CN110184393A - A kind of primer sets, purposes, kit and its detection method detecting African swine fever virus - Google Patents
A kind of primer sets, purposes, kit and its detection method detecting African swine fever virus Download PDFInfo
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Abstract
The invention belongs to African swine fever virus detection technique fields, and in particular to a kind of primer sets, purposes, kit and its detection method for detecting African swine fever virus.Primer sets include Bio-FIP, FITC-BIP, F3 and B3, and the nucleotide sequence of Bio-FIP is as shown in SEQ ID NO.1, and the nucleotide sequence of FITC-BIP is as shown in SEQ ID NO.2；The nucleotide sequence of F3 is as shown in SEQ ID NO.3, and the nucleotide sequence of B3 is as shown in SEQ ID NO.4.It is an advantage of the current invention that the primer group-specific is good, using the kit and its detection method specificity of primer sets preparation, good, high sensitivity, minimum to can detecte to 100.6The template of copy, 10000 times sensitiveer than conventional OIE-PCR, and detection method implementation process provided by the invention is simple to operation, can be used in the on-site test of ASFV, has great importance for the prevention and control of ASFV.
Description
Technical field
The invention belongs to swine fever detection technique fields, and in particular to a kind of primer sets for detecting African swine fever virus, simultaneously
It is related to purposes, the kit using primer sets preparation and the inspection using kit detection African swine fever virus of the primer sets
Survey method.
Background technique
African swine fever (African swine fever, ASF) is a kind of disease only propagated between domestic pig and wild boar
Viral disease, pathogen are African swine fever virus (African Swine fever virus, ASFV), are African swine fever disease
The unique member that malicious section, African swine fever virus belong to is a kind of large-scale, coated double-stranded DNA virus of film.Nineteen twenty-one, African swine fever
It is broken out in the domestic pig of Kenya for the first time, the country on the south the Sahara is then propagated to rapidly by domestic pig, and in 20th century 50
Age propagates to African western part, and the African Territories still on the south the Sahara are popular so far.2007, there is Africa in Georgia
Swine fever epidemic situation, then epidemic situation propagates to surrounding countries and further propagates to European east quickly.To 2018, African swine fever epidemic disease
Feelings propagate to the Russian Federation and its neighbouring country, including Ukraine, Poland, Latvia, Lithuania, love across Caucasus region
Sha Niya, Moldova, Czech Republic and Romania, and on the eastern region of the Russian Federation and the side of Mongolia
There is a situation where domestic pigs to infect African swine fever in border.2018, there is African swine fever epidemic situation in LiaoNing, China province, and sequencing result shows this
The p72 gene of strain is the same as 2007/1 plant of the Georgia of Georgia, 2012 strain of Krasnodar and Irkutsk of Russia
2017 strains and Estonian Estonia 2014isolated strain are identical, it was demonstrated that the African swine fever of China's outburst
Epidemic situation is probably spread by Russia.By on March 1st, 2019, China shared 28 provinces and breaks out 111 Africa altogether
Swine fever epidemic situation, under the stringent prevention and control measure in China, the African swine fever epidemic situation situation in China is gradually delayed at present, from January, 2019
Since part, 20 epidemic situations only occur, 99 of compared to parts of in August, 2018 to December, quantity sharp fall occurs for epidemic situation.
On the whole, African swine fever epidemic situation in China's is totally controllable, and prevention and control situation is gradually become better, and 100 Epidemic outbreak of disease regions have released
Block, the epidemic-stricken area in 18 provinces is all lifted a blockade.But the African swine fever situation that China faces is still to allow of no optimist,
Firstly, although African swine fever is not respiratory disease, since African swine fever virus survival ability is strong, for low temperature, drying
There is stronger resistivity, can survive in the environment up to the several months, so personnel, vehicle, feed, animal etc. can all become disease
The approach that poison is propagated, will completely cut through the propagation of African swine fever virus, need complete set, the bio-safety method of science and height
The procedure of effect, structuring operation.African swine fever epidemic situation in China's is still sporadicly broken out at present, with reference to African swine fever epidemic situation in Russia sieve
This, the long-term existence of the states such as Romania, show that fighting African swine fever will be a protracted war.
African swine fever between domestic pig have it is highly infectious, acute strain lethality is up to 100%.Due to currently without
Effective vaccine prevents African swine fever, so, for the prevention and control measure of African swine fever then based on slaughtering, block, and
A set of efficient detection method has very important significance for prevention and control tool.About the detection method of African swine fever, have very
More people study, and construct the monitoring method of a variety of viral genes.2003, King etc. used Taqman probe structure for the first time
The real time PCR detection method of detection African swine fever is built out, and was introduced into OIE handbook in 2012, this method is using VP72 as target
Point, detection are limited to 10-100 and copy every microlitre.2010, McKillen etc. used MGB probe, constructed by target spot of 9GL gene
New real time PCR detection method, detection are limited to 20 every microlitre of copies.Tignon in 2011 etc. uses beta-actin as in
Ginseng constructs new detection method, compared with the method that OIE is used, has higher sensitivity, detection is limited to be copied up to 5.7-57
Every microlitre of shellfish.2013, Fernandez-Pinero et al. was developed on the basis of already existing PCR method using general
Probe library (UPL) PCR method, this method will test limit and be reduced to 18 every microlitre of copies, further improve the sensitive of detection
Property.Although normal PCR and qPCR method have preferable specificity, and qPCR sensitivity with higher simultaneously, both
It needs to be reacted under accurate changing device, also, the monitoring needs of PCR product are realized by gel electrophoresis, these
Disadvantage limits application of the PCR method in real-time ASFV detection when participating in the cintest invariably.Isothermal amplification technique, earliest invention is in 2000
Year.The implementation of the technology only needs simple thermostatic equipment, such as water-bath, metal bath equipment etc..In recent years, for ASFV etc.
Warm amplification technique also has certain application.2010, the application such as James LAMP carried out ASFV detection, and detection is limited to 330 copies
Every microlitre or so.2017,Deng use PCLSR method, detection be limited to 720 every microlitre of copies.The same year,
J.Wang et al. constructs the isothermal amplification detection method using RPA method, and this method detection is limited to 100 every microlitre of copies.This
A little isothermal amplification methods can be rapidly performed by live ASFV detection, but sensitivity is lower.Therefore, be badly in need of develop one kind can be fast
The carry out scene ASFV detection of speed, and easy to operate, high sensitivity detection method.
Summary of the invention
In order to solve the above problems existing in the present technology, it is an object of that present invention to provide a kind of detection African swine fever viruses
Primer sets, while be related to the primer sets purposes, using the primer sets preparation kit and using the kit detect it is non-
The detection method of continent swine fever virus.
Specific technical solution is as follows:
The present invention provides a kind of primer sets for detecting African swine fever virus, and the primer sets include Bio-FIP, FITC-
BIP, F3 and B3, the nucleotide sequence of the Bio-FIP is as shown in SEQ ID NO.1, the nucleotide sequence of the FITC-BIP
As shown in SEQ ID NO.2；The nucleotide sequence of the F3 is as shown in SEQ IDNO.3, the nucleotide sequence of the B3 such as SEQ
Shown in ID NO.4.
The present invention also provides a kind of kits for detecting African swine fever virus, including primer sets above-mentioned, LAMP amplification
Reagent, positive quality control product and negative quality-control product, the positive quality control product are artificial synthesized African swine fever virus DNA, the yin
Property quality-control product be distilled water.
Preferably, the final concentration of the kit of above-mentioned detection African swine fever virus, the Bio-FIP and FITC-BIP is
1.6 μM, the final concentration of the F3 and B3 are 0.2 μM.
Preferably, the kit of above-mentioned detection African swine fever virus, the LAMP amplifing reagent include concentration be 10 ×
Isothermal Amplification Buffer, the LAMP amplifing reagent further includes MgSO4、Bst 2.0WarmStart
Archaeal dna polymerase, dATP, dGTP, dTTP and dCTP.
Preferably, the kit of above-mentioned detection African swine fever virus further includes LAMP reaction reagent, the LAMP reaction examination
Agent is HybriDetectAssay Buffer.
Preferably, the kit of above-mentioned detection African swine fever virus further includes lateral flow test strips.
The present invention also provides a kind of detection methods with aforementioned agents box detection African swine fever virus, including following step
It is rapid:
(1) prepare DNA sample to be measured: extracting the DNA of sample to be tested；
(2) reaction solution of three groups of LAMP is prepared: including detection group, negative control group and positive controls, three groups of LAMP's
In reaction solution include LAMP amplifing reagent, primer sets above-mentioned, template and distilled water, the detection group, positive controls and
The template of negative control group is followed successively by DNA sample, positive quality control product and distilled water to be measured respectively；
(3) amplified reaction: mixing centrifugation for three groups of LAMP reaction solutions prepared in step (2) respectively, expanded, point
Three groups of LAMP reaction solutions are not obtained；
(4) it detects: three groups of LAMP reaction solutions being detected respectively, judging result.
Specifically, the detection method of above-mentioned detection African swine fever virus, the reaction condition of amplified reaction in the step (3)
For 65 DEG C of isothermal reaction 40min.
Specifically, the detection method of above-mentioned detection African swine fever virus, the volume of LAMP reaction solution is in the step (2)
25μL；The sample-adding amount of each component or final concentration are as follows in every group of LAMP reaction solution: 10 × Isothermal
Amplification Buffer is 2.5 μ L, MgSO4For 2mM, Bio-FIP and FITC-BIP be 1.6 μM each, F3 and each 0.2 μ of B3
M, Bst 2.0WarmStart archaeal dna polymerase 8U, 1 μ l of template, each 1.4mM of dATP, dGTP, dTTP and dCTP, surplus are
ddH2O。
The present invention also provides a kind of aforementioned primer sets in detection African swine fever virus or preparation detection African swine fever virus
Kit in application.
The invention has the benefit that
Primer group-specific provided by the invention is good, utilizes the kit and its detection method specificity of primer sets preparation
Good, high sensitivity is minimum to can detecte to 100.6(4) template is copied, and detection method provided by the invention (LAMP-LFD) is real
It applies that process is simple to operation, can be used in the on-site test of ASFV, have great importance for the prevention and control of ASFV.
Detailed description of the invention
Fig. 1 is African swine fever virus B646L phylogenetic analysis.
Fig. 2 is the specific detection result of the detection method of African swine fever virus provided by the invention.
Fig. 3 is that the LAMP-LFD sensitivity of the detection method of OIE-PCR and African swine fever virus provided by the invention compares
As a result.
Specific embodiment
Test example does further explaination to the present invention with reference to the accompanying drawing and specifically.
Embodiment 1:
Two pairs of specific primers being designed to provide for detecting African swine fever virus of this test example.
According to the genome sequence of ASFV disclosed on NCBI, using 6 software of MEGA to 42 plants of ASFV whole genome sequences
(number of genome sequence used is shown in Fig. 1 and table 2) is compared, is then carried out using Simplot program 3.5.1
Similarity plotting is analyzed to determine the conservative region in ASFV genome.According to similarity plotting points
Analysis as a result, the conservative region in B646L gene is set by PrimerExplorer V4 software (Fujitsu, Tokyo, Japan)
Count LAMP primer.Design two pairs of primers altogether according to design of primers principle and LAMP amplification technique principle, including Bio-FIP and
FITC-BIP, F3 and B3, the nucleotide sequence of Bio-FIP is as shown in SEQ ID NO.1, and the nucleotide sequence of FITC-BIP is such as
Shown in SEQID NO.2；The nucleotide sequence of F3 is as shown in SEQ ID NO.3, the nucleotide sequence of B3 such as SEQID NO.4 institute
Show.Two pairs of specific primers of design are sent to biotech firm and are synthesized, Bio-FIP, FITC-BIP, F3 and B3 are respectively obtained, it is right
Obtained Bio-FIP, FITC-BIP, F3 and B3 carry out gene sequencing verifying confirmation.Bio-FIP, FITC-BIP, F3 and B3's
Sequence is specifically as shown in table 1:
1 list of primers of table
Test example 2
Being designed to provide for this test example is a kind of for detecting the kit of African swine fever virus.
It is a kind of for detecting the kit of African swine fever virus, primer sets, LAMP amplifing reagent, sun including test example 1
Property quality-control product and negative quality-control product, positive quality control product be DNA, virus liquid or the cDNA of African swine fever virus, negative quality-control product is
Distilled water.
Test example 3
Being designed to provide for this test example is a kind of for detecting the kit of African swine fever virus.
It is a kind of for detecting the kit of African swine fever virus, including following components:
(1) primer sets: Bio-FIP, FITC-BIP, F3 and B3, each primer sequence is referring to table 1, Bio-FIP and FITC-BIP
Final concentration be 1.6 μM, the final concentration of F3 and B3 are 0.2 μM；
(2) LAMP amplifing reagent: Isothermal Amplification Buffer (10 ×), MgSO4、
Bst2.0WarmStart archaeal dna polymerase, dATP, dGTP, dTTP and dCTP；
(3) LAMP reaction reagent: HybriDetectAssay Buffer (Milenia biotec, Gie β en,
Germany)；
(4) positive quality control product is artificial synthesized African swine fever virus DNA, and African swine fever virus is specially China/
2018/AnhuiXCGQ, Tengani 62, Warmbaths, Mkuzi 1979, NHV, Lil-20/1, Ken05/Tk1 and R7 (tool
Body information is shown in Table the 2) strain and other have been identified as the strain of African swine fever virus；
(5) negative quality-control product is distilled water；
(6) lateral flow test strips (LFD) (Milenia biotec, Gie β en, Germany).
Test example 4
A kind of detection method for being designed to provide African swine fever virus of this test example simultaneously verifies its specificity, that is, tests
The detection method of the detection African swine fever virus of kit described in example 3.
1 materials and methods
Strain and sequence used in 1.1 test examples
B646L (p72) gene for the 42 plants of ASFV strains announced on selection NCBI carries out phylogenetic analysis (specifying information
As shown in table 2), as a result as shown in Figure 1.Systematic evolution tree as shown in Figure 1 is it is found that 42 ASFV strains are divided into 4 branches.
Then, pick out be distributed in 4 branches 8 plants of strains (including China/2018/AnhuiXCGQ, Tengani 62,
Warmbaths, Mkuzi 1979, NHV, Lil-20/1, Ken05/Tk1 and R7), in Shanghai raw work it is artificial synthesized they
B646L gene order.In addition, the also artificial synthesized genome sequence of 1 PCV bacterial strain, for detection method in the present invention
Special Journal of Sex Research.It is separately added into BamH I and Sal I restriction enzyme site in the end 5' and 3' of all artificial synthesized sequences, and will
These artificial synthesized sequences are cloned into respectively in pEASY-T1 carrier (Transgen, China).
Table 2
Classic swine fever virus, porcine rotavirus disease used in this test example, Porcine epidemic diarrhea virus, pig transmissible stomach and intestine
Scorching virus, blue otopathy poison, Delt- coronavirus, Pseudorabies virus and pig parvoviral are respectively stored in -80 DEG C.
In addition, further to verify the detection method for the African swine fever virus that this test example provides and other pig viral templates
Between whether cross reaction can occur, that is, verify its specificity, this test example is also by classic swine fever virus, porcine rotavirus, pig
Epidemic diarrhea virus, transmissible gastro-enteritis virus, blue otopathy poison and Delt- coronavirus cDNA, porcine pseudorabies virus
It is used as template (table 3) with the DNA of pig parvoviral and the pig circular ring virus genome sequence of synthesis and carries out LAMP-LFD reaction,
CDNA and DNA preparation method is as shown in the present embodiment 1.2.
Other virus stains used in 3 the present embodiment of table
| Species | Strain | No. GenBank | Species | Strain | No. GenBank |
| CSFV | Thiverval | EU490425.1 | Delt-CoV | CH/SCMS/2017 | NA |
| RVA | NX | NA | PCV | MiSD-1 | KP282147.1 |
| PEDV | CV777 | NA | PRV | Bartha | JF797217.1 |
| TGEV | HUA | NA | PPV | N | HM989009.1 |
| PRRSV | SCcd17 | MG914067 |
Note:aCSFV, classic African swine fever virus；RVA, porcine rotavirus；PEDV, Porcine epidemic diarrhea virus；TGEV,
Transmissible gastro-enteritis virus；PRRSV, blue otopathy poison；Delt-CoV, Delt- coronavirus；PCV, pig circular ring virus；PRV,
Pseudorabies virus；PPV, pig parvoviral.NA is not committed to GenBank.
The extraction of 1.2 DNA, RNA and cDNA synthesis
DNA is mentioned using Universal Genomic DNA Kit (ComWin Biotech, Beijing, China)
It takes, the specific steps are as follows:
1. taking 200 μ l of virus liquid, 20 μ l Proteinase K are added thereto；
2. be added 200 μ l Buffer GL, be vortexed concussion mixes well, 56 DEG C water-bath 10 minutes；
3. of short duration be centrifuged to remove the droplet of cap wall, 200 μ l dehydrated alcohols are added, the concussion that is vortexed mixes well, short
Temporarily centrifugation；
4. acquired solution in step 3 is all added in the adsorption column (Spin ColumnsDM) for having been charged into collecting pipe,
If cannot once add solution, can be transferred to several times；12,000rpm centrifugations 1 minute, outwell the waste liquid in collecting pipe, will adsorb
Column places back in collecting pipe；
5. 500 μ l Buffer GW1 (whether preoperation inspection has been added dehydrated alcohol) are added into adsorption column, 12,
000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
6. 500 μ l Buffer GW2 (whether preoperation inspection has been added dehydrated alcohol) are added into adsorption column, 12,
000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
7. 2,000rpm centrifugations 2 minutes, outwell waste liquid in collecting pipe；Adsorption column is placed in several minutes of room temperature, thoroughly to dry in the air
It is dry；
8. adsorption column is placed in a new centrifuge tube, 100 μ l aqua sterilisas are vacantly added to the intermediate position of adsorption column,
5 minutes are placed at room temperature for, DNA solution, -20 DEG C of preservation DNA are collected in 12,000rpm centrifugations 1 minute.
RNA is extracted using TRIzol (Invitrogen, California, USA), the specific steps are as follows:
1. 300 μ L virus allantoic fluids and 4 DEG C of the 700 μ l RNAlso Plus being pre-chilled mildly are mixed, it is stored at room temperature 5min；
2. 200 μ l chloroforms are added, after oscillation mixes, it is stored at room temperature 15min, 12000r/min, 4 DEG C of centrifugation 10min；
3. abandoning precipitating, supernatant is moved in new sterile centrifugation tube, the isopropanol of 4 DEG C of 700 μ l pre-coolings is added, room temperature is quiet
Set 10min；
4. 7500r/min, 4 DEG C of centrifugation 10min；
5. supernatant is abandoned, after washing precipitating is resuspended with the ethyl alcohol of 1ml 75%, 12000r/min, 4 DEG C of centrifugation 15min；
6. after abandoning supernatant, centrifuge tube opening is air-dried precipitating in super-clean bench；
It is precipitated 7. being dissolved with suitable RNase-free water, -20 DEG C save backup.
The virus total RNA obtained using previous step is template, according to the PrimeScript RTreagent of TakaRa company
Kit reverse transcription reagent box standard step synthesizes cDNA, and reaction system is as shown in the table:
4 reverse transcription system of table
| Component | Volume |
| Total serum IgE | 8.4μl |
| 5×PrimeScript Buffer | 2μl |
| Oligo dT Primer | 0.5μl |
| Random 6mers | 0.5μl |
| PrimeScript RT Enzyme Mix1 | 0.5μl |
After reaction system mixes in PCR pipe, it is placed in PCR instrument, condition are as follows: 37 DEG C, 15min；85 DEG C, 5s.CDNA product
- 20 DEG C are placed in save backup.
1.3 LAMP-LFD reaction, the i.e. detection method of kit described in test example 3
(1) DNA or cDNA of sample to be tested are prepared by method shown in the present embodiment 1.2；
Sample to be tested include: 8 kinds of ASFV viruses (China/2018/AnhuiXC GQ, 62 Tengani, Warmbaths,
Mkuzi 1979, NHV, Lil-20/1, Ken05/Tk1 and R7, specifying information are as shown in table 2), classic swine fever virus, pig colyliform
Virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, blue otopathy poison and Delt- coronavirus, porcine pseudorabies
Poison, pig parvoviral and pig circular ring virus, specifically as shown in 1.1 part of the present embodiment；
(2) reaction liquid of LAMP-LFD is prepared:
(contain in the reaction liquid of LAMP-LFD containing 2.5 μ l 10 × Isothermal Amplification Buffer
There is 2mM MgSO4)、MgSO42mM, dNTP (dATP, dGTP, dTTP and dCTP) each 1.4mM, Bio-FIP and FITC-BIP is each
1.6 μM, F3 and B3 is 0.2 μM each, 1 μ l of Bst 2.0WarmStart archaeal dna polymerase 8U and template, then plus ddH2O is by reaction volume
Polishing is 25 μ l.Wherein, the template of sample to be tested group is the DNA or cDNA of ASFV and other viruses, the mould of negative control (NC)
Plate is distilled water.
Specific sample-adding system is as follows:
5 LAMP-LFD of table reacts sample-adding system
| Reagent | Sample-adding amount or final concentration |
| 10×Isothermal Amplification Buffer | 2.5μl |
| MgSO4 | 2mM |
| DNTP (dATP, dGTP, dTTP and dCTP) | Each 1.4mM |
| Bst 2.0WarmStart archaeal dna polymerase | 8U |
| F3/B3 | 0.2μM |
| Bio-FIP or FITC-BIP | 1.6μM |
| Template | 1μl |
| ddH2O | Polishing is to 25 μ l |
(3) amplified reaction: by LAMP reaction solution prepared in step (2) respectively at 65 DEG C of reaction 40min, LAMP is obtained
Reaction product takes each 20 μ l of reaction product respectively, and 80 μ lHybriDetectAssay Buffer (Milenia are added to it
Biotec, Gie β en, Germany), it is mixed in new centrifuge tube, then by lateral flow test strips (LFD) (Milenia
Biotec, Gie β en, Germany) it immerses in the mixture.After 3-5 minutes, i.e. observable result.
2 results
As a result as shown in Fig. 2, being followed successively by ASFV strain (ASFV strains), other viruses in Fig. 2 from left to right
(other Viruses) and negative control (NC).Wherein, ASFV strain (ASFV strains) is followed successively by China/ from left to right
2018/AnhuiXC GQ, 62 Tengani, Warmbaths, Mkuzi 1979, NHV, Lil-20/1, Ken05/Tk1 and R7；Its
It is popular that his viral (other Viruses) is followed successively by classic swine fever virus (CSFV), porcine rotavirus (RVA), pig from left to right
Property diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), blue otopathy poison (PPRSV), Delt- coronavirus (Delt-
CoV), pig circular ring virus (PCV), Pseudorabies virus (PRV) and pig parvoviral (PPV).It can be seen that after 3-5 minutes
Observe detection line.When ASFV sequence is as template, LAMP-LFD is LAMP- positive, that this test example provides as the result is shown
LFD primer can be reacted (ASFV strains) with the ASFV template of 8 plants of different genotypes, have versatility.And work as it
When his viral nucleic acid sequence is as template, false positive results are had no.It can be seen that LAMP-LFD has the special of height to ASFV
Property, it does not react with other viruses outside ASFV.
Test example 5
The purpose of this test example is the sensitivity of the detection method for the African swine fever virus that verification test example 3 provides.
Operating procedure is as follows:
(1) DNA sample to be measured is prepared:
The genetic fragment for the B646L that 10 times are serially diluted determines detection limit as template, according to DNA copy number.It will take
With B646L segmentCarrier is digested with BamH I and Sal I, and reaction system is as follows:
Table 6
| Reagent | Volume |
| Carry the carrier of B646L | 40μl |
| BamH I/Sal I | 1μl |
| 10×restriction enzymes Buffer | 5μl |
| ddH2O | 3μl |
| Total volume | 50μl |
Reaction condition: 37 DEG C of reaction 2h.Then, B646L segment and linearized vector are separated by 1% agarose electrophoresis,
And use TIANgel Midi Purification Kit (TIANGEN BIOTECH, Beijing, China) by B646L segment
It is extracted from Ago-Gel, the specific steps are as follows: DNA cuts purpose band after gel electrophoresis separates from gel, transfer
Into 1.5mL sterile centrifugation tube, weighing is added solution PN of its quality with respect to 3 times of volumes, places 10min in 50 DEG C of water-baths, to
Agar melts completely；Solution is transferred in adsorption column CA2 (CA2 is put in collecting pipe), is placed at room temperature for 2min, 12000r/min from
Heart 1min abandons waste liquid, and 600 μ L rinsing liquid PW, 12000r/min are added into CA2 and are centrifuged 1min, repeat to rinse primary；Abandon waste liquid
Afterwards, CA2 is put back in collecting pipe, 12000r/min is centrifuged 2min, and the remaining PW of reject is placed at room temperature for 5min；CA2 is transferred to nothing
In the 1.5mL centrifuge tube of bacterium, 20 μ L deionized waters are added, are placed at room temperature for 2min, 12000r/min is centrifuged 2min, the solution of collection
In i.e. contain corresponding target fragment；- 20 DEG C are placed in save backup.
Using the concentration of the B646L segment of NANO DROP 2000 (Thermo, Shanghai, China) measurement purifying, and
And the copy number of B646L segment: copy number (copies/ul)=[DNA concentration (DNA ng/ul)/segment is big is calculated according to the following formula
Small (bp)] × 9.12 × 1011.Determining B646L fragment concentrations after the recovery is 108.6Copy/μ l.Then, by purifying
B646L segment is from 108.6To 100.6Copy/μ l carries out gradient dilution, shown in table specific as follows:
Table 7
(2) PCR and LAMP-LFD reaction
The primer (being shown in Table 1) that this test example selects International Office of Epizootics (OIE) to recommend is in sensitivity test as reference side
Method compares the detection limit of the detection method (LAMP-LFD) of OIE-PCR and kit provided by the invention.
PCR reaction: the routine PCR reaction mixture that OIE recommends includes 12.5 μ 2 × M5Pfu of l PCRMasterMix
(Mei5, Beijing, China), each 10pmol of upstream and downstream primer (being specifically shown in Table 1), 1 μ l of DNA (template), surplus is distilled water
(ddH2O), final volume is 25 μ l.PCR parameter include 94 DEG C initial denaturation step 5 minutes, 94 DEG C be denaturalized 50 seconds, annealing 50
Second, 72 DEG C extend 10 seconds, carry out 32 circulations, and 72 DEG C extend 10 minutes.DNA (template) is the B646L segment through gradient dilution.
The template of negative control (NC) is distilled water.
LAMP-LFD reaction: contain 2.5 μ l 10 × Isothermal Amplification Buffer in reaction liquid
(contain 2mM MgSO4)、MgSO42mM, dNTP each 1.4mM, Bio-FIP and FITC-BIP are 1.6 μM each, F3 and B3 are 0.2 μM each,
1 μ l of Bst 2.0WarmStart archaeal dna polymerase 8U and template, then plus ddH2Reaction volume polishing is 25 μ l by O.Wherein, template
For the B646L segment through gradient dilution, the template of negative control (NC) is distilled water.By aforementioned prepared LAMP reaction solution point
Not in 65 DEG C of reaction 40min, LAMP reaction product is obtained, takes each 20 μ l of reaction product respectively, 80 μ are added to it
LHybriDetectAssay Buffer (Milenia biotec, Gie β en, Germany), mixes in new centrifuge tube, with
Lateral flow test strips (LFD) (Milenia biotec, Gie β en, Germany) are immersed in the mixture afterwards.After 3-5 minutes,
That is observable result.
(3) result
The target fragment size of the result figure that upper figure is OIE-PCR in Fig. 3, OIE-PCR amplification is 278bp, and Marker is
DNA molecular amount standard items, it is template used from left to right to be followed successively by 108.6Copy/μ l, 107.6Copy/μ l, 106.6Copy/μ l,
105.6Copy/μ l, 104.6Copy/μ l, 103.6Copy/μ l, 102.6Copy/μ l, 101.6Copy/μ l and 100.6Copy/μ l
B646L segment, the last one swimming lane are negative control (NC).The following figure is LAMP-LFD result figure in Fig. 3, from left to right mould used
Plate is followed successively by 108.6Copy/μ l, 107.6Copy/μ l, 106.6Copy/μ l, 105.6Copy/μ l, 104.6Copy/μ l, 103.6Copy/
μl、102.6Copy/μ l, 101.6Copy/μ l and 100.6Copy/μ l B646L segment, the last one lateral flow test strips are feminine gender
It compares (NC).
As a result as shown in figure 3, LAMP-LFD is minimum to can detecte 100.6A template is copied, OIE-PCR is minimum to be can detecte
104.6The template of copy.It can be seen that sensitive 10000 times of LAMP-LFD ratio OIE-PCR provided by the invention.
The present invention is not limited to above-mentioned optional embodiment, anyone can show that other are each under the inspiration of the present invention
The product of kind form.Above-mentioned specific embodiment should not be understood the limitation of pairs of protection scope of the present invention, protection of the invention
Range should be subject to be defined in claims, and specification can be used for interpreting the claims.
Sequence table
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Claims (10)
1. it is a kind of detect African swine fever virus primer sets, it is characterised in that: the primer sets include Bio-FIP, FITC-BIP,
The nucleotide sequence of F3 and B3, the Bio-FIP are as shown in SEQ ID NO.1, the nucleotide sequence of the FITC-BIP such as SEQ
Shown in ID NO.2；The nucleotide sequence of the F3 is as shown in SEQ ID NO.3, the nucleotide sequence of the B3 such as SEQ ID
Shown in NO.4.
2. a kind of kit for detecting African swine fever virus, it is characterised in that: including primer sets described in claim 1, LAMP
Amplifing reagent, positive quality control product and negative quality-control product, the positive quality control product are artificial synthesized African swine fever virus DNA, institute
Stating negative quality-control product is distilled water.
3. it is according to claim 2 detection African swine fever virus kit, it is characterised in that: the Bio-FIP and
The final concentration of FITC-BIP is 1.6 μM, and the final concentration of the F3 and B3 are 0.2 μM.
4. the kit of detection African swine fever virus according to claim 2 or 3, it is characterised in that: the LAMP amplification
Reagent include concentration be 10 × Isothermal Amplification Buffer, the LAMP amplifing reagent further includes
MgSO4, Bst 2.0WarmStart archaeal dna polymerase, dATP, dGTP, dTTP and dCTP.
5. the kit of detection African swine fever virus according to claim 4, it is characterised in that: further include LAMP reaction examination
Agent, the LAMP reaction reagent are HybriDetectAssay Buffer.
6. the kit of detection African swine fever virus according to claim 5, it is characterised in that: further include lateral flow examination
Paper slip.
7. a kind of detection method of the detection of the kit described in claim 2-6 any one African swine fever virus, feature
It is, comprising the following steps:
(1) prepare DNA sample to be measured: extracting the DNA of sample to be tested；
(2) reaction solution of three groups of LAMP is prepared: including detection group, negative control group and positive controls, the reaction of three groups of LAMP
Including primer sets, template and distilled water described in LAMP amplifing reagent, claim 1 in liquid, the detection group, the positive are right
It is followed successively by DNA sample, positive quality control product and distilled water to be measured respectively according to the template of group and negative control group；
(3) amplified reaction: mixing centrifugation for three groups of LAMP reaction solutions prepared in step (2) respectively, expanded, respectively
To three groups of LAMP reaction solutions；
(4) it detects: three groups of LAMP reaction solutions being detected respectively, judging result.
8. the detection method of detection African swine fever virus according to claim 7, it is characterised in that: in the step (3)
The reaction condition of amplified reaction is 65 DEG C；Isothermal reaction 40min.
9. the detection method of detection African swine fever virus according to claim 8, it is characterised in that: in the step (2)
The volume of LAMP reaction solution is 25 μ L；The sample-adding amount of each component or final concentration are as follows in every group of LAMP reaction solution: 10 ×
Isothermal Amplification Buffer is 2.5 μ L；MgSO4It is each 1.6 μM of 2mM, Bio-FIP and FITC-BIP；F3
Each 0.2 μM with B3；Bst 2.0WarmStart archaeal dna polymerase 8U；1 μ l of template；Each 1.4mM of dATP, dGTP, dTTP and dCTP；
Surplus is ddH2O。
10. a kind of primer sets described in claim 1 are in the examination of detection African swine fever virus or preparation detection African swine fever virus
Application in agent box.
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| CN110777220A (en) * | 2019-11-18 | 2020-02-11 | 华南农业大学 | Primer group, probe, RPA test strip kit and identification method |
| CN111270019A (en) * | 2020-04-07 | 2020-06-12 | 北京市动物疫病预防控制中心 | Primer group for detecting African swine fever virus, fluorescence visualization rapid detection kit and method |
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| CN111304361A (en) * | 2019-11-04 | 2020-06-19 | 浙江大学 | Kit for detecting African swine fever virus and method for detecting African swine fever virus |
| CN110777220A (en) * | 2019-11-18 | 2020-02-11 | 华南农业大学 | Primer group, probe, RPA test strip kit and identification method |
| CN111270019A (en) * | 2020-04-07 | 2020-06-12 | 北京市动物疫病预防控制中心 | Primer group for detecting African swine fever virus, fluorescence visualization rapid detection kit and method |
| CN113215320A (en) * | 2021-05-25 | 2021-08-06 | 龙岩学院 | Primer probe combination and kit for African swine fever virus and reference gene dual-fluorescence PCR detection |
| CN113981051A (en) * | 2021-11-23 | 2022-01-28 | 厦门倍博特医学科技有限公司 | Primer group, kit and method for detecting HLA-DPB 0202 gene |
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Application publication date: 20190830 |