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CN106957926A - A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox - Google Patents

A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox Download PDF

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CN106957926A
CN106957926A CN201710241966.8A CN201710241966A CN106957926A CN 106957926 A CN106957926 A CN 106957926A CN 201710241966 A CN201710241966 A CN 201710241966A CN 106957926 A CN106957926 A CN 106957926A
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primer
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csfv
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史喜菊
马贵平
柏亚铎
刘艳华
高峰
刘全国
李炎鑫
高志强
李冰玲
张玉红
郭敏卓
刘凌
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Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
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Abstract

The invention discloses a kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox.The primer and probe such as sequence table SEQ ID NO：1 to SEQ ID NO：Shown in 16.The invention also discloses it is quick, accurate, easy to use while detect and differentiate the one-step method multi-fluorescence RT PCR detection kits of classic swine fever, African swine fever and swine pox.The detection method of the present invention, which can be realized, is once sampled, and once analyzes, can detect and distinguish simultaneously 3 kinds of important viral purposes, not only reduces the workload and cost of detection, and the detection of epidemic disease can be completed within the most short time, is that disease prevention and cure gain time.

Description

A kind of inspection that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox Test agent box and primer and probe
Technical field
Can simultaneously be detected the present invention relates to one kind and differentiate classic CSFV (CSFV), African swine fever virus (ASFV) and The one-step method multi-fluorescence RT-PCR detection kit and primer and probe of swine vesicular disease virus (SVDV), can be achieved once to sample, Once analyze, while 3 kinds of viral purposes of detection and differentiation, belong to inspection and quarantine field.
Background technology
Classic CSFV (CSFV), African swine fever virus (ASFV) and swine vesicular disease virus (SVDV) are the acute height of pig Communicable disease, pathogeneticing characteristic is to generate heat, septic change based on, clinical symptoms are similar, defficulty in diagnosing, be harm cultivation Industry the most serious several cause of diseases.Swine fever, African swine fever and swine pox are defined as a variety of by OIE (OIE) It is animal infected, transboundary propagate cause of disease, be also important quarantine object in China's animal and animal's products international trade.
At present, different kinds of molecules detection technique, such as PCR and Fluorescence PCR assay are established for above-mentioned several cause of diseases, at this Important function has been played in the diagnosis and prevention and control of a little epidemic diseases.But the method set up at present is to be directed to a kind of single goal of cause of disease mostly Detection, it is actually detected it is middle needs repeatedly, could complete detect Different Kinds of Pathogens purpose, detect workload greatly and the time It is long, it is impossible to which that meet that high-volume animal quarantine speeds passage through customs is actually needed.And be difficult to largely to exist in actual sample it is mixed Close infection and the antidiastole of the similar epidemic disease of symptom.While single goal Pathogen test technology is set up, animal doctor mechanism of various countries Also have developed in succession can be while detect a variety of viral multiplex PCRs, but traditional PCR technique sensitivity is low, result is difficult to judge etc. Shortcoming limits its application in Multiple detection.
The characteristic such as special, sensitive that this research and utilization Fluorescence PCR assay has in terms of detection of nucleic acids, establish CSFV, The viral one-step method multi-fluorescence RT-PCR detections of ASFV and tri- kinds of SVDV and discrimination method are simultaneously assembled into kit, it is possible to achieve one Secondary sampling, is once analyzed, and can be detected and be distinguished simultaneously the purpose of 3 boars virus, not only reduce the workload and cost of detection, And the detection of epidemic disease can be completed within the most short time, it is that disease prevention and cure gain time.
The content of the invention
First purpose of the present invention be to provide high specificity, sensitivity it is high while detect and differentiate classic CSFV (CSFV), the multi-fluorescence RT-PCR primer and probe of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV).
It is a further object to provide it is quick, accurate, easy to use while detect and differentiate classic hog cholera Malicious (CSFV), African swine fever virus (ASFV) and swine vesicular disease virus (SVDV) one-step method multi-fluorescence RT-PCR detection reagents Box.
To achieve the above object, the present invention uses following technical scheme：
On the basis of sequence alignment analysis, for the classic UTR of CSFV 5 ' genes, African swine fever virus VP72 genes With the UTR genes of swine vesicular disease virus 5 ', most of separation strains can be covered and be adapted to Multiple detection again and can enter by separately designing and filtering out Primer and probe that row differentiates (sequence is shown in Table 1).Classic CSFV (CSFV) detection primer to by 3 forward primers, 2 it is anti- To primer and 2 probe compositions, African swine fever virus (ASFV) primer pair is by 2 forward primers, 3 reverse primers and 1 spy Pin is constituted, and swine vesicular disease virus (SVDV) primer pair is made up of 1 forward primer, 1 reverse primer and 1 probe, multiple anti- The mixture that primer mixture in system is this 12 primers compositions is answered, probe is the mixture of this 4 probes compositions, by one Footwork multi-fluorescence RT-PCR reacts, it is possible to achieve detects and differentiates while three kinds of cause of diseases.
The one-step method multi-fluorescence RT-PCR detections of the classic CSFV of table 1, African swine fever virus and swine vesicular disease virus Primer
Note：Cytimidine (C), guanine (G), adenine (A), thymidine (T).Degeneracy base W represents A or T.Probe 5 ' end is marked with luminous group, and ' end is marked with quenching group to probe 3.
We are established using multiple fluorescence PCR technology while detecting and differentiating classic CSFV, African swine fever virus With the detection method of swine vesicular disease virus and assemble kit.
Detection and the one-step method multi-fluorescence RT- for differentiating classic CSFV, African swine fever virus and swine vesicular disease virus PCR detection kit, including above-mentioned multi-fluorescence RT-PCR detection primers and probe.Further, the kit has also included Material and reagent needed for being reacted into multi-fluorescence RT-PCR, for example, reaction buffer, enzyme, positive control, negative control etc..
In the specific embodiment of the present invention, the reaction solution is Multiplex RT-PCR reaction buffers； The enzyme is Multiplex enzymes；The negative control is free nucleic acid aqua sterilisa；The positive control is respectively classic CSFV (CSFV) the in-vitro transcription cRNA of 5 ' UTR genes, the extracorporeal recombinant DNA of African swine fever virus (ASFV) VP72 genes, pig bubble The extracorporeal recombinant DNA of virus (SVDV) 5 ' UTR genes.
Present invention also offers it is a kind of detect and differentiate classic CSFV, African swine fever virus, swine vesicular disease virus One-step method multi-fluorescence RT-PCR detection method, comprises the following steps：
(1) viral total nucleic acid is extracted；
(2) RT-PCR reaction systems are prepared, wherein, above-mentioned multi-fluorescence RT-PCR primer is included in the reaction system And probe；
(3) one-step method multi-fluorescence RT-PCR reactions are carried out；
(4) result judgement；
1. quality control standard：
Positive control has S-shaped amplification curve, and CT values are about 20 or so, and negative control is without amplified signal.
This condition is met, is considered as and this time test effectively.
2. result judges：
There is amplification curve, and judgement of the CT values below 30 is the positive；
There are amplification curve, but 30<CT values<35, belong to doubtful interval, it is necessary to reaffirm；
There is no amplification curve or CT values to be more than 35, be determined as feminine gender.
It is an advantage of the invention that：1) multiplicity：It can simultaneously detect and distinguish classic CSFV, African swine fever virus, pig Vesicular disease virus, has saved detection time and cost, is conducive to making a definite diagnosis in time for epidemic disease particularly mixed infection.2) it is sensitive：This Multi-fluorescence RT-PCR systems can detect three kinds of cause of diseases simultaneously, and its is specific suitable with single fluorescence RT-PCR.3) it is special：If Meter primer and interference that may be present between cause of disease has been taken into full account during probe, the special of primer and probe is ensure that by screening Property and exclusiveness.3) covering property and versatility are good：The primer and probe designed in the present invention is all most protected for each virus respectively The gene design kept, for each virus, its primer and probe are all a plurality of, and contain degenerate primer, are substantially covered Existing this kind viral all sequences in Genebank.4) easy, easily automation：The design of all primers and probe be all from Multiple detection consideration can be carried out, although have DNA virus and RNA virus, but the one-step method program can be used, eliminated anti- Transcription, it is time saving and energy saving.
Brief description of the drawings
Fig. 1：The mono- fluorescence RT-PCR standard curves of CSFV.
Fig. 2：The mono- fluorescent PCR standard curves of ASFV.
Fig. 3：The mono- fluorescence RT-PCR standard curves of SVDV.
Fig. 4：CSFV, ASFV and SVDV multi-fluorescence RT-PCR standard curves.
Fig. 5：The comparison of the mono- fluorescence RT-PCRs of CSFV and multi-fluorescence RT-PCR.
Fig. 6：The comparison of the mono- fluorescence RT-PCRs of ASFV and multi-fluorescence RT-PCR.
Fig. 7：The comparison of the mono- fluorescence RT-PCRs of SVDV and multi-fluorescence RT-PCR.
Embodiment
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, and this should not be limited with this The protection domain of invention.
Method therefor is conventional method, test material used, such as without special unless otherwise instructed in following embodiments Illustrate, be routine biochemistry reagent suppliers and be commercially available.
The composition of the kit of embodiment 1 and use
1. the preparation composition of kit
The composition of the kit of table 2
Wherein 2 × Multiplex RT-PCR buffer solutions and Multiplex enzymes are purchased from AB Products Path-ID TM Multiplex one-step RT-PCR kit。
3 forward primers of primer mixture including CSFV, 2 reverse primers, ASFV 2 forward primers, 3 reversely 1 forward primer and 1 reverse primer of primer and SVDV, primer sequence such as table 1, the working concentration of mix primer is 400nM, the precious biotech firm's synthesis in primer commission Dalian.
2 probes, ASFV and the SVDV that CSFV probe mixtures include CSFV have 1 probe, probe sequence such as table respectively 1, concentration is respectively 200nM, the precious biotech firm's synthesis in probe commission Dalian.
Negative control is free nucleic acid aqua sterilisa.
CSFV positive controls are the in-vitro transcription cRNA of classic CSFV (CSFV) 5 ' UTR genes；ASFV positive controls For the extracorporeal recombinant DNA of African swine fever virus (ASFV) VP72 genes；SVDV positive controls are swine vesicular disease virus (SVDV) 5 ' The extracorporeal recombinant DNA of UTR genes.
The classic UTR gene in-vitro transcriptions RNA of CSFV (CSFV) 5 ' preparation：Reclaim the UTR genes of CSFV 5 ' RT-PCR amplified productions, length 400bp is attached with pGEM-T carriers (being purchased from Promega companies), conversion JM109 impressions State cell, alkaline lysis method of extracting plasmid DNA obtains positive recombinant plasmid after PCR and digestion identification, is named as pGEM-CSFV- 5’UTR.Using the plasmid of purifying as template, after plasmid linearization, with the Ribo MAXTM Large of Promega companies Scale RNA Production System-T7 kits carry out in-vitro transcription；In-vitro transcription product is removed it with DNAse In DNA profiling after extracted through TRIZOL after be measured after, that is, obtain the in-vitro transcription RNA of the UTR genes of CSFV 5 ', order Entitled CSFV-5 ' UTR-cRNA；SEQ ID NO in the UTR of CSFV 5 ' gene orders such as sequence table：Shown in 17.
The preparation of African swine fever virus (ASFV) VP72 gene extracorporeal recombinant DNAs：External synthesis African swine fever virus VP72 Gene, length is 500bp, is attached with pGEM-T carriers (being purchased from Promega companies), converts JM109 competent cells, alkali Cracking process extracts DNA, obtains positive recombinant plasmid after PCR and digestion identification, pGEM-ASFV-VP72 is named as, through pure Concentration mensuration is carried out after change, that is, obtains the extracorporeal recombinant DNA of African swine fever virus VP72 genes, is named as ASFV-VP72-DNA； SEQ ID NO in African swine fever virus VP72 gene orders such as sequence table：Shown in 18.
The preparation of the UTR gene extracorporeal recombinant DNAs of swine vesicular disease virus (SVDV) 5 '：External synthesis swine vesicular disease virus 5 ' UTR genes, length is 500bp, is attached with pGEM-T carriers (being purchased from Promega companies), conversion JM109 competence is thin Born of the same parents, alkaline lysis method of extracting plasmid DNA obtains positive recombinant plasmid after PCR and digestion identification, is named as pGEM-SVDV-5 ' UTR, purified rear progress concentration mensuration, that is, obtain the extracorporeal recombinant DNA of the UTR genes of swine vesicular disease virus 5 ', be named as SVDV- 5’UTR-DNA；SEQ ID NO in the UTR of swine vesicular disease virus 5 ' gene orders such as sequence table：Shown in 19.
2. the application method of kit
1) extraction of viral total nucleic acid (RNA/DNA)
The extraction of viral nucleic acid uses virus genom DNA/RNA extracts kits (TIANamp Virus DNA/RNA Kit), step is as follows：
The preparation of Carrier RNA solutions：
(1) Carrier RNA storing liquids：310 μ L are added into the pipe equipped with 310 μ g Carrier RNA freeze-dried powders RNase-Free ddH2O, Carrier RNA are thoroughly dissolved, and obtain final concentration of 1 μ g/ μ L solution, and dispense, be placed in- 20 DEG C of storages.
(2) Carrier RNA working solutions：Buffer solution GB and the Carrier RNA solution according to needed for being calculated the quantity of sample Volume (computing formula is as follows), mix buffer solution GB and Carrier RNA solutions are reverse, that is, obtain Carrier RNA works Make liquid；There is foaming phenomena to avoid solution, vortex oscillation please don't be used.
Computing formula：
N × 0.22mL=y mL
The μ L/mL=z μ L of y mL × 28
The sample number that n=is extracted simultaneously in formula, y=needs to add buffer solution GB volume, and z=needs to add The volume of Carrier RNA solutions.
Operating procedure：
(1) 20 μ L Proteinase K are added in a clean 1.5ml centrifuge tube with pipettor.
(2) tissue that 200 μ L plasma/serums/lymph or 25mg grind is added into centrifuge tube, and (sample need to be balanced to room Temperature), if sample volume is less than 200 μ L, 0.9%NaCl solution supplement can be added.
(3) 200 μ L Carrier RNA working solutions are added, lid is covered, vortex oscillation 15sec is mixed.
(4) 56 DEG C of incubation 15min, brief centrifugation is attached to the liquid of tube wall and lid to collect.
(5) 250 μ L absolute ethyl alcohols are added, vortex oscillation 15sec is thoroughly mixed, in (15-25 DEG C) placement 5min of room temperature.
(6) brief centrifugation, tube wall and the liquid of lid are attached to collect.
(7) solution in centrifuge tube and flocculent deposit are fully transferred to RNase-Free adsorption columns CR2 (adsorption column is placed on In collecting pipe), lid is covered, centrifugation 1min in 8,000rpm (~6,000 × g) abandons waste liquid, adsorption column is put back in collecting pipe.
(8) it is careful to open adsorption column lid, add 500 μ L solution GD and (please first checked whether before use and added anhydrous second Alcohol), lid is covered, centrifugation 1min in 8,000rpm (~6,000 × g) abandons waste liquid, adsorption column is put back into collecting pipe.
(9) it is careful to open adsorption column lid, add 600 μ L solution PW and (please first checked whether before use and added anhydrous second Alcohol), lid is covered, 2min, 8,000rpm (~6,000 × g) centrifugation 1min is stood, abandons waste liquid, adsorption column is put back into collecting pipe.
(10) repeat step (9).
(11) it is careful to open adsorption column lid, 500 μ L absolute ethyl alcohols are added, lid is covered, 8,000rpm (~6,000 × G) 1min is centrifuged, waste liquid is abandoned.
(12) adsorption column is put back in collecting pipe, centrifugation 3min in 12,000rpm (~13,400 × g) makes adsorbed film complete It is dried, abandons waste liquid.
(13) adsorption column is put into a new RNase-Free centrifuge tube (1.5ml), the careful lid for opening adsorption column Son, room temperature places 3min, is completely dried up adsorbed film.20-150 μ L RNase- are vacantly added dropwise to the middle part of adsorbed film Free ddH2O, closes the lid, and room temperature places 5min.
(14) 12,000rpm (~13,400 × g) centrifuge 1min, and what is be collected into is viral total nucleic acid (DNA or RNA).
2) fluorescence RT-PCR is detected
Detection sample, positive negative control are taken respectively, and reaction system is prepared according to table 3 below：
The one-step method multi-fluorescence RT-PCR reaction systems of table 3：Cumulative volume 20uL
After mixed liquor is sufficiently mixed, template is eventually adding, brief centrifugation, according to following response procedures, carries out one-step method Fluorescence RT-PCR reacts, and is shown in Table 4：
The multi-fluorescence RT-PCR response procedures of table 4
3) result description and judgement
1. quality control standard：
Positive control has S-shaped amplification curve, and CT values are about 20 or so, and negative control is without amplified signal.
This condition is met, is considered as and this time test effectively.
2. result judges：
There is amplification curve, and judgement of the CT values below 30 is the positive；
There are amplification curve, but 30<CT values<35, belong to doubtful interval, it is necessary to reaffirm；
There is no amplification curve or CT values to be more than 35, be determined as feminine gender.
The sensitivity tests of embodiment 2, kit
1st, material
Classic CSFV is preserved by this laboratory, and African swine fever virus and swine vesicular disease virus are vitro recombination plasmids.
2nd, method
1) preparation of positive criteria product
Method as described in Example 1.
2) standard items are quantitative determined
The aqua sterilisa of the in-vitro transcription cRNA prepared and extracorporeal recombinant DNA without RNase is taken to make 200 times of dilutions respectively, Determine its 260nm and 280nm absorbance (OD260 and OD280) with ultraviolet spectrometer, calculate the concentration of testing sample with it is pure Degree.Pure dna：(＞ 1.9 shows there are RNA pollutions to OD260/OD280 ≈ 1.8；＜ 1.6, shows there is the pollution such as protein, phenol；＞ Show there may be isothiocyanic acid remaining when 2.0)；Pure rna：1.7 ＜ OD260/OD280 ＜ 2.0 (show there is protein during ＜ 1.7 Or phenol pollution；Show there may be isothiocyanic acid remaining during ＞ 2.0).The concentration (μ g/ μ L) of DNA sample：OD260 × extension rate ×50/1000；The concentration (μ g/ μ L) of RNA sample：OD260 × extension rate × 40/1000, and copy is calculated as follows Number (Copies/ μ L)：
Specification Curve of Increasing：
Will CSFV Standard in vitro product carry out 10 times of gradient dilutions after carry out fluorescence RT-PCR, with template concentrations it is reciprocal with it is corresponding Fluorescence CT values do concentration standard curve, PCR amplification efficiency E values, coefficient R2Value and slope of curve Slope values all fall can In the range of receiving, illustrate that amplification efficiency is good.See Fig. 1.
Will ASFV Standard in vitro product carry out 10 times of gradient dilutions after carry out fluorescence RT-PCR, with template concentrations it is reciprocal with it is corresponding Fluorescence CT values do concentration standard curve, PCR amplification efficiency E values, coefficient R2Value and slope of curve Slope values all fall can In the range of receiving, illustrate that amplification efficiency is good.See Fig. 2.
Will SVDV Standard in vitro product carry out 10 times of gradient dilutions after carry out fluorescence RT-PCR, with template concentrations it is reciprocal with it is corresponding Fluorescence CT values do concentration standard curve, PCR amplification efficiency E values, coefficient R2Value and slope of curve Slope values all fall can In the range of receiving, illustrate that amplification efficiency is good.See Fig. 3.
After CSFV, ASFV and SVDV Standard in vitro product mixed in equal amounts, then 10 times of gradient dilutions are carried out, carry out multi-fluorescence RT-PCR, with corresponding fluorescence CT values concentration standard curve, PCR amplification efficiency E values, coefficient R are done with template concentrations are reciprocal2 Value and slope of curve Slope values all fall in tolerance interval, illustrate not interfere with each other between multiplex PCR, amplification efficiency is good It is good.See Fig. 4.
3) determination of one-step method multi-fluorescence RT-PCR method test limit
Above-mentioned quantitative RNA solution is adjusted to concentration roughly the same, it is every kind of respectively to take 10 μ L, after fully mixing, sun is made Property standard items.After 10 times of gradient dilutions of the standard items, fluorescence RT-PCR detection, highest extension rate institute are carried out by optimum condition The concentration as minimum detection limit corresponding to CT values that can be detected.
3rd, result
1) external DNA/RNA solution copy number measurement results
By the extracorporeal recombinant DNA prepared or reverse transcription RNA, its 260nm and 280nm is determined with ultraviolet spectrometer respectively Absorbance and extrapolate copy number, refer to table 5 below.
The DNA/RNA purity of table 5 and assay
2) determination of multi-fluorescence RT-PCR method test limit
Using the multi-fluorescence RT-PCR detection method of foundation, 10 times of positive criteria products being serially diluted are detected, As a result this method can the highest CT values that arrive of stable detection be 35, corresponding standard concentration reaches 1-10 copy numbers/reaction.
The specific test of embodiment 3, kit
1 material
Used virus such as table 6 in this experiment.
The virus and nucleic acid being applied in the specific test research process of table 6
Virus Source
Classic CSFV (CSFV) This laboratory is preserved
African swine fever virus (ASFV) United States Department of Agriculture's exotic disease center
Swine vesicular disease virus vitro recombination plasmid It is prepared by this laboratory
Porcine circovirus 2 type (PCV-2) This laboratory is preserved
Pseudorabies virus (PRV) This laboratory is preserved
Pig parvoviral (PPV) This laboratory is preserved
Infectious gastroenteritis virus (TGEV) This laboratory is preserved
2nd, method
2.1 respectively with the primer and probe of classic CSFV, African swine fever virus and swine vesicular disease virus in form Other 6 kinds of viral nucleic acids carry out fluorescence RT-PCR detections, to verify the specificity of primer and probe.
2.2 are detected using the multi-fluorescence RT-PCR detection method of foundation to 7 kinds of viruses in form, are verified multiple The specificity of fluorescence RT-PCR method.
2.3 carry out the comparison of single fluorescence RT-PCR and multi-fluorescence RT-PCR.
3rd, result
3.1 every kind of viral primers and probe can only amplify curve from the viral nucleic acid of oneself, other viral templates Amplification be feminine gender, show that designed primer and probe specificity are good.
3.2 using foundation multi-fluorescence RT-PCR method in form 6 virus detect, as a result CSFV, African swine fever virus and swine pox vitro recombination plasmid have amplification curve, and other viruses are without specific amplified signal, table Bright set up method specificity is good.
The specific detection result of table 7
3.3 by after CSFV, ASFV and SVDV Standard in vitro product mixed in equal amounts, then carries out 10 times of gradient dilutions, carries out respectively The mono- fluorescent PCRs of multi-fluorescence RT-PCR and CSFV, the corresponding fluorescence CT values of same template concentration are compared, and solve recurrence side Journey, therebetween coefficient R2Value has reached 0.9988, meets between instruction sheet fluorescence RT-PCR and multi-fluorescence RT-PCR Property it is good, multi-fluorescence RT-PCR does not produce interference, does not influence amplification efficiency.See Fig. 5.
After CSFV, ASFV and SVDV Standard in vitro product mixed in equal amounts, then 10 times of gradient dilutions are carried out, carried out respectively multiple Fluorescence RT-PCR and the mono- fluorescent PCRs of ASFV, the corresponding fluorescence CT values of same template concentration are compared, and solve regression equation, Therebetween coefficient R2Value has reached 0.9978, and accordance is good between instruction sheet fluorescence RT-PCR and multi-fluorescence RT-PCR Good, multi-fluorescence RT-PCR does not produce interference, does not influence amplification efficiency.See Fig. 6.
After CSFV, ASFV and SVDV Standard in vitro product mixed in equal amounts, then 10 times of gradient dilutions are carried out, carried out respectively multiple Fluorescence RT-PCR and the mono- fluorescent PCRs of SVDV, the corresponding fluorescence CT values of same template concentration are compared, and solve regression equation, Therebetween coefficient R2Value has reached 0.9976, and accordance is good between instruction sheet fluorescence RT-PCR and multi-fluorescence RT-PCR Good, multi-fluorescence RT-PCR does not produce interference, does not influence amplification efficiency.See Fig. 7.
Embodiment 4：The laboratory report that kit is detected to clinical sample
1st, material
25 parts of the known CSFV positives of laboratory collection, 15 parts of PRV positives, 10 parts of PPV positives, FMDV 35 parts of 8 parts of positive, 9 parts of PRRSV positives and other unknown samples.
2nd, method
Multi-fluorescence RT-PCR detections are carried out to above-mentioned sample, with the Sensitivity and Specificity of actual sample verification method.
3rd, result
The testing result of clinical sample such as table 8.As shown in Table 8, CSFV positives testing result is known to 25 parts CSFV is positive, and the detection of other samples is all feminine gender, coincidence rate 100%.Regrettably, it can not find Africa on domestic market Swine fever and swine pox positive, but the detection coincidence rate of vitro recombination plasmid is also 100%.
The testing result of the clinical sample of table 8
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair Row of the obvious change or variation that bright technical scheme is extended out still in protection scope of the present invention.
SEQUENCE LISTING
<110>Inspection and Quarantine Technology Center, Beijing Entry-Exit Inspection and Q
<120>A kind of detection kit and primer that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox
And probe
<130>
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial synthesized primer
<400> 1
agcccacctc gagatgcta 19
<210> 2
<211> 21
<212> DNA
<213>Artificial synthesized primer
<400> 2
agcccacctc gatatgctat g 21
<210> 3
<211> 21
<212> DNA
<213>Artificial synthesized primer
<400> 3
agctcacctc gagatgctat g 21
<210> 4
<211> 23
<212> DNA
<213>Artificial synthesized primer
<400> 4
ctatcaggtc gtactcccat cac 23
<210> 5
<211> 21
<212> DNA
<213>Artificial synthesized primer
<400> 5
tatcaggtcg tacccccatc a 21
<210> 6
<211> 19
<212> DNA
<213>Artificial synthesized probe
<400> 6
acgagggcaw gcccaagac 19
<210> 7
<211> 17
<212> DNA
<213>Artificial synthesized probe
<400> 7
acgagggcac gcccaag 17
<210> 8
<211> 24
<212> DNA
<213>Artificial synthesized primer
<400> 8
gcgatgatga ttacctttgc tttg 24
<210> 9
<211> 24
<212> DNA
<213>Artificial synthesized primer
<400> 9
cgatgatgat taccttcgct ttga 24
<210> 10
<211> 21
<212> DNA
<213>Artificial synthesized primer
<400> 10
cgataccaca agatcagccg t 21
<210> 11
<211> 22
<212> DNA
<213>Artificial synthesized primer
<400> 11
ctgataccac aagatcagcc gt 22
<210> 12
<211> 20
<212> DNA
<213>Artificial synthesized primer
<400> 12
gataccacaa gatcggccgt 20
<210> 13
<211> 22
<212> DNA
<213>Artificial synthesized probe
<400> 13
cacgggagga ataccaaccc ag 22
<210> 14
<211> 17
<212> DNA
<213>Artificial synthesized primer
<400> 14
tcctccggcc cctgaat 17
<210> 15
<211> 21
<212> DNA
<213>Artificial synthesized primer
<400> 15
acacccaaag tagtcggttc c 21
<210> 16
<211> 20
<212> DNA
<213>Artificial synthesized probe
<400> 16
caccagtggg cagtctgtcg 20
<210> 17
<211> 400
<212> DNA
<213>CSFV 5'UTR gene orders
<400> 17
gtatacgagg ttagttcatt ctcgtatgca tgattggaca aattaaaatt tcaatttgga 60
tcagggcctc cctccagcga cggccgaact gggctagcca tgcccacagt aggactagca 120
aacggaggga ctagccgtag tggcgagctc cctgggtggt ctaagtcctg agtacaggac 180
agtcgtcagt agttcgacgt gagcagaagc ccacctcgag atgctatgtg gacgagggca 240
tgcccaagac acaccttaac cctagcgggg gtcgctaggg tgaaatcaca ccacgtgatg 300
ggagtacgac ctgatagggt gctgcagagg cccactatta ggctagtata aaaatctctg 360
ctgtacatgg cacatggagt tgaatcattt tgaactttta 400
<210> 18
<211> 500
<212> DNA
<213>ASFV VP72 gene orders
<400> 18
aatcctcatc aacaccgaga ttggcacaag ttcggacatg ttgttaacgc cattatgcag 60
cctactcacc acgcagagat aagctttcag gatagagata cagctcttcc agacgcatgt 120
tcatctatat cggatattag ccccgttacg tatccgatca cattacctat tattaaaaac 180
atttccgtaa ctgctcatgg tatcaatctt atcgataagt ttccatcaaa gttctgcagc 240
tcttacatac ccttccacta cggaggcaat gcaattaaaa cccccgatga tccgggtgcg 300
atgatgatta cctttgcttt gaagccacgg gaggaatacc aacccagtgg tcatattaac 360
gtatccagag caagagaatt ttatattagt tgggacacgg attacgtggg gtctatcact 420
acggctgatc ttgtggtatc ggcatctgct attaactttc ttcttcttca gaacggttca 480
gctgtgctgc gttacagtac 500
<210> 19
<211> 500
<212> DNA
<213>SVDV 5'UTR gene orders
<400> 19
cccggactga gtaccaatag gctgctcacg cggctgaagg ggaaaccgtt cgttacccga 60
ctaactactt cgagaaacct agtaccacca tgaaagttgc gcagcgtttc gctccgcaca 120
accccagtgt agatcaggtc gatgagtcac cgcaaacccc acgggcgacc gtggcggtgg 180
ctgcgctggc ggcctgccca tggggcaact catgggacgc ttcaatactg acatggtgcg 240
aagagtctat tgagctagtt ggtagtcctc cggcccctga atgcggctaa tcctaactgc 300
ggagcagata cccacgcacc agtgggcagt ctgtcgtaat gggcaactct gcagcggaac 360
cgactacttt gggtgtccgt gtttcctttt gttcttatac tggctactta tggtgacaat 420
tgagagattg taaccatatt gctattggat tggccacccg gcgacgaata gaacagttgc 480
ttacctgttt gttagtctcg 500

Claims (7)

1. one group is used to detecting and differentiating that the one-step method of classic CSFV, African swine fever virus and swine vesicular disease virus is more simultaneously Weight fluorescence RT-PCR primer and probe, it is characterised in that the primer and probe are respectively：
3 forward primers for detecting classic CSFV, such as sequence table SEQ ID NO：1 to SEQ ID NO：Shown in 3,2 Bar reverse primer, such as sequence table SEQ ID NO：4 to SEQ ID NO：Shown in 5, and 2 probes, such as sequence table SEQ ID NO：6 To SEQ ID NO：Shown in 7；
2 forward primers for detecting African swine fever virus, such as sequence table SEQ ID NO：8 to SEQ ID NO：Shown in 9,3 Bar reverse primer, such as sequence table SEQ ID NO：10 to SEQ ID NO：Shown in 12, and 1 probe, such as sequence table SEQ ID NO：Shown in 13；
1 forward primer for detecting swine vesicular disease virus, such as sequence table SEQ ID NO：Shown in 14,1 reverse primer, such as Sequence table SEQ ID NO：Shown in 15, and 1 probe, such as sequence table SEQ ID NO：Shown in 16.
2. one-step method multi-fluorescence RT-PCR primer according to claim 1 and probe, it is characterised in that described to be used to examine Survey 5 ' end flag F AM, 3 ' end mark BHQ1 of the probe of classic CSFV；The probe for being used to detect African swine fever virus 5 ' end mark HEX, 3 ' end mark BHQ1；5 ' the end mark Cy5,3 ' end marks for being used to detect the probe of swine vesicular disease virus Remember BHQ2.
3. it is a kind of while the kit of detection and the classic CSFV of discriminating, African swine fever virus and swine vesicular disease virus, it is special Levy and be, including one-step method multi-fluorescence RT-PCR primer and probe described in claim 1 or 2.
4. kit according to claim 3, it is characterised in that the kit also include RT-PCR reaction buffers, Enzyme, positive control and negative control.
5. kit according to claim 4, it is characterised in that the positive control is respectively classic CSFV 5 ' The in-vitro transcription cRNA of UTR genes, the extracorporeal recombinant DNA of African swine fever virus VP72 genes, the UTR genes of swine vesicular disease virus 5 ' Extracorporeal recombinant DNA.
6. kit according to claim 3, it is characterised in that the RT-PCR reaction buffers are Multiplex RT-PCR reaction buffers, the enzyme is Multiplex enzymes.
7. it is a kind of while detection and differentiate the detection method of classic CSFV, African swine fever virus and swine vesicular disease virus, its It is characterised by：This method includes：
(1) viral total nucleic acid is extracted；
(2) RT-PCR reaction systems are prepared, wherein, the multi-fluorescence described in claim 1 or 2 is included in the reaction system RT-PCR primer and probe；
(3) one-step method multi-fluorescence RT-PCR reactions are carried out；
(4) result judgement.
CN201710241966.8A 2017-04-14 2017-04-14 A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox Pending CN106957926A (en)

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CN110760620A (en) * 2019-12-12 2020-02-07 黑龙江八一农垦大学 Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method
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Application publication date: 20170718