it is purified after connect
It is connected in PMD-20T carriers, and is transformed into DH5 α competent cells.White recombinant bacterium, which is picked out, through the screening of blue hickie expands training
Upgrading grain after supporting, through the sequencing identification again of Beijing Hua Da gene after digestion identification.So that successful positive plasmid is sequenced as in vitro
The standard items of transcription, it is purified to obtain RNA standard items with T7 in-vitro transcription kit rna transcription sheets, pass through ultra micro after purification
Amount spectrophotometer Nanodrop 2000 is determined, and the RNA standard concentrations of extraction are for 839.3ng/ μ L, OD260/OD280
1.96, -70 DEG C save backup.
4th, the foundation of quantitative fluorescent PCR reaction system and reaction condition
It is comprehensive using gradient experiment optimization fluorescence quantification PCR primer, probe and Taq polymerase concentration and elongating temperature
Ct values, fluorescence intensity and repeatability are considered, it is determined that optimal quantitative fluorescent PCR reaction condition.
The optimal primer pair filtered out using one-step method PCR kit for fluorescence quantitative is：Forward primer is PEDV-Q-F, its
Gene order is 5'-ACTACCTCGGAACAGGACCTCA-3'；Reverse primer is PEDV-Q-R, and its gene order is 5'-
AGACGCCTTTCTGACACCCA-3'；Probe is Probe-Q, and its gene order is 5'-FAM-
CGCCGACCTCCGCTATAGGACTCGT-BHQ1-3'；The concentration of primer and probe is respectively 0.2mol/L and 0.15mol/L.
Amplification reaction system：2 × RT-PCR b μ ffer, E × Taq HS, reverse transcriptase mixture, forward primer, reversely
Primer, probe, without RNase water；
Experiment is optimized to amplification reaction system,
The dosage of each material is as shown in table 2；
The dosage of each material in the amplification reaction system Optimal Experimental of table 2
Compared by the gradient that concentration is added to reaction system middle probe, it is determined that optimal concentration and probe concentration is 0.15mol/
L, primer concentration is then using empirical value 0.2mol/L as optium concentration.As shown in Table 2, optimal amplification reaction system is：2×RT-
10 μ L, E × Taq HS of PCR buffer 0.4 μ L, the μ L of reverse transcriptase mixture 0.4, the μ L of forward primer 0.4, reverse primer 0.4
μ L, probe 0.6 μ L, no μ L of RNase water 5.8；
Take the μ L of the RNA 2 and μ L of negative control 2 to be separately added into the amplification reaction system that total amount is 20 μ L, reactant will have been configured
The PCR pipe of system preheats 5min, 95 DEG C of pre-degeneration 10s in 42 DEG C, 95 DEG C of denaturation 5s, 57 DEG C of annealing extension 20s, denaturation with it is annealed
Cheng Yanshen totally 40 circulations；
(4) result detects：Software is carried by quantitative real time PCR Instrument and reads corresponding Ct values, judges that testing sample is positive
It is or negative.
5th, the foundation of fluorescent quantitation standard curve
The concentration of rna transcription sheet after purification is determined, utilizes copy number calculation formula：Copy concentrations=[6.023 × 1023 ×
(concentration ng/ μ L × 10-9)]/base number × 660, its copy number is calculated as 2.4 × 1012Copies/ μ L are dilute through 10 times of gradients
Release, with 2.4 × 109、2.4×108、2.4×107、2.4×106、2.4×105、2.4×104Copies/ μ L standard items are mould
Plate carries out quantitative fluorescent PCR, obtains detecting PEDV amplification kinetic curve, as shown in figure 3, corresponding standard curve is counted out,
As shown in Fig. 2 the concentration of PEDV quantitative fluorescent PCR standard items is 2.4 × 108~2.4 × 102Have in the range of copies/ μ L good
Good linear, Y=-3.365 × lg (x)+49.36, its amplification efficiency are 98.2%, coefficient R2=1.000.
6th, sensitivity tests
By rna transcription this through 10 times of gradient dilutions, with 2.4 × 108、2.4×107、2.4×106、2.4×105、2.4×
104、2.4×103、2.4×102、2.4×101Copies/ μ L standard items are template, carry out quantitative fluorescent PCR reaction, it is determined that
The sensitivity of this method, if detection sample Ct values are less than or equal to 35.0, and it is then the positive that curve, which has obvious Exponential growth stage,；If
Detect sample Ct values and be more than 35.0, and the sample of typical amplification curve occur, then carry out repeating experiment；If it can't detect sample Ct
It is worth and without obvious amplification curve then to be negative.The Ct values for detecting sample are less than 35 for the positive, and result of the test is as shown in figure 4, knot
Close Fig. 2, it can be seen that when the copy number of standard items is 2.4 × 102Remain to detect during copies/ μ L, and blank control is without expansion
Increase, illustrate the lowest detection limit of the detection method up to 240copies/ μ L.
7th, specific test
With RNA extracts kits extraction porcine reproductive and respiratory syndrome virus (PRRSV), swine foot-and-mouth disease virus (FMDV),
Pig ridge virus (PKV), transmissible gastro-enteritis virus (TGEV) and the RNA of pig Delta coronavirus (PDCoV) and reverse transcription
Into cDNA, performing PCR amplification is entered with respective specific primer, obtains corresponding specific fragment, it was demonstrated that 5 viruses are positive poison
Strain, and reverse transcription obtain cDNA can be expanded normally.PRRSV, FMDV, PKV, TGEV, PDCoV RNA is taken to enter for template
Row fluorescent quantitative PCR, while negative control is set.As a result it is as shown in Figure 5, it can be seen that PRRSV, FMDV, PKV, TGEV,
The quantitative fluorescent PCR of PDCoV and negative control does not have amplification curve, only occurs good amplification curve with PEDV.Say
Bright the established TaqMan real-time fluorescence quantitative PCR detection method of experiment has good specificity.
8th, replica test
(1) replica test between criticizing：Take 2.4 × 107、2.4×106With 2.4 × 1053 various concentrations of copies/ μ L
RNA standard samples, 8 repetitions are carried out under identical conditions and are tested；Result of the test is as shown in table 2：
Tab.2 Inter-assay reproducibility of are repeated between the PEDV real-time fluorescence quantitative PCRs batch of table 2
PEDV TaqMan real-time fluorescent quantitative PCR
As shown in Table 2,2.4 × 107、2.4×106With 2.4 × 105The coefficient of variation of copies/ μ L RNA standard samples
Respectively 0.59%, 0.75%, 0.24%, respectively less than 1%, show this experiment establish TaqMan real-time fluorescence quantitative PCRs inspection
The method for surveying PEDV has good repeatability between criticizing.
(2) replica test in criticizing：Take 2.4 × 107、2.4×106With 2.4 × 1053 various concentrations of copies/ μ L
RNA standard samples, 8 repetitions are done in once testing and are tested, and testing result is tested and analyzed.Calculate its variation lines
Number, the coefficient of variation (β)=standard deviation (SD)/average (x), as a result as shown in table 3：
Crowd interior repetition Tab.3 Intra-assay reproducibility of of the PEDV real-time fluorescence quantitative PCRs of table 3
PEDV TaqMan real-time fluorescent quantitative PCR
As shown in Table 3,2.4 × 107、2.4×106With 2.4 × 105The coefficient of variation of copies/ μ L RNA standard samples
Respectively 0.60%, 0.76%, 0.55% is respectively less than 1%, shows the TaqMan real-time fluorescence quantitative PCRs detection that this experiment is established
PDDV method has good repeatability in criticizing.
9th, the detection of clinical sample
185 parts of the ground swine excrement samples such as 2015-2017 collections Tianshui city, Xinjiang, Henan, Hainan, Xianyang, Hunan
Product, the method established with the present invention detects to the swine excrement sample gathered, while also carries out conventional RT-PCR to sample
Method detects, and sets up negative and positive control.Testing result is compareed with conventional RT-PCR, conventional RT-PCR specificity
Primer is carried out, and specific method illustrates to carry out referring to RT-PCR kit operating method.Testing result is as shown in table 4：
Testing result Tab.4 ResuLts of clinical of the PEDV real-time fluorescence quantitative PCRs of table 4 to clinical sample
sample detection by PEDV TaqMan real-time fluorescent quantitative PCR
As shown in Table 4：50 parts of positives are detected using conventional RT-PCR detection method, positive rate 27.56%, used
TaqMan real-time fluorescence quantitative RT-PCRs detection method of the present invention detects 71 parts of positives, positive rate 38.37%.Illustrate this
The established TaqMan real-time fluorescence quantitative RT-PCR detection methods of invention have higher sensitiveness.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (4)
1. Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kits, it is characterised in that the kit, which includes, to be drawn
Thing and probe；
The sequence of the primer is：
Forward primer：5'-ACTACCTCGGAACAGGACCTCA-3'；
Reverse primer：5'-AGACGCCTTTCTGACACCCA-3'；
The sequence of the probe is：
5'-FAM-CGCCGACCTCCGCTATAGGACTCGT-BHQ1-3'。
2. Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kits according to claim 1, its feature exist
In the concentration of the primer is 0.2mol/L, and the concentration of probe is 0.15mol/L.
3. Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR detecting methods, it is characterised in that the detection method makes
With the Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kits described in claim 1 or 2, detection method include with
Under several steps：
(1) RNA in testing sample is extracted；Negative control is set；
(2) RNA standard items are prepared；
(3) TaqMan fluorescence quantitative RT-RCRs amplified reaction：
Primer, the probe used during amplification be respectively：
Forward primer：5'-ACTACCTCGGAACAGGACCTCA-3'；
Reverse primer：5'-AGACGCCTTTCTGACACCCA-3'；
Probe：5'-FAM-CGCCGACCTCCGCTATAGGACTCGT-BHQ1-3'；
Amplification reaction system：2 × RT-PCR buffer, 10 μ L, E × Taq HS 0.4 μ L, the μ L of reverse transcriptase mixture 0.4, just
To the μ L of primer 0.4, the μ L of reverse primer 0.4, probe 0.6 μ L, no μ L of RNase water 5.8；
Take the μ L of RNA 2 in step (1) and the μ L of negative control 2 to be separately added into the amplification reaction system that total amount is 20 μ L, will match somebody with somebody
The PCR pipe for having put reaction system preheats 5min in 42 DEG C, 95 DEG C of pre-degeneration 10s, 95 DEG C of denaturation 5s, 57 DEG C of annealing extension 20s, becomes
Property with annealing process extension totally 40 circulation；
(4) result detects：Software is carried by quantitative real time PCR Instrument and reads corresponding Ct values, judges that testing sample is positive or cloudy
Property.
4. Porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR detecting methods according to claim 3, its feature exists
In the criterion of the testing sample positive or negative is：
(1) it is positive：Sample Ct value≤35.0 are detected, and curve has obvious Exponential growth stage；
(2) it is suspicious：Sample Ct values ＞ 35.0 is detected, and the sample of typical amplification curve occurs, carries out repeating experiment；
(3) it is negative：Sample Ct values are can't detect, and without obvious amplification curve.
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| CN108998568A (en) * | 2018-07-10 | 2018-12-14 | 广西壮族自治区兽医研究所 | A kind of the duplex RT-PCR detection primer and kit of quick differentiation PEDV and PReoV |
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| CN111440902A (en) * | 2020-05-29 | 2020-07-24 | 华南农业大学 | Porcine epidemic diarrhea virus detection primer, kit and application thereof |
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| CN112391497A (en) * | 2020-11-30 | 2021-02-23 | 山东绿都生物科技有限公司 | Primer probe set, application thereof and kit for detecting African swine fever virus and porcine epidemic diarrhea virus |
| CN113943831A (en) * | 2021-10-14 | 2022-01-18 | 泰州蕾灵百奥生物科技有限公司 | Multiple fluorescent quantitative primer and probe combination capable of diagnosing three high-incidence pathogens of porcine diarrhea disease simultaneously and application thereof |
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