CN110438265B - Rapid differential diagnosis method for African swine fever virus gene type I and type II - Google Patents
Rapid differential diagnosis method for African swine fever virus gene type I and type II Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention provides a method for differential diagnosis of African swine fever virus genotype I and II based on a probe-guided recombinase-mediated isothermal amplification technology, which can be used for rapidly diagnosing and distinguishing the African swine fever virus genotype I and II. The probe for detecting different genotype specific differential sites based on the probe-oriented recombinase-mediated isothermal amplification method is characterized in that on the basis of the probe for the recombinase-mediated isothermal amplification method, the probe carries out enzymolysis on the last base of a tetrahydrofuran substitution site or the last base of a specific differential site complementary site, exonuclease III is carried out on the tetrahydrofuran substitution site until the cutting is finished, the cut base modified by a fluorophore generates a fluorescent signal in a system, and the rest probe part plays a role of a downstream primer. The probe simplifies the components of a downstream primer in the detection process, and the downstream primer and the probe are used in combination, so that the occurrence of non-specific amplification of the probe and the primer is reduced.
Description
Technical Field
The invention belongs to the technical field of poultry disease detection, and particularly relates to a rapid differential diagnosis method for African swine fever virus genotype I and type II, namely, a probe-oriented recombinase-mediated isothermal amplification technology is applied to detection of specific differential sites of the African swine fever virus genotype I and type II and the rapid differential diagnosis method.
Background
African Swine Fever (ASF) is an acute, contact infectious disease of pigs caused by African Swine Fever Virus (ASFV), which is mainly characterized by high fever, cyanosis of the skin and severe bleeding of the lymph nodes and internal organs with a mortality rate of up to 100%, which is classified as a class A infectious disease by the world animal health Organization (OIE). Due to the special immune evasion mechanism of the virus, no effective treatment means exists at present. Therefore, the detection and quarantine of the virus are very important.
The African swine fever virus has only 1 serotype, but the existing epidemic strains can be divided into 24 genotypes by using the B646L gene encoding the vp72 protein. According to the spreading trend of African swine fever virus genotype I and II in other continents outside Africa, the distribution and epidemic trend of the strains in corresponding areas can be further known and the epidemic source and possible propagation mode can be traced back by quickly identifying and analyzing the genotype difference so as to take countermeasures in time.
Disclosure of Invention
In view of the above, the present invention aims to provide a rapid differential diagnosis method for the type I and type II of african swine fever virus gene, which is based on a probe-oriented recombinase-mediated isothermal amplification method for detecting specific differential sites, wherein the probe can be used as a probe for generating a fluorescent signal and also can be used as a primer, thereby simplifying primer components required by an RAA detection method.
The invention provides a probe for detection by a recombinase-mediated isothermal amplification method, wherein the latter base of a specific differential site to be detected in the probe is replaced by tetrahydrofuran, the distance between the 5 'end of the probe and the tetrahydrofuran site is at least 30 bases, and the distance between the 3' end of the probe and the tetrahydrofuran site is at least 15 bases; the probe is modified with a fluorescent group and a quenching group; the fluorescent group and the quenching group are respectively modified on T bases at the upstream and downstream of a tetrahydrofuran locus;
the fluorescent group is modified on any T base in the 3 'end direction of tetrahydrofuran, and the quenching group is modified on any T base in the 5' end direction of the specific differential site;
the interval between the T base modified by the fluorescent group and the T base modified by the quenching group is 2 to 5 bases;
preferably, the fluorophore is modified at the T base immediately above the tetrahydrofuran site.
Preferably, the quenching group is modified at the T base next to the specific differential site.
Preferably, the fluorescent group comprises 6-carboxyfluorescein or hexachloro-6-methylfluorescein; the quenching group comprises BHQ quenching group.
Preferably, the 3' terminal base of the probe is modified with a phosphate blocker.
When the detection sequence is SEQ ID No:4, the probes comprise two probes which are respectively provided with the sequence shown in SEQ ID No:1 and a nucleotide sequence having a sequence shown in SEQ ID No:2, the specific differential site is 220 nucleotides, and is A or G (R).
The invention also provides a diagnostic method for detecting specific differential sites based on a probe-oriented recombinase-mediated isothermal amplification method, which comprises 10-100 mu mol/L of upstream primer and 10-100 mu mol/L of the probe; the upstream primer is used for amplifying a nucleic acid segment containing a specific difference site together with the probe;
one specific upstream primer is a nucleotide sequence shown as SEQ ID No.3 in a sequence table.
Preferably, the diagnostic method further comprises enzyme mixture, reaction buffer, 280mmol/L magnesium acetate; the enzyme cocktail included 800 ng/. Mu.L of single-stranded binding protein, 60 ng/. Mu.L of bacteriophage UvsX protein, 50 ng/. Mu.L of LDNA polymerase and 40 ng/. Mu.L of exonuclease III.
The invention provides a probe for detecting specific differential sites based on a probe-oriented recombinase-mediated isothermal amplification method, which is characterized in that on the basis of the probe for the recombinase-mediated isothermal amplification method, the last base of the specific differential sites is replaced by tetrahydrofuran, exonuclease III cuts from the 3' end of the probe until the tetrahydrofuran sites are cut off, and the base modified by the cut-off fluorescent group generates a fluorescent signal in a system to play the role of the probe; the remaining probe portion acts as a downstream primer, and when the probe for the specific differential site is properly matched with the specific differential site in the sample, the remaining probe acts as a downstream primer for amplification extension, and the incorrectly matched bases cannot be normally amplified. The probe can be used as a probe to generate a fluorescent signal and also can be used as a primer, so that the necessary downstream primer component in the detection process is saved, and the non-specific amplification of the probe and the primer is avoided. Meanwhile, the probe provided by the invention is used in combination with the downstream primer, so that the previous design idea of pairing the 3' end of the upstream primer with the specific differential site is broken through, the detection of the specific differential site is realized, and the genotype of a sample can be accurately and quickly obtained.
Drawings
FIG. 1 is a schematic diagram of the RAA probe design principle of the present invention;
FIG. 2 shows the amplification of primer probe combinations of different genotypes and concentrations in the A and G reactions; wherein, the figure 2-1 shows the amplification condition of ASFV gene type I sample in A and G reactions; FIG. 2-2 shows the amplification of ASFV gene type II sample in reactions A and G; FIGS. 2 to 3 show the amplification of ASFV genotype I in reactions with primer probe combinations A and G of different concentrations; FIGS. 2 to 4 show the amplification of ASFV gene type II in reactions with primer probe combinations A and G of different concentrations.
FIG. 3 is an evaluation of the accuracy of RAA detection of different concentrations of plasmid in example 1, wherein FIG. 3-1 shows the amplification of different concentrations of ASFV type I plasmid in A reaction; FIG. 3-2 shows the amplification of ASFV type I plasmid at different concentrations in the G reaction; FIGS. 3-3 show the amplification of different concentrations of ASFV type II plasmid in the G reaction; FIGS. 3 to 4 show the amplification of ASFV type II plasmids at different concentrations in the A reaction.
FIG. 4 shows the specific detection of A-reaction and G-reaction in the method of example 2 on African Swine Fever Virus (ASFV) type I, type II, foot and Mouth Disease Virus (FMDV), porcine Parvovirus (PPV), pseudorabies virus (PRV), porcine Circovirus (PCV), epidemic encephalitis B virus (JEV), porcine reproductive and respiratory syndrome virus (PRRV), classical Swine Fever Virus (CSFV), transmissible gastroenteritis virus (TGEV), and negative control water; wherein, FIG. 4-1 is the specificity detection of A reaction, only the ASFV gene type I appears obvious amplification line, which shows that the specificity of A reaction detection ASFV gene type I is good; FIG. 4-2 shows the detection of the specificity of the G reaction, and only the ASFV gene type II shows a distinct amplification line, which indicates that the specificity of the G reaction for detecting the ASFV gene type II is good.
FIG. 5 shows the results of RAA method and sequencing for ASFV type I and II clinical specimens in example 3.
Detailed Description
The terms related to the present invention are described as follows:
1. single nucleotide polymorphism (Single nucleotide polymorphism, specific differential SNP) mainly refers to DNA sequence polymorphism caused by variation of a Single nucleotide at the genome level, and accounts for more than 90% of all known polymorphisms. The specific difference site is a genetic marker formed by variation of a single nucleotide on a genome, including transition, transversion, deletion and insertion, and has a large quantity and rich polymorphism.
2. The Recombinase-mediated isothermal amplification (RAA) uses a Recombinase obtained from bacteria or fungi, which can be tightly bound to a primer DNA at a constant temperature of 39 ℃ to form an aggregate of the enzyme and the primer, and when the primer searches for a sequence completely complementary to the primer on the template DNA, the template DNA is melted with the help of a single-stranded DNA binding protein (SSB), and a new DNA complementary strand is formed by the action of a DNA polymerase, and the reaction product is also exponentially increased. Detection is usually completed within 30min, and it is also possible to detect real-time amplification of the product by adding a probe carrying a fluorescent label. However, although the RAA detection method has high sensitivity and specificity, the conventional RAA detection method relies on designing a pair of amplification primers and a probe, and avoids non-specific amplification of the primers and non-specific binding of the primers to the probe, which requires high primer and probe requirements.
The invention provides a probe for detecting specific differential sites based on a probe-oriented recombinase-mediated isothermal amplification method, wherein the length of the probe is 46-52 nt, the last base of the specific differential site is replaced by tetrahydrofuran, the distance between the 5 'end of the probe and the tetrahydrofuran site is at least 30 bases, and the distance between the 3' end of the probe and the tetrahydrofuran site is at least 15 bases; the probe is modified with a fluorescent group and a quenching group, the fluorescent group is modified on a T base at the 3 'end of a tetrahydrofuran locus, and the quenching group is modified on a T base at the 5' end of a specific differential locus; the base modified by the fluorescent group and the base modified by the quenching group are separated by 2-5 bases.
In the present invention, the design concept of the probe is shown in FIG. 1. In the present invention, the nucleotide sequence of the probe is arranged in the direction from the 5 'end to the 3' end. The fluorophore modification is preferably at the next T base of the tetrahydrofuran site. The quenching group preferably modifies the T base immediately above the specific differential site.
In the present invention, the fluorescent group preferably includes FAM (6-carboxyfluorescein) or HEX (hexachloro-6-methylfluorescein); the quencher group comprises BHQ.
In the present invention, the 3' -terminal base of the probe is preferably modified with a phosphate blocker. The role of the phosphate blocker modification is to prevent amplification that might be directed by the polymerase.
In the invention, bases modified by fluorescent groups and quenching groups in the probes are required to be T bases, and the probes preferably comprise forward probes and reverse probes according to the distribution of A and T bases before and after specific difference sites in positive and negative chains of the template. The forward probe binds to the negative strand of the template. The forward probe is designed to contain specific differential sites. The reverse probe binds to the positive strand of the template. The reverse probe is designed to contain a specific differential site complementary site. A basic group exists in front of and behind the specific difference site or the specific difference complementary site in the template, so that the construction of the probe can be met.
In the invention, when the detection gene is ASFV B646L gene sequence and the specific differential site shows A/G polymorphism, the complementary base of the specific differential site is corresponding to T/C, which meets the construction of a reverse probe. The reverse probe for detecting the specific differential site of the ASFV gene type I (specific differential site is A) and type II (specific differential site is G) is preferably a reverse probe with the nucleotide sequence shown in SEQ ID No:1 and a nucleotide sequence having a sequence shown in SEQ ID No:2 under the condition of high nucleotide sequence. The ASFV B646L gene sequence template has the sequence shown in the sequence table as SEQ ID No: 4. The sequences of the probe and the upstream primer are specifically shown in the following table 1:
wherein a represents a forward primer used for the reaction A and G; b represents a specific probe for the A reaction; c represents a specific probe for the G reaction; d represents the modification of the probe, and FAM (6-carboxyfluorescein) represents a fluorescent group; THF (tetrahydrofuran); BHQ represents a quenching group;
the reaction principle of the probe is explained by taking the detection of the ASFV B646L gene sequence (SEQ ID No: 4) as an example. When the sample is ASFV gene type I, A reaction occurs, two probes are added to two amplification reaction systems, 627-A-P is added b ,d The complementary base of the specific differential site in the probe is T, and can be complementarily paired with the base A in the template, the exonuclease III cuts to the THF site and is interrupted, and the 3' end of the rest probe can be strictly complementarily paired with the template, so that the rest probe is used as a downstream primer for extension and expansion to generate an amplification curve; and 628-G-P added in another system c,d The complementary base of the specific differential site of the probe is C, which can not be strictly complementary paired with the A base of the specific differential site in the template, and compared with the complementary base T, the complementary base T can not be normally amplified. When ASFV gene type II is present in the sample, the addition628-G-P c,d The probe system can be normally amplified, and G reaction is carried out to generate an amplification curve. Addition of 627-A-P b,d The probe system (2) cannot normally amplify the complementary base C. Therefore, the genotype is judged based on the presence or absence of an amplification curve or the appearance and the type of the probe used.
The invention provides a diagnostic method for detecting specific differential sites between type I and type II of African swine fever virus genes based on a probe-directed recombinase-mediated isothermal amplification method, which comprises 10-100 mu mol/L of an upstream primer and 10-100 mu mol/L of a downstream primer/probe.
In the invention, the upstream primer and the downstream primer/probe are designed by using templates with different positive and negative chains, namely when the downstream primer/probe is designed by using a negative chain as a template, the upstream primer is designed by using a positive chain as a template. The method for designing the primer is not particularly limited, and a primer design method known in the art may be used. The upstream primer synthesis in the present invention is not particularly limited, and a primer synthesis company known in the art may be used. In the present example, the probe was synthesized by Shanghai Biotechnology engineering, inc.
The concentration of the upstream primer is preferably 15. Mu. Mol/L. When detecting the specific difference site of the ASFV B646L gene sequence, the upstream primer preferably has the nucleotide sequence shown in SEQ ID No.3 in the sequence table.
In the present invention, the diagnostic method includes the probe. The downstream primer/concentration of the probe is independently preferably 15. Mu. Mol/L. The number of the probes is two, and the probes are preferably independently subpackaged.
In the present invention, the diagnostic method preferably further comprises an enzyme mixture, a reaction buffer and 280mmol/L magnesium acetate; the enzyme cocktail included 800 ng/. Mu.L of single-stranded binding protein, 60 ng/. Mu.L of bacteriophage UvsX protein, 50 ng/. Mu.L of LDNA polymerase and 40 ng/. Mu.L of exonuclease III. The sources of the enzyme mixture, the reaction buffer and the magnesium acetate are not particularly limited in the present invention, and those well known in the art may be used. In the embodiment of the invention, the enzyme mixture, the reaction buffer solution and the magnesium acetate are respectively selected from RAA basic reaction unit kits produced by Jiangsu Qitian GenBank.
The detection principle of the diagnosis method is improved on the basis of the traditional RAA detection method, and a new detection method, namely a probe-oriented recombinase-mediated isothermal amplification method (RAA), is obtained by using unhydrolyzed partial probes as downstream primers for amplification.
In the present invention, the method of using the diagnostic method comprises the steps of:
1) Mixing an enzyme mixture, a reaction buffer solution, ribozyme-free water, magnesium acetate, template DNA and upstream primers, uniformly dividing the obtained mixed solution into 3 parts, respectively adding one probe into each of 2 parts of the mixed solution, and adding ribozyme-free water into the other part of the mixed solution to serve as a control;
2) And carrying out isothermal amplification on 3 parts of the mixed solution to obtain the amplification condition.
In the present invention, the concentration of the forward primer and the backward primer/probe is preferably 15. Mu. Mol/L. The concentration of the template DNA is preferably 10 to 75 ng/. Mu.L. The concentration of the magnesium acetate is preferably 280mmol/L. The apparatus for isothermal amplification is not particularly limited, and any apparatus for isothermal amplification known in the art may be used. In the present example, the apparatus for isothermal amplification was a QT-RAA-F7200 fluorescence detector manufactured by Kingsu Qitian Kogyo. The reaction temperature is preferably 39 ℃. The time for the isothermal amplification is preferably 25min.
FIG. 2 shows the amplification conditions of the primer probe combinations with different genotypes and concentrations in the A and G reactions; wherein, FIG. 2-1 shows the amplification condition of ASFV gene type I sample in A and G reactions, when the sample is ASFV type I, the amplification line of the A reaction is the first-out; FIG. 2-2 shows the amplification of ASFV genotype II samples in the reactions A and G, and when the sample is ASFV genotype II, the amplification line of the reaction G begins at first; FIG. 2-3 shows the amplification of ASFV gene type I in the primer probe combination A and G reactions with different concentrations, when the sample is ASFV type I, the concentration of the primer probe is selected to be 15 μ M, the amplification lines of the A reaction and the G reaction are distinguished in sequence; FIGS. 2-4 show the amplification of ASFV gene type II in the primer probe combination A and G reactions with different concentrations, when the sample is ASFV type II, the concentration of the primer probe is selected to be 15 μ M, and the amplification lines of the G reaction and the A reaction are distinguished in sequence.
The probe and the diagnostic method for detecting specific differential sites by the probe-directed recombinase-mediated isothermal amplification method provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
In order to further explore the influence of the sample concentration on the specificity of the RAA method, the ASFV gene type I and type II plasmids are constructed and are respectively subjected to a series of dilutions including 10 5 ,10 4 ,10 3 ,10 2 ，10 1 The copies/reactions were then tested using the RAA method (A and G reactions), respectively (FIG. 3).
Wherein FIG. 3-1 shows the amplification of ASFV type I plasmid at different concentrations in the A reaction, and the concentration is 10 2 -10 5 The ASFV I type plasmid has obvious amplification gradient in A reaction; FIG. 3-2 shows the amplification of ASFV type I plasmid at different concentrations in the G reaction, and the concentration is 10 1 -10 5 The amplification gradient of the ASFV type I plasmid in the G reaction is not obvious, and is only 10 4 -10 5 The amplification line is more obvious and is shown after the amplification line of the reaction A in figure 3-1; FIGS. 3-3 show the amplification of different concentrations of ASFV type II plasmid in the G reaction at a concentration of 10 1 -10 5 The ASFV II type plasmid has obvious amplification gradient in G reaction; FIGS. 3-4 show the amplification of ASFV type II plasmids at different concentrations in the A reaction, and the amplification gradient is not obvious, but only 10 4 -10 5 The amplification line of (2) is more distinct and is shown after the amplification line of the G reaction in FIGS. 3-3. For the plasmids, the lower limit of the detectable template for both A and G reactions was found to be 10 2 Copy/reaction (fig. 3). Suggesting that the RAA method may be at a template copy number of 10 2 ～10 5 Has high specificity.
Example 2
To investigate the specificity of the a-and G-responses, RAA method (a and G-responses) assays were performed using nucleic acids of African Swine Fever (ASFV) type I, type II genes, foot and Mouth Disease (FMD), porcine Parvovirus (PPV), pseudorabies virus (PRV), porcine Circovirus (PCV), epidemic encephalitis b (JEV), porcine reproductive and respiratory syndrome (PRRV), classical Swine Fever Virus (CSFV), transmissible gastroenteritis virus (TGEV) as templates (fig. 4).
Wherein, FIG. 4-1 is the specificity detection of the A reaction, only the ASFV genotype I appears obvious amplification line, which indicates that the specificity of the A reaction for detecting the ASFV genotype I is good; FIG. 4-2 shows the specificity detection of the G reaction, in which only the ASFV gene type II appears as a distinct amplification line, indicating that the specificity of the G reaction for detecting the ASFV gene type II is good.
Example 3
1. Collection of clinical specimens
Extracting DNA in the pig tissue and the whole blood sample by an automatic nucleic acid extractor of the Tianlong, and storing at-20 ℃ for later use. The DNA concentration of the obtained specimen ranged from 10 to 75 ng/. Mu.L.
2. RAA assay was performed on 20 clinical specimens
The RAA fluorescence method basic reaction unit kit produced by Jiangsu Qitian gene company is adopted. A. The C reactions were all carried out in a 0.2ml reaction tube containing a previously lyophilized enzyme mixture (SSB, uvsX, DNA polymerase, exonaclease III), followed by the addition of 25. Mu.L of reaction buffer, 17. Mu.L of ribozyme-free water, 2.1. Mu.L of forward primer (15. Mu.M), 1.4. Mu.L of the corresponding specific reverse probe (15. Mu.M), 2. Mu.L of template (10-75 ng/. Mu.L), 2.5. Mu.L of magnesium acetate (280 mM). After the solution preparation is finished, the solution is transferred to a QT-RAA-F7200 fluorescence detector produced by Qitianjus of Jiangsu, and the detection is carried out, and the reaction is carried out for 25min at the temperature of 39 ℃. Water without ribozyme served as a negative control for each reaction.
3. Results and analysis of RAA
The positive reaction of A and G is judged by the sequence of the specific curve appearing in the reaction, and the reaction time is 25min. For example, if a reaction first develops a specificity curve, it is judged as ASFV genotype I; if a specific curve is first developed in the G reaction, the ASFV gene type II is judged.
Comparative examples
1. Direct sequencing of 4 of the clinical specimens tested
A25. Mu.l PCR reaction system containing 16.375. Mu.l ribozyme-free water, 2.5. Mu.l 10 XPCR reaction buffer, 2. Mu.l dNTP, 0.125. Mu.l Taq enzyme, 1. Mu.l (10. Mu. Mol/L) of the corresponding upstream and downstream primers, and 3. Mu.l template (50-150 ng/. Mu.l) was prepared by the PCR diagnostic method of TaKaLa Ex TaqHot Start version (Dalianbao bioengineering Co., ltd.). And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; entering into circulation: denaturation at 94 ℃ for 30s, denaturation at 58 ℃ for 30s, denaturation at 72 ℃ for 30s, and 40 cycles; 7min at 72 ℃; finally, the mixture is stored at 4 ℃. The sequencing company Borneo, qingdao, was assigned to perform Sanger sequencing after PCR of all samples.
2. Sequencing results and analysis
FIGS. 5-1 and 5-2 show the nucleic acid RAA and sequencing results of clinical samples of ASFV type I and type II, respectively, and the accuracy of the nucleic acid RAA reaction-detected ASFV type I and the nucleic acid ASFV type II reaction-detected G is consistent with that of the sequencing results.
Sequence listing
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Claims (4)
1. A probe for detecting gene I or II African swine fever virus by a recombinase-mediated isothermal amplification method is characterized in that the sequence of the gene I probe is
5′-TCATCGCACCCGGATCATCGGGGGTTTTAATT(THF)CATTGCCTCCGTAGTGGA-3′，
The sequence of the gene II type probe is 5 '-TCATCGCACCCGGATATCGGGGTTTTAATC (THF) CATTGCCTCCGTAGGTGGA-3';
wherein, the 32 th site of the probe is a specific differential site to be detected, the later base is replaced by Tetrahydrofuran (THF), the 31 th site T of the probe is modified with a fluorescent group, and the 36 th site T is modified with a quenching group.
2. The probe of claim 1, wherein the fluorophore comprises 6-carboxyfluorescein or hexachloro-6-methylfluorescein; the quenching group comprises a BHQ quenching group.
3. The probe of claim 1, wherein the 3' terminal base of said probe is modified with a phosphate blocker.
4. A method for detecting African swine fever virus type I or II, which is not a disease diagnosis and treatment purpose, wherein the probe and the upstream primer of claim 1 are used; and reagents for recombinase-mediated isothermal amplification; the sequence of the upstream primer is SEQ ID NO. 3.
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| CN112831597A (en) * | 2019-11-22 | 2021-05-25 | 洛阳普泰生物技术有限公司 | Real-time fluorescent PCR amplification primer pair and probe primer for gene identification and detection of African swine fever virus and prepared kit |
| CN111621603A (en) * | 2020-06-23 | 2020-09-04 | 广州欧密伽畜牧有限公司 | Specific primer for identifying African swine fever virus, and identification method and detection kit thereof |
| CN111676327B (en) * | 2020-07-21 | 2021-05-14 | 河南省农业科学院 | Dual fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for African swine fever virus wild virus infection and gene deletion strain |
| CN115976273B (en) * | 2022-09-13 | 2024-04-23 | 青岛嘉智生物技术有限公司 | Dual fluorescence PCR detection kit for identifying African swine fever virus genes type I and type II |
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| CN106947838B (en) * | 2017-05-31 | 2020-12-01 | 广州海关技术中心 | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method |
| CN108060213B (en) * | 2018-01-19 | 2020-07-17 | 中国疾病预防控制中心病毒病预防控制所 | Probe and kit for detecting SNP (single nucleotide polymorphism) sites by recombinase-mediated isothermal amplification method based on probe guidance |
| CN109593893A (en) * | 2019-02-03 | 2019-04-09 | 郑州中道生物技术有限公司 | African hog cholera virus fluorescent PCR quick detection kit |
-
2019
- 2019-08-13 CN CN201910745127.9A patent/CN110438265B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| African swine fever virus isolate Irkutsk 2017 major capsid protein P72 gene, partial cds;Titov,I.等;《Genbank》;20171212;全文 * |
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