CN116064930B - Multiplex fluorescence PCR primer probe set, method and application for detecting type I and type II African swine fever virus - Google Patents
Multiplex fluorescence PCR primer probe set, method and application for detecting type I and type II African swine fever virus Download PDFInfo
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Abstract
The invention discloses a multiplex fluorescence PCR primer probe group, a method and application for detecting type I and type II African swine fever viruses, wherein the fluorescence PCR primer probe group comprises primer pairs shown as SEQ ID No. 1 and SEQ ID No. 2, a first specific probe shown as SEQ ID No. 3 and a second specific probe shown as SEQ ID No. 4. The multiplex fluorescence PCR primer probe group, the kit and the detection method provided by the invention solve the problems of high cost, insufficient sensitivity, complex detection method and the like of ASFVI type and II type identification detection in the prior art, realize high sensitivity, and can accurately identify ASFVI type and II type strains and have strong specificity.
Description
Technical Field
The invention relates to the technical field of animal pathogen molecular diagnosis, in particular to a multiplex fluorescence PCR primer probe group, a kit, a method and application for detecting I type and II type African swine fever viruses.
Background
African swine fever (African swine fever, ASF) is an acute contagious disease characterized by high morbidity and mortality due to African swine fever virus (African swine fevervirus, ASFV). The disease is listed by the world animal health Organization (OIE) as a legal report of animal epidemic disease, which is listed in China as a class of animal infectious diseases. The acute ASF is clinically manifested by high fever, skin cyanosis and hemorrhage of various organs, the death rate is as high as 100 percent, the harm to the pig industry is huge, and the international trade of the pig industry is seriously impacted. At present, no vaccine and therapeutic drug for effectively controlling the disease exist, and the spread of the disease is controlled mainly through detection and combination of killing.
The result of the early sequence analysis shows that the first ASFV isolate in China is of the gene type II. Subsequently we further performed evolutionary analysis at the genomic level, and the results show that the current global ASFV isolates can be divided into 3 evolutionary lineages (lineages), and that chinese isolates belong to both Lineage I and genotype II (p 72 gene-based typing system). All asian isolates belong to both pedigree I and genotype II, all european strains also belong to pedigree I, but have two genotypes (genotype I and II). The evolutionarily intermixed Asian isolates with some European isolates suggests that they may have a common source. There are multiple genotypes in africa, which may be due to the multiple transmission cycles of pigs-boars, boars-soft ticks, soft ticks-domestic pigs, and boars-soft ticks-boars in africa regions, which may lead to viral transmission between different hosts and genomic variation.
Therefore, establishing a new method for identifying the type I and type II Chinese epidemic strains of African swine fever virus, so that the reliable identification and detection of the type I and type II strains of the currently popular ASFVI can be realized, and the method is a problem to be solved in the invention.
Disclosure of Invention
Aiming at the problems of high cost, insufficient sensitivity, complex detection method and the like of the identification detection of ASFVI type and II type in the prior art, the invention provides a multiplex fluorescence PCR primer probe group, a kit and a detection method which have high sensitivity, can accurately identify ASFVI type and II type strains and are used for detecting I type and II type African swine fever viruses.
In order to achieve the above object, the present invention provides a multiplex fluorescence PCR primer probe set for detecting type I and type II African swine fever virus, the fluorescence PCR primer probe set comprising a primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2, a first specific probe as shown in SEQ ID No. 3, and a second specific probe as shown in SEQ ID No. 4.
Preferably, the 5' ends of the first specific probe and the second specific probe are respectively marked with a fluorescent report group;
the 3' ends of the first specific probe and the second specific probe are respectively marked with a minor groove conjugate and a non-fluorescence quenching group; and, in addition, the method comprises the steps of,
the fluorescent reporter group is selected from VIC and/or FAM.
The invention also provides a multiplex fluorescence PCR kit for detecting the I type and II type African swine fever virus, which comprises the multiplex fluorescence PCR primer probe set and the DNase mixed solution.
Preferably, the dnase mixture comprises an enzyme diluent, a hot start Taq enzyme and deoxyribonucleotides.
Preferably, the deoxyribonucleotide is selected from one or more of dATP, dUTP, dGTP and dCTP.
Preferably, the concentration of each of the primer pair, the first specific probe and the second specific probe is 0.1 to 1.5. Mu. Mol/L.
The invention also provides a detection method for the type I and type II African swine fever virus, which adopts the multiplex fluorescence PCR kit.
Preferably, the detection method comprises:
s100, extracting DNA of a sample to be detected;
s200, performing fluorescent amplification on the DNA extracted in the step S100 by adopting a multiplex fluorescent PCR kit;
s300, judging whether the sample to be detected contains the type I and/or type II African swine fever virus according to a fluorescence amplification result.
Preferably, the detection method further comprises: preparing a positive control plasmid; diluting the prepared positive control plasmid according to a gradient to obtain a standard sample; and respectively carrying out fluorescent amplification on the obtained standard samples, then measuring Ct values, and constructing a concentration-Ct standard curve.
Preferably, step S300 further includes detecting a Ct value of the sample to be measured, and quantitatively detecting the concentration of the sample to be measured according to a concentration-Ct standard curve.
The invention also provides an application of the multiplex fluorescence PCR kit or the detection method in detecting type I and type II African swine fever viruses.
Through the technical scheme, the primer probe group provided by the invention can specifically identify and detect the type I and type II Chinese epidemic strains of the African swine fever virus; the kit provided by the invention can simultaneously identify and diagnose the type I and type II Chinese epidemic strains of the African swine fever virus in a system, double amplification is realized on the premise of not influencing the amplification efficiency, the change of the amplification product quantity of each cycle in the PCR amplification reaction is detected in real time by utilizing the change of fluorescent signals, the quantitative analysis can be carried out on the sample to be detected by the obtained relationship between the Ct value of the sample to be detected and a standard curve, the detection result is more accurate and visual, the time and the labor are saved, the cost is lower, the time is shortened to 1 to 1.5 hours from the original 3 to 4 hours, and the kit has higher sensitivity than that of the ordinary PCR, and can be used for detecting the African swine fever virus in the sample with low micro content. The kit prepared based on the invention is not only suitable for qualitative and quantitative analysis of scientific research units, but also suitable for pathogen detection analysis of various levels of prevention and control units, basic veterinary stations, large and medium-sized farms and the like, and has good application prospect.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 shows a multiplex fluorescence PCR primer probe set provided by the invention; wherein, the middle virtual frame is the probe design position of the African swine fever virus type I and type II Chinese epidemic strains.
FIG. 2A is an amplification curve of double fluorescence quantitative PCR for A1 (i.e., african swine fever virus type I positive control plasmid) in test example 1;
FIG. 2B is an amplification curve of the double fluorescence quantitative PCR for A2 (i.e., african swine fever virus type II positive control plasmid) in test example 1;
FIG. 3A is a graph showing the concentration of African swine fever virus type I-Ct standard curve obtained in test example 4;
FIG. 3B is a graph showing the concentration of African swine fever virus type II-Ct standard curve obtained in test example 4;
FIG. 4 shows amplification curves of double fluorescent quantitative PCR obtained in detection example 1 and control example.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
It should be noted that the african swine fever viruses identified by the present invention include african swine fever viruses of the type I and the type II, and the multiplex fluorescent PCR primer probe set corresponds to highly conserved fragments of p72 genes of the type I and the type II of the african swine fever viruses. Specifically, the gene I type African swine fever virus is the Chinese epidemic strain Pic/HeN/ZZ-P1/2021 and Pic/SD/DY-I/2021 described in GenBank under the numbers MZ945536.1 and MZ 945537.1. The African swine fever virus type II is Chinese epidemic strains Pic/HLJ/2018 and Pic/Heilongjiang/HRB 1/2020 which are described in GenBank and are numbered MK333180.1 and MW 656282.1. Wherein, the amplified target nucleotide sequence of the highly conserved fragment of the gene I type African swine fever virus strain p72 gene is shown as SEQ ID No. 5; the amplified target nucleotide sequence of the highly conserved fragment of the gene II type African swine fever virus strain p72 gene is shown as SEQ ID No. 6. The dnase mixture herein may be of a type conventionally understood and formulated by those skilled in the art, and for example, may be a conventionally formulated mixture comprising an enzyme diluent, a hot start enzyme and deoxyribonucleotides, wherein the deoxyribonucleotides herein may be selected from one or more of dATP, dUTP, dGTP and dCTP. Of course, the DNase mixture may be a conventional commercial product, and for example, a conventional commercial product manufactured by Japan under the brand name TaKaRa may be selected.
The invention is further illustrated by the following specific examples.
Preparation example 1, preparation of primer probe set:
the primer probe group is obtained by synthesizing the primer pairs shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, the first specific probe and the second specific probe. As shown in fig. 1, specifically:
forward primer: 5'-GGGGATAAAATGACTGGATA-3';
reverse primer: 5'-CATCGGTAAGAATAGGTTTG-3';
first specific probe: 5'-FAM-CACTTGGTCGGCCA-NFQ-MGB-3';
second specific probe: 5'-VIC-CACTTGGTTGGCCA-NFQ-MGB-3'.
Preparation example 2 preparation of positive control plasmid
The P72 sequence of the African swine fever virus type I Chinese epidemic strain Pic/SD/DY-I/2021 (GenBank is MZ 945537.1) and the P72 sequence of the African swine fever virus type II Chinese epidemic strain Pic/HLJ/2018 (GenBank is MK 333180.1) are respectively synthesized artificially to obtain corresponding synthetic fragments; the synthesized fragments are respectively connected with pUC57 cloning vectors according to the ratio of 1:1 by means of recombinant DNA technology to construct recombinant plasmids; after DH5 alpha competent cells are transformed by the obtained recombinant plasmid, culturing the transformed competent cells, after single bacterial colonies grow out, picking single bacteria, amplifying and extracting to obtain positive control plasmids which are respectively marked as A1 (corresponding to GenBank number MZ 945537.1) and A2 (corresponding to GenBank number MK 333180.1).
Detection example 1
The PCR system was constructed according to the following conditions:
the total PCR system was 50. Mu.L: the first and second specific probes (concentration 10. Mu. Mol/L each) were 4. Mu.L each; 4. Mu.L each of the forward primer and the reverse primer (concentration of 10. Mu. Mol/L each) in the primer pair; ROX reference dye 4. Mu.L; premix Ex Taq Probe qPCR (a conventional commercial product manufactured by Japan under the brand name TaKaRa) 25. Mu.L, 5. Mu.L of a sample to be measured, and the balance ddH 2 O; added to a 200. Mu.L reaction tube.
The PCR amplification conditions were: the pre-denaturation temperature is 95 ℃ for 5min; denaturation temperature 95 ℃,35s, annealing and extension temperature 59 ℃,35s,45 cycles; after each cycle, a fluorescent signal is detected.
5. Mu.L of each of the African swine fever type I plasmid (corresponding to A1) and the African swine fever type II plasmid (corresponding to A2) obtained in preparation example 2 was taken as a sample to be tested, and the sample was added to the reaction tube, centrifuged at 12000rpm for 5-30s, amplified according to the PCR amplification conditions, and the amplification result was observed on a real-time quantitative amplification apparatus, and the obtained results are shown in Table 1. Amplification curves are shown in FIGS. 2A and 2B.
TABLE 1
As can be seen from Table 1, the technical scheme of the invention can detect the type I and type II Chinese epidemic strains of African swine fever virus simultaneously and detect the two strains in a discrimination way. Meanwhile, the Ct value of the detection is lower, the detection limit is lower, and the sensitivity is higher.
Detection example 2
The PCR system was constructed according to the following conditions:
the total PCR system was 20. Mu.L: the first and second specific probes (concentration 10. Mu. Mol/L each) were each 0.4. Mu.L; the forward primer and the reverse primer (concentration 10. Mu. Mol/L each) in the primer pair were each 0.4. Mu.L; ROX reference dye 0.4. Mu.L; premix Ex TaqProbe qPCR (a conventional commercial product manufactured by Japan under the brand name TaKaRa) 10. Mu.L, 1. Mu.L of a sample to be measured, and the balance ddH 2 O; is added into the reaction tube.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 5s, and fluorescence quantitative PCR test (for selecting annealing temperature) at 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃ according to the Tm value of the primer, wherein the annealing time is 45s,45 cycles, and FAM and VIC channel fluorescence signals are collected after each cycle is finished.
Respectively under the amplification conditions of pair 10 3 Standard positive plasmids of copies/. Mu.L (i.e.A1 and A2 in preparation 2 were each diluted according to a 10-fold gradient, 10 in this case) 3 The fluorescence quantitative amplification (three sets of parallel experiments at each annealing temperature) was performed after the copies/. Mu.L was serially diluted three times according to a 10-fold gradient, and Ct values thereof were recorded, and the obtained results are shown in Table 2.
TABLE 2
As can be seen from Table 2, when the annealing temperature was 59 ℃,10 3 The average Ct values of the copies/. Mu.L standard positive plasmids were 30.390 and 28.978, respectively, with the best amplification efficiency, and therefore, 59℃was chosen as the annealing temperature for real-time fluorescent quantitative PCR.
Detection example 3
The total PCR system was 50. Mu.L: the first and second specific probes (concentration 10. Mu. Mol/L each) were 4. Mu.L each; 4. Mu.L each of the forward primer and the reverse primer (concentration of 10. Mu. Mol/L each) in the primer pair; ROX reference dye 4. Mu.L; premix Ex Taq Probe qPCR (a conventional commercial product manufactured by Japan under the brand name TaKaRa) 25. Mu.L, 5. Mu.L of a sample to be measured, and the balance ddH 2 O; added to a 200. Mu.L reaction tube.
The PCR amplification conditions were: the pre-denaturation temperature is 95 ℃ for 5min; denaturation temperature 95 ℃,5s, annealing and extension temperature 59 ℃,45s,45 cycles. The amplification result is directly observed on a real-time quantitative amplification instrument.
The concentrations of A1 and A2 obtained in preparation example 2 were quantitatively measured by a nucleic acid analyzer, and then diluted by TE buffer in a 10-fold gradient to obtain 1X 10 9 The negative control and the standard sample (three parallel experiments were performed on each gradient of the standard sample) were taken at 5. Mu.L each for each of the samples from copies/. Mu.L to 1 copy/. Mu.L, and the samples were placed in the reaction tube, centrifuged at 12000rpm for 30s, and then placed in an amplification apparatus for amplification.
For the obtained concentration of 10 4 、10 5 、10 6 Fluorescence quantitative PCR was performed simultaneously on the samples (three replicates per concentration) of the copies/. Mu.L, the standard deviation and Coefficient of Variation (CV) in the group were calculated from the Ct values obtained by the detection, and the reproducibility of the method was evaluated based on the results of the coefficient of variation in the group, and the results were shown in Table 3.
TABLE 3 Table 3
From the amplification results, the dilution was 10 -9 (i.e., corresponding to 1 copy/standard sample of reaction), the amplification result is in the critical value range, the detection is negative in three repetitions, positive in detection, and unreliable in result, so the lowest detection limit of the method is 10 -8 The dilution was 50 copies/reaction, as shown in FIG. 3.
From the results in Table 3, it can be seen that the intra-group variation coefficient of the African swine fever virus type I real-time fluorescent quantitative PCR detection method is between 0.129% and 0.414%; the intra-group variation coefficient of the African swine fever virus II type real-time fluorescence quantitative PCR detection method is between 0.362 and 0.712 percent.
Detection example 4
The PCR system and PCR amplification conditions of detection example 3 were used for detection, except that the sample to be tested was 1X 10 in 10-fold increase in concentration 9 Positive control plasmids A1 and A2 of the copies/. Mu.L to 10 copies/. Mu.L were detected for Ct values, respectively, and a concentration-Ct standard curve was constructed based on the concentration and Ct values, the obtained African swine fever virus type I concentration-Ct standard curve is shown in FIG. 3A, and the obtained African swine fever virus type II concentration-Ct standard curve is shown in FIG. 3B, wherein the abscissa is the logarithm of the concentration, and the ordinate is the Ct value.
Comparative example
The PCR total system and PCR amplification conditions of detection example 3 were used, except that the samples to be tested were selected from the group consisting of Porcine Parvovirus (PPV), porcine circovirus type2 (Porcine circovirustype, PCV 2) and PRVSC strain (storage condition-80 ℃ C.) provided by the high technology research laboratory for veterinary biopharmaceuticals in Jiangsu province. Wherein the sample to be detected is obtained by repeatedly freezing and thawing the virus DNA for three times at-80 ℃ and extracting with a DNA extraction kit.
No fluorescent signal was detected by any of PCV2, PPV and PRVSC strains. The comparison result is shown in FIG. 4.
In summary, the qualitative detection judgment standard can be obtained according to the detection example:
judging the result according to the cycle number Ct value of the fluorescent signal reaching a set threshold value: the ASFV-positive control has obvious exponential amplification curve, the Ct value of the two channels is less than or equal to 35, the negative control detection result is that no Ct value is displayed or the Ct value is more than 35, the test can be judged to be established only when the negative control detection result is established, otherwise, the test is invalid. Judging according to the amplification result: FAM is selected independently, the Ct value is less than or equal to 35, the African swine fever virus I type is positive, and the Ct value is more than 35, the African swine fever virus I type is negative; and (3) independently selecting VIC, wherein the Ct value is less than or equal to 35, judging that the African swine fever virus II type is negative, and the Ct value is more than 35. When the Ct value of the detection result of the sample to be detected is greater than 40 or the unreported Ct value is greater than 36, the result is invalid, and the repeated detection of the sample is required.
Further, the fluorescent signal can be quantitatively detected according to the cycle number Ct value of the fluorescent signal reaching a set threshold, and the specific method is as follows:
judging the result according to the cycle number Ct value of the fluorescent signal reaching a set threshold value: the ASFV-positive control has obvious exponential amplification curve, the Ct value of the two channels is less than or equal to 35, the negative control detection result is that no Ct value is displayed or the Ct value is more than 35, the test can be judged to be established only when the negative control detection result is established, otherwise, the test is invalid. Judging according to the amplification result: FAM is selected independently, the Ct value is less than or equal to 35, the African swine fever virus I type is positive, and the Ct value is more than 35, the African swine fever virus I type is negative; and (3) independently selecting VIC, wherein the Ct value is less than or equal to 35, judging that the African swine fever virus II type is negative, and the Ct value is more than 35. When the Ct value of the detection result of the sample to be detected is greater than 40 or the unreported Ct value is greater than 36, the result is invalid, and the repeated detection of the sample is required.
Accordingly, the invention has the following beneficial effects:
1. the multiplex fluorescence PCR kit can be suitable for the differential diagnosis of the African swine fever virus type I and type II Chinese epidemic strains, as long as the nucleotide sequences of the African swine fever virus type I and type II Chinese epidemic strain P72 gene fragments can be completely matched with the primer probes of the method, namely the parent strains of the gene deletion strains can provide correct DNA amplification templates for the primer probes. Therefore, the method is suitable for differential diagnosis of ASFVI type and II type Chinese epidemic strains.
2. The kit can simultaneously diagnose the type I and type II Chinese epidemic strains of the African swine fever virus in a system, realizes double amplification on the premise of not influencing the amplification efficiency, detects the change of the amplification product quantity of each cycle in the PCR amplification reaction in real time by utilizing the change of a fluorescent signal, quantitatively analyzes an initial template through the relation between a Ct value and a standard curve, calculates the copy number of the sample to be detected, has more accurate and visual result, saves time and labor, has lower cost, shortens the time from the original 3-4 hours to 1-1.5 hours, has higher sensitivity than the common PCR, and can be used for detecting the African swine fever virus in a low-micro-content sample.
3. Compared with the fluorescent quantitative PCR technology of SYBR and other dye methods, the TaqmanqPCR has better specificity and can shorten the reaction time; the quenching group NFQ used in the invention is non-fluorescent dye and does not emit fluorescence, so that the probe has low fluorescence background, larger signal-to-noise ratio and higher detection sensitivity; meanwhile, the MGB is used in the invention, so that the Tm value of the probe can be improved, the length of the probe can be shortened, the probe design is facilitated, the difference of Tm values between paired templates and unpaired templates is improved, and the experimental result is more stable and accurate.
4. The kit is not only suitable for quantitative analysis of scientific research units, but also suitable for pathogen detection analysis of various levels of prevention and control units, basic-level veterinary stations, large and medium-sized farms and the like, and has a good application prospect.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. A multiplex fluorescence PCR primer probe group for detecting type I and type II African swine fever viruses is characterized by comprising a primer pair shown as SEQ ID No. 1 and SEQ ID No. 2, a first specific probe shown as SEQ ID No. 3 and a second specific probe shown as SEQ ID No. 4.
2. The multiplex-fluorescent PCR primer probe set of claim 1, wherein the 5' ends of the first and second specific probes are each labeled with a fluorescent reporter group; the 3' ends of the first specific probe and the second specific probe are respectively marked with a minor groove conjugate and a non-fluorescence quenching group; and, the fluorescent reporter group is selected from VIC or FAM.
3. A multiplex fluorescence PCR kit for detecting type I and type II african swine fever virus, wherein the multiplex fluorescence PCR kit comprises the multiplex fluorescence PCR primer set of claim 1 or 2 and a dnase mixed solution.
4. The multiplex-fluorescence PCR kit according to claim 3, wherein the DNase mixture comprises an enzyme diluent, a hot start Taq enzyme, and deoxyribonucleotides.
5. The multiplex fluorescent PCR kit according to claim 4, wherein the deoxyribonucleotides are dATP, dUTP, dGTP and dCTP.
6. The multiplex fluorescence PCR kit according to any one of claims 3-5, wherein the concentration of each of the primer pair, the first specific probe and the second specific probe is 0.1-1.5. Mu. Mol/L.
7. A method for the non-disease diagnostic detection of type I and type II african swine fever virus, characterized in that the multiplex fluorescent PCR kit according to any one of claims 3 to 6 is employed.
8. The method of claim 7, wherein the method comprises:
s100, extracting DNA of a sample to be detected;
s200, performing fluorescent amplification on the DNA extracted in the step S100 by adopting a multiplex fluorescent PCR kit;
s300, judging whether the sample to be detected contains the type I and/or type II African swine fever virus according to a fluorescence amplification result.
9. The method of claim 8, further comprising: preparing a positive control plasmid; diluting the prepared positive control plasmid according to a gradient to obtain a standard sample; and respectively carrying out fluorescent amplification on the obtained standard samples, then measuring Ct values, and constructing a concentration-Ct standard curve.
10. The method according to claim 9, wherein the step S300 further comprises detecting a Ct value of the sample to be tested, and quantitatively detecting the concentration of the sample to be tested according to a concentration-Ct standard curve.
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| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
| CN114058740A (en) * | 2021-12-18 | 2022-02-18 | 扬州大学 | Fluorescent PCR amplification primer probe set capable of identifying genotype I and genotype II African swine fever viruses and detection kit |
| CN114350848A (en) * | 2021-11-30 | 2022-04-15 | 福建傲农生物科技集团股份有限公司 | Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit |
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2022
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
| CN114350848A (en) * | 2021-11-30 | 2022-04-15 | 福建傲农生物科技集团股份有限公司 | Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit |
| CN114058740A (en) * | 2021-12-18 | 2022-02-18 | 扬州大学 | Fluorescent PCR amplification primer probe set capable of identifying genotype I and genotype II African swine fever viruses and detection kit |
Non-Patent Citations (1)
| Title |
|---|
| A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains;Shinuo Cao等;Frontiers in Veterinary Science;1-7 * |
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