CN110777220A - Primer group, probe, RPA test strip kit and identification method - Google Patents
Primer group, probe, RPA test strip kit and identification method Download PDFInfo
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Abstract
The invention belongs to the technical field of virus strain identification methods, and particularly relates to a primer group and a probe for detecting a wild strain of African swine fever virus, which comprise a first primer group and a first probe; the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification; the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2; the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3. Meanwhile, a primer group and a probe for detecting MGF360-505R gene deletion strains are also provided; the primer group and the probe group have strong specificity and high sensitivity, and simultaneously, the invention also provides an RPA test strip kit and an identification method based on the RPA-LFD technology and the primer group and the probe group.
Description
Technical Field
The invention relates to the technical field of virus strain identification methods, in particular to a primer group, a probe, an RPA test strip kit and an identification method.
Background
African Swine Fever (ASF) is an acute and highly-contagious infectious disease of domestic pigs and wild pigs caused by African Swine Fever Virus (ASFV), is clinically characterized by high fever, loss of appetite and bleeding of skin and internal organs, generally dies 2-10 days after illness, and the death rate can reach 100% in severe cases. The disease belongs to a group A animal epidemic disease which must be reported by the requirements of the world animal health Organization (OIE), and is classified as an animal epidemic disease in China. African swine fever is prevalent in tens of countries in africa, europe and america, causing significant economic losses and has spread to many asian countries bordering europe.
Since the first African swine fever outbreaks in our country in 2018, the African swine fever causes significant loss to the pig industry in China, and the healthy development of the pig industry in China is seriously influenced. Domestic and foreign researches show that after the African swine fever virus MGF360-505R gene is knocked out, the virus toxicity can be obviously reduced, and the African swine fever live virus with the two knocked-out genes is expected to become a vaccine. At present, CN201910348878.7 and CN 201910700685.3 respectively disclose a gene-deleted attenuated African swine fever virus which can be used as a vaccine, the vaccine and a construction method thereof, and the virulence gene of the African swine fever virus is deleted by a genetic engineering technology to obtain the gene-deleted virus with MGF360-505R deletion and combined deletion of CD2V and MGF 360-505R. Experiments show that the two strains can provide 100 percent immune protection for the Chinese epidemic virulent strain of the African swine fever, can be used as a safe and effective vaccine for preventing and controlling the epidemic situation of the Chinese African swine fever, and has great social value. However, the use of the vaccine can lead the result that the conventional method for detecting the African swine fever virus is jun positive, and the result that the African swine fever virus is positive due to wild virus infection or positive due to vaccination cannot be distinguished.
At present, ASF diagnosis methods include direct immunofluorescence experiments, indirect immunofluorescence experiments, methods for detecting viral nucleic acids and the like. Among them, the nucleic acid detection method is widely used because of its simple operation and high sensitivity. The Recombinase Polymerase Amplification (RPA) technique can accurately detect ASFV in a shorter time than these conventional detection techniques. RPA technology relies primarily on three enzymes: recombinase (recombination), single-stranded binding protein, and DNA polymerase. The whole RPA reaction process is very fast, and a large amount of target products can be amplified after the reaction is carried out for 20 minutes at a constant 37 ℃. The RPA reaction product can be rapidly detected by a flow-through immunodipstick (LFD). The RPA-LFD technology has the advantages of strong specificity, simple operation process, no initial heating step, high sensitivity, rapid detection and the like, and provides a possibility for detecting the African swine fever in the field.
Therefore, the technical problem to be solved by the application is as follows: how to provide a primer group, a probe, an RPA test strip kit and an identification method for identifying African swine fever virus wild strains and MGF360-505R gene deletion strains based on an RPA-LFD technology.
Disclosure of Invention
The invention aims to provide an RPA detection primer group and a probe group for distinguishing an African swine fever virus wild strain and an MGF360-505R gene deletion strain, the primer group and the probe group have strong specificity and high sensitivity, and meanwhile, the invention also provides an RPA test strip kit and an identification method based on the RPA-LFD technology and the primer group and the probe group.
Unless otherwise specified, all the% and parts in the present invention are weight percentages and parts, and M represents mol/L.
In order to achieve the purpose, the invention provides the following technical scheme:
a primer group and a probe for detecting a wild strain of African swine fever virus comprise a first primer group and a first probe;
the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1, the upstream primer we named: ASFV-MGF 360-505R-F1;
ASFV-MGF360-505R-F1：5’TGTGCTATTGCCCATAAGGATCTACATCTAT3’(SEQ ID NO：1)；
the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2; this downstream primer we named: ASFV-MGF 360-505R-R1;
ASFV-MGF360-505R-R1：5’Biotin-CATACTCAGAATGCCTATTATATTTGTTGAATTG 3’(SEQ ID NO：2)；
the first probe is a probe which is provided with carboxyl fluorescein at the 5 'end and C3-spacer group at the 3' end and has the sequence shown in SEQ ID NO: 3. The probe is named ASFV-MGF 360-505R-P1;
ASFV-MGF360-505R-P1：5’[FAM]TAACAGAATCGTACCCGATAAGTATCATCA[THF]TTTAGATATTCGCAT[C3-spacer]3’(SEQ ID NO：3)；
wherein biotin represents biotin, FAM represents carboxyfluorescein, THF represents a tetrahydrofuran linker, and C3-spacer means 3 methylene groups for preventing chain extension.
Meanwhile, the invention provides a primer group and a probe for detecting MGF360-505R gene deletion strains, which comprise a second primer group and a second probe;
the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification; the upstream primer is named as ASFV-MGF360-505 RQS-F2;
ASFV-MGF360-505RQS-F2：5’TATTTAATCATTTAGAGAAGGTCATCATAGGAG 3’(SEQ IDNO：4)；
the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure; the downstream primer is named as ASFV-MGF360-505 RQS-R2;
ASFV-MGF360-505RQS-R2：5’Biotin-CAGGATACGATTCACTACAATAGTGAGTAC 3’(SEQID NO：5)；
the second probe is a probe which has a carboxyl fluorescein at the 5 'end and a C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6; this second probe we named ASFV-MGF360-505 RQS-P2;
ASFV-MGF360-505RQS-P2:5’[FAM]TTCAACGAGCAGGAAACAACTGTGTGCTTA[THF]TACAGCAACATACCC[C3-space r]3’(SEQ ID NO：6)。
wherein biotin represents biotin, FAM represents carboxyfluorescein, THF represents a tetrahydrofuran linker, and C3-spacer means 3 methylene groups for preventing chain extension.
Meanwhile, the invention provides an RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains, which comprises:
a first primer set and a first probe, a second primer set and a second probe;
the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;
the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;
the first probe is a probe which is provided with carboxyl fluorescein at the 5 'end and C3-spacer group at the 3' end and has the sequence shown in SEQ ID NO: 3, a DNA molecule shown in seq id no;
the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;
the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;
the second probe is a probe which has a carboxyl fluorescein at the 5 'end and a C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.
In addition, the kit further comprises: a positive control and a negative control;
preferably, the positive control is plasmid DNA containing African swine fever virus MGF360-505R gene; the negative control is deionized water.
The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain further comprises a DNA extraction reagent, an RPA amplification reagent and a lateral flow test strip.
The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain further comprises magnesium acetate and ddH
2O、5×Extration Buffer。
Finally, the invention also provides a rapid distinguishing method for identifying the African swine fever virus wild strain and the MGF360-505R gene deletion strain by the RPA-LFD technology, which comprises the following steps:
s1: extracting viral DNA from the sample;
s2: placing the extracted virus DNA into an RPA reaction system by using the first primer group and the first probe, and the second primer group and the second probe respectively as described in claim 3 to perform RPA reaction, and obtaining amplification products A1 and A2 respectively
S3, fully reacting the amplification products A1 and A2 in the step S2 with the 5 × extraction Buffer respectively to obtain products B1 and B2;
s4, respectively dripping the products B1 and B2 obtained in the step 3 into the sample adding holes (P1 and P2 test strips) of the lateral flow test strip of claim 3, carrying out LFD reaction, observing the result and determining the virus type.
And respectively dripping the amplification products B1 and B2 onto the sample adding holes of the test strip, wherein the detection results are as follows:
1) when the test strip detection area of the P1 shows color, the quality control area also shows color; the detection area of P2 is colored, and the quality control area is also colored, so that the virus in the sample to be detected is the mixed infection of wild strains and MGF360-505R gene deletion strains;
2) when the test strip detection area of the P1 shows color, the quality control area also shows color; the detection area of P2 does not develop color, and the quality control area develops color, so that the virus in the sample to be detected is infected by wild strains;
3) when the test strip detection area of the P1 strip does not develop color, and the quality control area develops color; the detection area of P2 is developed, and the quality control area is also developed, so that the virus in the sample to be detected is MGF360-505R gene deletion strain;
4) when the test strip detection area of the P1 strip does not develop color, and the quality control area develops color; and (3) the detection area of the P2 does not develop color, and the quality control area develops color, so that the sample to be detected does not contain viruses.
5) When the quality control area does not develop color, the test strip is ineffective, and a new test strip is needed for re-determination.
In the above method, step S2 specifically includes:
taking 2.1 mu L of each of the upstream primer and the downstream primer in the first primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of first probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template, adding the mixture into an RPA enzyme tube after mixing, shaking and uniformly mixing, adding 2.5 mu L of magnesium acetate, and carrying out initial reaction to obtain an amplification product A1;
taking 2.1 mu L of each of the upstream primer and the downstream primer in the second primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of second probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template are mixed, added into an RPA enzyme tube and uniformly shaken, and then 2.5 mu L of magnesium acetate is added to start the reaction to obtain an amplification product A2.
In the above method, in the step S3, the volumes of the amplification product a1 and the amplification product a2 are 10 μ L; the 5 × extraction Buffer was 80 μ L.
In the above method, in the step S4, the volume of the products B1 and B2 is 75 μ L, and the result is read after the sample is loaded into the sample loading well for at least 5 min.
In the above method, in step S4, the RPA enzyme tube contains recombinase uvs X, single-strand binding protein, and DNA polymerase; the RPA reaction conditions in step S2 are: the reaction system is placed in a water bath kettle at 37 ℃ for reaction for 20min without high-temperature denaturation, annealing and extension. .
Compared with the prior art, the invention has the beneficial effects that:
1. recombinase Polymerase Amplification (RPA) is a method of amplifying a target fragment using a specific primer pair and a probe under isothermal conditions. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes. The flow measurement immune test strip technology (LFD) is used for carrying out test strip detection on an RPA amplification product, the detection result of the amplification product on a lateral flow test strip can be observed within a few minutes, complex instruments and equipment are not needed, and the LFD is suitable for on-site rapid detection.
2. According to the project, the regions of the primers and the probes are skillfully designed, so that whether a detected sample is infected by the African swine fever virus or not and whether an infected strain has gene deletion or not can be simply, conveniently and rapidly identified, whether mixed infection of a wild strain and a gene deletion strain exists or not can be judged, and the result can be observed by naked eyes through detection of a test strip.
3. The method has the advantages of simple operation, strong specificity, high sensitivity and the like, only generates specific amplification reaction on the DNA of African Swine Fever Virus (ASFV), and has no amplification reaction on nucleic acids of swine fever virus (CSFV), porcine pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Parvovirus (PPV) and the like.
4. The kit provides a new means for detecting the African swine fever virus in China, is convenient and fast to apply and operate clinically, has strong practicability, can be used for epidemic situation monitoring, differential diagnosis and epidemic disease purification of the African swine fever virus in production practice, can also be used for rapid identification of the African swine fever virus strain in a professional laboratory, and can provide technical support for improving the comprehensive prevention and control level of the African swine fever virus in China.
Drawings
FIG. 1 shows the results of African Swine Fever Virus (ASFV) RPA-LFD assay of example 2.
FIG. 2 shows the results of electrophoretic validation of the African Swine Fever Virus (ASFV) RPA product of example 2.
FIG. 3 shows the results of the African Swine Fever Virus (ASFV) RPA-LFD specificity test of example 3.
FIG. 4 shows the results of electrophoresis of the African Swine Fever Virus (ASFV) RPA product specificity of example 3.
FIG. 5 shows the results of African Swine Fever Virus (ASFV) RPA-LFD susceptibility assay of example 4.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Primer design
Through comparing a large number of 360-fold 505R genes of African swine fever virus MK333180.1(ASFV) published in a GenBanK database of NCBI (American national center for Biotechnology information) and the sequences of the front and the back parts thereof are highly conserved and have specific regions, the design of the front and the back 250bp of the ASFV 360-fold 505R genes and the ASFV 360-fold 505R genes are taken as specific primer pairs and probe sets.
Therefore, the RPA primer and the probe for identifying the ASFV wild strain and the 360-fold 505R gene deletion strain are provided, and specifically comprise the following steps:
primer set for detecting wild strains and probe 1:
primer set 1:
ASFV-MGF360-505R-F1：5’TGTGCTATTGCCCATAAGGATCTACATCTAT3’(SEQ ID NO：1)；
ASFV-MGF360-505R-R1：5’Biotin-CATACTCAGAATGCCTATTATATTTGTTGAATTG 3’(SEQ ID NO：2)；
1, probe 1:
ASFV-MGF360-505R-P1:5’[FAM]TAACAGAATCGTACCCGATAAGTATCATCA[THF]TTTAGATATTCGCAT[C3-spacer]3’(SEQ ID NO：3)；
the primer group and probe 2 for detecting MGF360-505R gene deletion strain are as follows:
primer set 2:
ASFV-MGF360-505RQS-F2：5’TATTTAATCATTTAGAGAAGGTCATCATAGGAG 3’(SEQ IDNO：4)；
ASFV-MGF360-505RQS-R2：5’Biotin-CAGGATACGATTCACTACAATAGTGAGTAC 3’(SEQID NO：5)；
and (3) probe 2:
ASFV-MGF360-505RQS-P2:5’[FAM]TTCAACGAGCAGGAAACAACTGTGTGCTTA[TH F]TACAGCAACATACCC[C3-spacer]3’(SEQ ID NO：6)。
wherein biotin represents biotin, FAM represents carboxyfluorescein, THF represents a tetrahydrofuran linker, and C3-spacer means 3 methylene groups for preventing chain extension.
The primer group and the probe 1 are used for amplifying a 360-fold 505R fragment of an ASFV wild strain by an RPA method, and the length of the fragment is 437bp and 381bp respectively.
The MGF360-505R gene deletion strain is amplified by the primer group and the probe 1 through an RPA method, and a target segment cannot be amplified.
MGF360-505R gene deletion strains are amplified by the primer group and the probe 2 through an RPA method, and the lengths of the fragments are 415bp and 263bp respectively.
The primer group and the probe 2 can amplify the 360-fold 505R gene of the ASFV wild strain by the RPA method, and can not amplify the target fragment.
Example 2
RPA-LFD detection method
Materials and methods
1.1 primers of example 1
1.2 sample DNA extraction
The DNA extraction has no special requirements, and can be extracted by a conventional method or a DNA extraction kit. The extracted DNA was stored at-20 ℃ for future use or immediately used for RPA amplification.
1.3 Positive plasmids
Artificially synthesizing a gene by using a part of sequence of deletion 360-plus 505R gene of ASFV published in a GenBanK database, connecting the gene with a pUC57 vector, transforming an escherichia coli competent cell DH5 α, coating the cell on an LB medium plate containing 50mg/L kanamycin sulfate, culturing for 12-16h at 37 ℃, selecting, screening, sequencing and identifying bacteria, expanding and culturing positive bacteria liquid, extracting a plasmid, and naming the positive plasmid as pUC 57-360-plus 505 RQS.
After artificially comparing the published 360-charge 505R gene and partial sequences on two sides thereof, designing a primer ASFV-360-charge 505R-F1/R1 to amplify a segment of sequence containing the 360-charge 505R gene, connecting the segment of sequence with a pJET1.2 cloning vector, transforming an Escherichia coli competent cell DH5 α, coating the transformed Escherichia coli competent cell DH5 α on an LB medium plate containing 100mg/L ampicillin, culturing for 12-16h at 37 ℃, extracting plasmids after amplifying and culturing positive bacteria liquid after screening, sequencing and identifying bacteria, and respectively naming the positive plasmids as pJET1.2-360-charge 505R.
1.4 RPA-LFD assay
The RPA-LFD experiment was performed as follows
S1, extracting virus nucleic acid from a sample according to the instruction of a nucleic acid extraction kit;
s2, taking nucleic acid as a template, and respectively carrying out RPA reaction on a sample by using the primer groups 1 and 2 and the probes 1 and 2 described in the embodiment 1 to obtain amplification products; a50. mu.L reaction was used: 2.1. mu.L of each of the upstream primer and the downstream primer (10. mu. mol/L), 0.6. mu.L of RPA probe (10. mu. mol/L), 29.5. mu.L of regeneration Buffer, 12.2. mu.L of ddH2O 12.2.2 of RNase Inhibitor and 1. mu.L of DNA template, mixing, adding into an RPA enzyme tube, shaking and mixing uniformly, adding 2.5. mu.L of magnesium acetate, and initiating the reaction. Wherein, the RPA enzyme tube contains recombinase uvs X, single-strand binding protein and DNA polymerase. The reaction conditions for the RPA amplification reaction were: the reaction was carried out at 37 ℃ for 20min in a heater.
S3, fully reacting two products of the S2 amplification reaction with the 5 × extraction Buffer described in claim 5. The reaction system comprises: mu.L of the amplification product, 80. mu.L of 5 × extraction Buffer, were mixed well.
And S4, dropwise adding the two products in the S3 to a lateral flow test strip sample adding hole, observing the result, and determining the virus type. The reaction system is as follows: and (3) dropwise adding 75 mu L of the solution into a reaction hole of lateral flow test paper, carrying out LFD detection, and reading the result within 5 min.
The constructed positive plasmid samples were tested as described above. C represents a quality control band; t represents a detection zone. 1: primer group and probe 1+ negative control; 2: a primer group and a probe 1+ sample to be detected; 3: a primer group and a probe 2+ sample to be detected; 4: primer set and probe 2+ negative control.
FIG. 1- (a) the sample to be tested is a wild strain without gene deletion;
FIG. 1- (b) shows that the sample to be tested has the deletion of the gene 360-505R;
FIG. 1- (c) the sample to be tested is a mixture of the 360-fold 505R gene deletion strain and the wild strain.
The experimental results are shown in the attached figures 1- (a), 1- (b) and 1- (c).
1.5 nucleic acid electrophoresis verification experiment of RPA reaction product
The following procedure was followed for the RPA electrophoresis experiment
S1, amplifying the RPA amplification product of S2 in 1.4 according to the volume ratio of 1: 1, adding phenol chloroform solution (phenol: chloroform: isopropanol volume ratio is 25:24:1) for purification, centrifuging at 12000rmp/min for 8min, and taking supernatant.
S2, taking 10 mu L of the supernatant obtained in the step S1, adding 1 mu L of 10Xloading buffer, mixing, spotting into a 2% agarose gel hole, carrying out electrophoresis in 1 XTAE electrophoresis buffer solution at a voltage of 120V, and observing the result by a gel imaging system.
The experimental results are shown in figure 2; note: m: DL2000 marker; 1: primer group and probe 1+ negative control; 2: a primer group and a probe 1+ sample to be detected; 3: a primer group and a probe 2+ sample to be detected; 4: primer set and probe 2+ negative control.
FIG. 2- (a) the sample to be tested is a wild strain without gene deletion; FIG. 2- (b) shows that the sample to be tested has the deletion of the gene 360-505R; FIG. 2- (c) the sample to be tested is a mixture of the 360-fold 505R gene deletion strain and the wild strain.
The result of the RPA electrophoresis verification is consistent with the result of the RPA-LFD experiment.
Example 3
Specificity test
The RPA-LFD detection method established according to 1.4 in example 2 was performed on the basis of the following 1: 360-505R gene positive plasmid; 2: hog cholera virus (CSFV); 3: porcine pseudorabies virus (PRV); 4: porcine Reproductive and Respiratory Syndrome Virus (PRRSV); 5: porcine Parvovirus (PPV); 6: negative control (deionized water) was tested.
C represents a quality control band; t represents a detection zone. The results are shown in FIG. 3.
The RPA product was identified by the nucleic acid gel electrophoresis method established in 1.5 of example 2. Note: 1: 360-505R gene positive plasmid; 2: hog cholera virus (CSFV); 3: porcine pseudorabies virus (PRV); 4: porcine Reproductive and Respiratory Syndrome Virus (PRRSV); 5: porcine Parvovirus (PPV); 6: negative control (deionized water).
C represents a quality control band; t represents a detection zone. The results are shown in FIG. 4.
As can be seen from FIGS. 3 and 4, the corresponding 360-fold 505R gene positive plasmid test strip is positive, and clear bands can be seen in the positions of the corresponding fragment sizes in the lane; while other pigs are often infected, such as 2: hog cholera virus (CSFV); 3: porcine pseudorabies virus (PRV); 4: porcine Reproductive and Respiratory Syndrome Virus (PRRSV); 5: porcine Parvovirus (PPV); and 6: the test strip corresponding to the negative control (deionized water) was negative and none of the lanes showed a band. The detection method has better specificity.
Example 4
Sensitivity test
Diluting the positive template pJET1.2-360-505R to 10
0、10
1、10
2、10
3、10
4、10
5、10
6Copy/. mu.L, etc. of 7 concentration gradients.
The concentration of the different templates was determined according to the RPA-LFD method established in 1.4 of example 2. The results are shown in FIG. 5. As can be seen in FIG. 5, the negative control (DI water), 10
0The test strip only has a quality control zone for color development, and a detection zone does not have color development; 10
1-10
6The test strip and the quality control strip of the copy are colored, which shows that the sensitivity of the method can reach 10
1Copies/. mu.L.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Sequence listing
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Zhaoqingdahua agricultural biopharmaceutical Co Ltd
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Claims (10)
1. A primer group and a probe for detecting a wild strain of African swine fever virus are characterized by comprising a first primer group and a first probe;
the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;
the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;
the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3.
2. A primer group and a probe for detecting MGF360-505R gene deletion strains are characterized by comprising a second primer group and a second probe;
the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;
the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;
the second probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.
3. An RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains is characterized by comprising: a first primer set and a first probe, a second primer set and a second probe;
the upstream primer in the first primer group is shown as a sequence SEQ ID NO: 1 is shown in the specification;
the downstream primer in the first primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 2;
the first probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 3, a DNA molecule shown in seq id no;
the upstream primer in the second primer group is shown as a sequence SEQ ID NO: 4 is shown in the specification;
the downstream primer in the second primer group is a primer with biotin at the 5' end and shown as a sequence SEQ ID NO: 5, a DNA molecule shown in the figure;
the second probe is a probe which is provided with carboxyfluorescein at the 5 'end and C3-spacer group at the 3' end and has a sequence shown in SEQ ID NO: 6.
4. The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain as claimed in claim 3, further comprising a DNA extraction reagent, an RPA amplification reagent and a lateral flow test strip.
5. The RPA test strip kit for distinguishing the African swine fever virus wild strain from the MGF360-505R gene deletion strain as claimed in claim 3, further comprising magnesium acetate and ddH
2O、5×Extration Buffer。
6. A rapid distinguishing method for identifying African swine fever virus wild strains and MGF360-505R gene deletion strains by an RPA-LFD technology is characterized by comprising the following steps:
s1: extracting viral DNA from the sample;
s2: placing the extracted virus DNA into an RPA reaction system by using the first primer group and the first probe, and the second primer group and the second probe respectively as described in claim 3 to perform RPA reaction, and obtaining amplification products A1 and A2 respectively
S3, fully reacting the amplification products A1 and A2 in the step S2 with the 5 × extraction Buffer respectively to obtain products B1 and B2;
s4, respectively dripping the products B1 and B2 obtained in the step 3 into a sample adding hole of the lateral flow test strip as claimed in claim 3, carrying out LFD reaction, observing the result, and determining the virus type.
7. The method according to claim 6, wherein step S2 is specifically:
taking 2.1 mu L of each of the upstream primer and the downstream primer in the first primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of first probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template, adding the mixture into an RPA enzyme tube after mixing, shaking and uniformly mixing, adding 2.5 mu L of magnesium acetate, and carrying out initial reaction to obtain an amplification product A1;
taking 2.1 mu L of each of the upstream primer and the downstream primer in the second primer group, wherein the concentration of the upstream primer and the concentration of the downstream primer are 10 mu mol/L; 0.6 mu L of second probe with the concentration of 10 mu mol/L, 29.5 mu L of reduction Buffer, 12.2 mu L of ddH2O 12.2.2 mu L of RNase Inhibitor and 1 mu L of DNA template are mixed, added into an RPA enzyme tube and uniformly shaken, and then 2.5 mu L of magnesium acetate is added to start the reaction to obtain an amplification product A2.
8. The method according to claim 6, wherein in the step S3, the volume of the amplification product A1 and the amplification product A2 is 10 μ L; the 5 × extraction Buffer was 80 μ L.
9. The method of claim 6, wherein in step S4, the volume of products B1 and B2 is 75 μ L, and the result is read after the products are loaded into the loading wells for at least 5 min.
10. The method according to claim 6, wherein in step S4, the RPA enzyme tube comprises the recombinase uvs X, a single-strand binding protein, and a DNA polymerase; the RPA reaction conditions in step S2 are: the reaction system is placed in a water bath kettle at 37 ℃ for reaction for 20min without high-temperature denaturation, annealing and extension.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN111286559A (en) * | 2020-03-06 | 2020-06-16 | 广东海大畜牧兽医研究院有限公司 | Primer, probe and kit for detecting African swine fever virus |
| CN111635962A (en) * | 2020-05-25 | 2020-09-08 | 成都海之元生物科技有限公司 | Novel coronavirus RPA test strip detection kit |
| CN111912984A (en) * | 2020-08-12 | 2020-11-10 | 江苏省农业科学院 | Test strip for detecting African swine fever virus CD2v and MGF360 mucosal antibody and application thereof |
| CN112322796A (en) * | 2020-12-10 | 2021-02-05 | 广东省妇幼保健院 | Visual kit for detecting coxsackievirus A group 6 type nucleic acid and application |
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| CN114672576A (en) * | 2022-04-20 | 2022-06-28 | 山西医科大学 | Method for rapidly detecting body fluid spots based on RT-RPA-LFD technology |
| CN114703177A (en) * | 2022-03-29 | 2022-07-05 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA (reverse transcriptase amplification) isothermal amplification and immunochromatography technology |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399910A (en) * | 2011-12-13 | 2012-04-04 | 华南农业大学 | Primers and method for identifying swine fever virus vaccine strains and wild strains |
| CN109735657A (en) * | 2019-02-02 | 2019-05-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of reagent, detection method and application for African swine fever virus detection |
| CN109797246A (en) * | 2019-02-23 | 2019-05-24 | 军事科学院军事医学研究院军事兽医研究所 | A kind of quickly detection African swine fever virus RPA amplimer and detection kit |
| CN110093324A (en) * | 2019-04-26 | 2019-08-06 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | The attenuation African swine fever virus of gene delection and its application as vaccine |
| CN110184393A (en) * | 2019-06-25 | 2019-08-30 | 四川大学 | A kind of primer sets, purposes, kit and its detection method detecting African swine fever virus |
| CN110184390A (en) * | 2019-06-10 | 2019-08-30 | 河南省动物疫病预防控制中心 | For identifying the double FQ-PCR detection kit of African swine fever and wild strains of classical swine fever virus |
-
2019
- 2019-11-18 CN CN201911129895.8A patent/CN110777220B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399910A (en) * | 2011-12-13 | 2012-04-04 | 华南农业大学 | Primers and method for identifying swine fever virus vaccine strains and wild strains |
| CN109735657A (en) * | 2019-02-02 | 2019-05-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of reagent, detection method and application for African swine fever virus detection |
| CN109797246A (en) * | 2019-02-23 | 2019-05-24 | 军事科学院军事医学研究院军事兽医研究所 | A kind of quickly detection African swine fever virus RPA amplimer and detection kit |
| CN110093324A (en) * | 2019-04-26 | 2019-08-06 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | The attenuation African swine fever virus of gene delection and its application as vaccine |
| CN110184390A (en) * | 2019-06-10 | 2019-08-30 | 河南省动物疫病预防控制中心 | For identifying the double FQ-PCR detection kit of African swine fever and wild strains of classical swine fever virus |
| CN110184393A (en) * | 2019-06-25 | 2019-08-30 | 四川大学 | A kind of primer sets, purposes, kit and its detection method detecting African swine fever virus |
Non-Patent Citations (2)
| Title |
|---|
| FAMING MIAO等: "Rapid and Sensitive Recombinase Polymerase Amplification Combined With Lateral Flow Strip for Detecting African Swine Fever Virus", 《FRONTIERS IN MICROBIOLOGY》 * |
| FAMING MIAO等: "Rapid and Sensitive Recombinase Polymerase Amplification Combined With Lateral Flow Strip for Detecting African Swine Fever Virus", 《FRONTIERS IN MICROBIOLOGY》, vol. 10, 15 May 2019 (2019-05-15), pages 2 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN111286559A (en) * | 2020-03-06 | 2020-06-16 | 广东海大畜牧兽医研究院有限公司 | Primer, probe and kit for detecting African swine fever virus |
| CN111635962A (en) * | 2020-05-25 | 2020-09-08 | 成都海之元生物科技有限公司 | Novel coronavirus RPA test strip detection kit |
| CN111912984A (en) * | 2020-08-12 | 2020-11-10 | 江苏省农业科学院 | Test strip for detecting African swine fever virus CD2v and MGF360 mucosal antibody and application thereof |
| CN111912984B (en) * | 2020-08-12 | 2024-04-05 | 江苏省农业科学院 | Test strip for detecting African swine fever virus CD2v and MGF360 mucous membrane antibody and application thereof |
| CN112322796A (en) * | 2020-12-10 | 2021-02-05 | 广东省妇幼保健院 | Visual kit for detecting coxsackievirus A group 6 type nucleic acid and application |
| CN113718060A (en) * | 2021-09-28 | 2021-11-30 | 安徽中起生物科技有限公司 | Primer group and method for detecting African swine fever virus natural variant strain based on RPA-LFD method |
| CN114015813A (en) * | 2021-12-17 | 2022-02-08 | 苏州荧莱生物工程有限公司 | Method and kit for identifying African swine fever virus based on RPA (reverse transcriptase amplification) isothermal amplification technology |
| CN114703177A (en) * | 2022-03-29 | 2022-07-05 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA (reverse transcriptase amplification) isothermal amplification and immunochromatography technology |
| CN114703177B (en) * | 2022-03-29 | 2023-08-11 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA isothermal amplification and immunochromatography technology |
| CN114672576A (en) * | 2022-04-20 | 2022-06-28 | 山西医科大学 | Method for rapidly detecting body fluid spots based on RT-RPA-LFD technology |
| CN114672576B (en) * | 2022-04-20 | 2023-10-13 | 山西医科大学 | Method for rapidly detecting body fluid spots based on RT-RPA-LFD technology |
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