CN112301163A - Kit for detecting and identifying capripoxvirus viruses and use method thereof - Google Patents
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Abstract
The invention relates to a virus detection kit and a use method thereof, in particular to a kit for detecting and identifying capripoxvirus viruses (capripoxvirus, ovipoxvirus and bovine sarcoidosis viruses) and a use method for non-disease diagnosis. The kit for detecting and identifying the capripoxvirus virus (capripoxvirus, ovipoxvirus and bovine sarcoidosis virus) comprises specific primers with sequences of SEQ ID No.1 and SEQ ID No. 2. The invention has high specificity and high sensitivity; the method has high cost performance, can screen unknown mutation on any amplicon by only one pair of specific primers, greatly reduces the cost and is simple and convenient to operate compared with a Taq-man probe method.
Description
Technical Field
The invention relates to a virus detection kit and a use method thereof, in particular to a kit for detecting and identifying capripoxvirus viruses (capripoxvirus, ovipoxvirus and bovine sarcoidosis viruses) and a use method thereof.
Background
Capripoxvirus (CPV) belongs to the poxviridae, Chordopoxvirinae, and includes three members of Capripoxvirus (gatopox Virus, GTPV), ovipoxvirus (SPPV), and bovine sarcoidosis Virus (LSDV). The members of the genus capripoxvirus can cause diseases of animals such as bovine sarcoid skin disease (Lumpy skin disease), capripoxvirus (Goatpox), and sheep poxvirus (Sheeppox). The bovine sarcoidosis, also known as bovine sarcoidosis or bovine sarcoidosis, is a disease characterized by widespread nodules, swollen lymph nodes, skin edema and local formation of hard nodules or ulcers on the skin, mucous membranes and the surfaces of internal organs of sick cattle clinically. A sick cow has a reduced appetite, is listened, has damaged skin, has a reduced milk production of the cow, and is temporarily or permanently infertile. The disease is a legal report infectious disease specified by the world animal health Organization (OIE), is a first class infectious disease in the famous book of epidemic diseases of imported animals of the people's republic of China, and is managed by the second class of animal epidemic diseases temporarily in China. The disease is mainly endemic in africa, europe and central asia. Once the nodular skin disease of the cattle is developed in a large scale, the nodular skin disease of the cattle can bring very serious influence on cattle raising industry in China and also bring great pressure on prevention and control work. Goat pox and sheep pox are collectively called goat pox, also called sheep' smallpox, and are acute, febrile and contact infectious diseases of sheep. The world animal health Organization (OIE) classified this disease as a legal communicable infectious disease, which is classified as a type of animal disease in our country. Clinically, it is mainly characterized by fever, erythema, pimple, herpes, blisters, etc. on the skin or mucous membrane of the area without or with little hair. The fatality rate of susceptible flocks of sheep can reach 10-100%, and the fatality rate of lambs can reach 100%. The sheep pox is the most serious epidemic disease in all animal pox diseases, and causes huge economic loss due to reduction of milk production of a milch goat, weight loss of a mutton sheep, reduction of fur quality and abortion of a ewe after infection. The disease is distributed worldwide, and in recent years, the disease is also generated in Guangxi, Guizhou, Heilongjiang, Gansu and other places in China. In view of the serious threat of the members of the capripoxvirus to the breeding industry of cattle and sheep, the establishment of a simple and rapid detection method for distinguishing the capripoxvirus, the sheep poxvirus and the bovine sarcoidosis is urgent and especially necessary.
A kit and a method for detecting or identifying a capripoxvirus virus by a hydrolysis probe method (TaqMan or TaqMan-MGB) have been disclosed, for example, Chinese patent Nos. 201911342156.7 and 201410394083.7. When the method is used for differential diagnosis of the capripoxvirus virus, 1 pair of primers and 3 probes are required to be added, so that the cost is high and the operation steps are complicated.
Disclosure of Invention
The invention provides a kit which overcomes the defects of the prior art, can be used for detecting and identifying goat pox virus, sheep pox virus and bovine nodular skin disease virus, and has the advantages of strong specificity, high sensitivity, simple method and low detection cost, and a using method thereof.
The kit for detecting and identifying the capripoxvirus virus (capripoxvirus, ovipoxvirus and sarcoidosis virus) comprises specific primers with sequences of SEQ ID No.1 and SEQ ID No. 2.
Preferably, the kit for detecting and identifying the capripoxvirus virus (capripoxvirus, ovipoxvirus, and sarcoidosis virus) of the invention further comprises standard control GTPV nucleotide sequence of SEQ ID NO.3, standard control SPPV nucleotide sequence of SEQ ID NO.4 and standard control LSDV nucleotide sequence of SEQ ID NO. 5.
In order to facilitate use, the kit for detecting and identifying the goat pox virus (goat pox virus, sheep pox virus and bovine nodular skin disease virus) is also filled with a saturated fluorescent dye and qPCR reaction premixed solution.
More preferably, the saturation dye in the kit for detecting and identifying the capripoxvirus virus (capripoxvirus, ovine poxvirus, bovine sarcoidosis dermatosis virus) is any one of Eva Green, LC Green Plus or SYTO 9.
The detection method of the kit of the invention for non-disease diagnosis purposes comprises the following steps: extracting the genome DNA of a sample to be detected, preparing a reaction system, carrying out qPCR amplification, collecting a fluorescence signal at a detection temperature, comparing a dissolution curve of the sample to be detected with a dissolution curve of a positive control, and detecting and identifying the virus type of the sample to be detected.
The invention relates to a kit and a detection method for detecting by using a high-resolution melting curve. High-resolution melting (HRM) is a new gene analysis technique that forms melting curves of different forms based on different melting temperatures of mononucleotides. The double-stranded DNA can be gradually melted and releases the fluorescent dye combined with the double-stranded DNA in the temperature rising process, so that the fluorescence intensity is reduced, and the melting curve of the DNA sequence can be generated by monitoring the change of the fluorescence intensity in the temperature rising process in real time. When the temperature resolution in the fluorescent signal recording process reaches 0.02-0.3 ℃/s, the drawn fluorescent curve is called as an HRM curve. The characteristics of DNA fragments such as sequence length and base content are highly correlated with the formation of melting curve, and thus are relatively specific. The high resolution melting curve can be used for analyzing sequence difference of low to single base in an amplicon due to the extremely high temperature scanning resolution (lower than 0.3 ℃/s), so that more information of a DNA double strand can be distinguished.
SYBR Green I is commonly used as a dye in the conventional fluorescent quantitative PCR, and belongs to a non-saturated dye. The unsaturated dye SYBR Green I has an inhibiting effect on PCR amplification at high concentration, and in the process of DNA double strand melting, SYBR Green I molecules are easy to rearrange, namely, dye molecules separated from a melted DNA fragment are combined with double strand DNA which is not melted, so that the result is distorted. Therefore, the melting curve obtained by this system cannot truly reflect the melting of DNA, and the resolution of detection is affected. The invention adopts saturation dyes, in particular to the saturation dyes of LC Green, LC Green Plus, SYTO 9 and Eva Green. The dye has stronger DNA binding capacity, has very low inhibition effect on PCR amplification, and does not generate molecular rearrangement in the process of DNA melting. The dissolution curve obtained by the system can truly reflect the melting condition of DNA, and the detection resolution is obviously improved, so that the HRM technology can distinguish the difference of single base.
The primers for virus detection in the kit are 34 full-length genome sequences of the capripoxvirus virus downloaded from NCBI, wherein 10 full-length genome sequences of GTPV, 9 full-length genome sequences of SPPV and 15 full-length genome sequences of LSDV. The sequences are aligned by using multiple sequences to design 1 pair of common primers, which can simultaneously amplify target fragments in GTPV, SPPV and LSDV genomes, but the amplification products can respectively represent different viruses. The specific primers are an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2.
The using method of the invention is as follows:
(1) extraction of viral DNA: a commercially available virus DNA extraction kit is selected to extract a sample to be detected or goat pox virus negative bovine epithelial cell genome DNA to obtain a template DNA to be detected and a negative control DNA.
(2) Preparing an amplification reaction system. The amplification reaction system in the embodiment of the invention is as follows: 18 mu L of qPCR premixed reaction liquid, and sequentially adding 2 mu L of negative control, 2 mu L of template DNA to be detected and 2 mu L of positive control respectively; the total volume of the reaction system was 20. mu.L.
(3) Amplification by qPCR was performed. The specific method comprises the following steps: carrying out qPCR amplification on the amplification reaction system prepared in the step (2), wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, one cycle; 5s at 95 ℃, 10s at 56 ℃ and 15s at 72 ℃; 42 cycles, collecting fluorescence signals at 72 ℃ for 15 s; collecting fluorescence signals at the temperature of 60-95 ℃ at the temperature of 0.01-0.2 ℃/0.1sec in one cycle of 95 ℃ for 10s, 60 ℃ for 60s, 95 ℃ for 15s and 60 ℃ for 15 s.
(4) And (4) judging a result: if the positive control has an obvious amplification curve and the Ct value is less than or equal to 22; negative contrast has no amplification curve and Ct value is larger than 34 or has no numerical value, and the test is judged to be true if the negative contrast and the Ct value are both true, otherwise, the test is invalid.
1) If the sample to be detected has an obvious amplification curve, the Ct value is less than or equal to 34, and the dissolution curve graph is completely coincided with the GTPV positive standard dissolution curve, the sample is reported to be goat pox virus (GTPV) nucleic acid positive, the sample is completely coincided with the SPPV positive standard dissolution curve, the sample is reported to be sheep pox virus (SPPV) nucleic acid positive, and the sample is completely coincided with the LSDV positive standard dissolution curve, the sample is reported to be bovine sarcoidosis virus (LSDV) nucleic acid positive.
2) If the sample to be detected has an obvious amplification curve, the Ct value is more than 30 and less than or equal to 34, and the dissolution curve is suspicious when being matched with the GTPV, SPPV and LSDV positive standard dissolution curve part, the sample needs to be processed and detected again; if the sample is still suspicious, the sample is judged to be positive. If the Ct value of the repeated detection is more than 35 and the dissolution curve is not matched with the GTPV, SPPV and LSDV positive standard dissolution curve completely, the test result is negative.
3) If the sample to be detected has no obvious amplification curve, the Ct value is more than 35 or has no numerical value and the dissolution curve has no peak diagram, or the dissolution curve is completely not matched with the GTPV, SPPV and LSDV positive standard dissolution curve, the sample to be detected is judged to be negative.
The invention has high specificity and high sensitivity; the method has high cost performance, can screen unknown mutation on any amplicon by only one pair of specific primers, and has greatly reduced cost and simple and convenient operation compared with a Taq-man probe method.
Drawings
FIG. 1 is a diagram showing the results of setting the reaction conditions of the kit for detecting and identifying a capripoxvirus virus according to the embodiment of the present invention.
FIG. 2 is a schematic diagram showing the results of the kit for detecting and identifying a capripoxvirus virus according to the embodiment of the present invention.
FIG. 3 is a diagram showing the result of a specific test of the kit for detecting and identifying a capripoxvirus virus according to the embodiment of the present invention, wherein the left diagram is an amplification curve and the right diagram is a lysis curve.
FIG. 4 is a diagram showing the results of the sensitivity test for detecting caprine pox virus using the kit for detecting and identifying caprine pox virus according to the present invention, wherein the left diagram is an amplification curve and the right diagram is a standard curve.
FIG. 5 is a diagram showing the result of the sensitivity test for detecting vaccinia virus using the kit for detecting and identifying capripoxvirus in the example of the present invention, wherein the left diagram is an amplification curve and the right diagram is a standard curve.
FIG. 6 is a graph showing the results of the sensitivity test for detecting bovine sarcoidosis, a skin disease virus using the kit for detecting and identifying capripoxvirus in the present invention, wherein the left graph is an amplification curve and the right graph is a standard curve.
Detailed Description
The present invention will be explained in detail with reference to examples.
First, design and screening of primers
Based on the design of fluorescence quantitative PCR amplification primers for identifying the capripoxvirus virus by a high resolution melting curve method (HRM), 34 full-length genome sequences of the capripoxvirus virus are downloaded from NCBI, wherein 10 full-length genome sequences of GTPV, 9 full-length genome sequences of SPPV and 15 full-length genome sequences of LSDV. And performing multiple sequence alignment on the sequences by utilizing Megalign, searching a section with common conserved sequences at two ends and obvious difference of middle sequences in GTPV, SPPV and LSDV genomes, and designing a common primer in the section by adopting primer 3.0. The primer can simultaneously amplify target fragments in GTPV, SPPV and LSDV, but the amplification products thereof can respectively represent different viruses. By analyzing 34 whole genomes of the goat pox virus, a pair of fluorescent quantitative amplification primers is designed, wherein the upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and the downstream primer with a nucleotide sequence shown as SEQ ID NO.2 are synthesized by biological engineering (Shanghai) GmbH.
The amplification condition in the reaction process is monitored in real time by using a BioRad CFX96 PCR instrument, parameters such as primer concentration, reaction program, maximum amplification rate and the like are analyzed, and parameters of an optimal amplification system and reaction conditions are screened out.
Preparation of a Positive control
The upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and the downstream primer with a nucleotide sequence shown as SEQ ID NO.2 are utilized to respectively clone gene segments with target sequences of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in GTPV, SPPV and LSDV, the gene segments are constructed to a PUC57 plasmid, after the sequencing is correct, an enterobacter coli Top10 competent cell is transformed, the enterobacter Top10 competent cell is subjected to amplification culture, and the plasmid is extracted to be used as a GTPV, SPPV and LSDV positive standard control.
Thirdly, preparation of reaction system
(1) Preparation of Single negative control reaction System (20.0. mu.L)
qPCR reaction premix 18.0. mu.L;
negative control 2.0 μ L;
total reaction system totaled 20.0. mu.L
(2) Preparation of Single sample reaction System to be tested (20.0 μ L)
qPCR reaction premix 18.0. mu.L;
2.0 mu L of template DNA to be detected;
total reaction system totaled 20.0. mu.L
(3) Preparation of Single Positive control reaction System (20.0. mu.L)
qPCR reaction premix 18.0. mu.L;
positive control 2.0 μ L;
total reaction system totaled 20.0. mu.L
Fourth, kit for detecting and identifying capripoxvirus virus and use method
(1) Extraction of viral DNA: and extracting the genome DNA of the sample to be detected by using a commercially available virus DNA extraction kit to obtain the template DNA to be detected.
(2) Preparing a reactant system: comprises a negative control, a sample to be detected and a positive control, and the total volume of the reaction system is 20 mu L.
(3) Amplification of qPCR: carrying out qPCR amplification on the amplification reaction system prepared in the step (2), wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, one cycle; collecting fluorescence signals at 95 ℃ for 5s, 56 ℃ for 10s, 72 ℃ for 15s, and 42 cycles at 72 ℃ for 15 s; 10s at 95 ℃ and 60s at 60 ℃; the fluorescence signals are collected at the temperature of between 60 ℃ and 95 ℃ at the temperature rising rate of 0.01-0.1 ℃/0.1sec in one cycle of 95 ℃ and 15s at 60 ℃ for 15s (see figure 1).
(4) Result determination (see fig. 2): if the positive control has an obvious amplification curve and the Ct value is less than or equal to 22, and the negative control has no amplification curve and the Ct value is greater than 34 or has no numerical value, the test can be judged to be true if the positive control and the negative control both have the amplification curve, and the test is invalid if the negative control and the Ct value are both true.
1) If the sample to be detected has an obvious amplification curve, the Ct value is less than or equal to 34, and the dissolution curve graph is completely coincided with the GTPV positive standard dissolution curve, the sample is reported to be goat pox virus (GTPV) nucleic acid positive, the sample is completely coincided with the SPPV positive standard dissolution curve, the sample is reported to be sheep pox virus (SPPV) nucleic acid positive, and the sample is reported to be bovine sarcoidosis virus (LSDV) nucleic acid positive if the sample is completely coincided with the LSDV positive standard dissolution curve.
2) If the sample to be detected has an obvious amplification curve, the Ct value is more than 30 and less than or equal to 34, and the dissolution curve is suspicious when being matched with the GTPV, SPPV and LSDV positive standard dissolution curve part, the sample needs to be processed and detected again; if the sample is still suspicious, the sample is judged to be positive. If the Ct value of the repeated detection is more than 34 and the dissolution curve is not matched with the GTPV, SPPV and LSDV positive standard dissolution curve completely, the test result is negative.
3) If the sample to be detected has no obvious amplification curve, the Ct value is more than 35 or has no numerical value and the dissolution curve has no peak diagram, or the dissolution curve is completely not matched with the GTPV, SPPV and LSDV positive standard dissolution curve, the sample to be detected is judged to be negative.
Fifth, the kit for detecting and identifying the goat pox virus (50 pieces)
TABLE 1 kit composition list for detecting and identifying capripoxvirus virus
The qPCR reaction premix, positive control, reference fluorescent Dye (ROX Dye I, ROX Dye II) and negative control were packaged in an external packaging box in the amounts required in Table 1, and labeled (including identification name, lot number, production date, storage life, and production unit information).
Sixth, kit specificity test for detecting and identifying goat pox virus
And (3) carrying out specificity test by adopting the reaction systems and reaction conditions in the third and fourth steps, wherein the adopted templates are respectively negative control, bovine nodular skin disease virus, goat pox virus, sheep aphtha virus, foot and mouth disease virus O type virus, bovine viral diarrhea virus and positive control. The test results show in figure 3, according to the kit result judgment method, the bovine sarcoidosis virus, the caprine poxvirus and the sheep pox virus are specifically amplified, and the three viruses can be identified by a high resolution melting curve (HRM). Has no cross reaction with sore mouth virus, foot and mouth disease virus O type virus and bovine viral diarrhea virus. Seventh, kit sensitivity test for detecting and identifying goat pox virus
By adopting the reaction systems and reaction conditions in the third and fourth steps and performing a sensitivity test by diluting the positive control in example 2 by 10 times with a DNA diluent, the lowest detectable lower limit of the established method is as follows: GTPV is 1.35X 101Copy/. mu.L, SPPV 1.8X 101Copy/. mu.L, LSDV 1.29X 101Copies/. mu.L of DNA samples, where GTPV results are shown in FIG. 4, the amplification plots from left to right represent template concentrations after dilution by fold of 1.35X 107Copy/. mu.L, 1.35X 10/6Copy/. mu.L, 1.35X 10/5Copy/. mu.L, 1.35X 10/4Copy/. mu.L, 1.35X 10/3Copy/. mu.L, 1.35X 10/2Copy/. mu.L, 1.35X 101Copies/. mu.L. The amplification efficiency in the standard curve is 104.3%, the correlation coefficient is 0.999, and the experiment has high amplification efficiency, good correlation between the fluorescence curve and the concentration of the detected target gene and high accuracy. The SPPV results are shown in FIG. 5, which shows the template concentrations after dilution by multiple ratios from left to right in the amplification plot as 1.8 × 107Copy/. mu.L, 1.8X 106Copy/. mu.L, 1.8X 105Copy/. mu.L, 1.8X 104Copy/. mu.L, 1.8X 103Copy/. mu.L, 1.8X 102Copy/. mu.L, 1.8X 101Copies/. mu.L. The amplification efficiency in the standard curve is 105.3%, the correlation coefficient is 0.996, and the experiment has the advantages of high amplification efficiency, good correlation between the fluorescence curve and the concentration of the detected target gene and high accuracy. LSDV results are shown in FIG. 6, which shows the template concentration after dilution from left to right of the amplification curve as 1.29X 107Copy/. mu.L, 1.29X 106Copy/. mu.L, 1.29X 105Copy/. mu.L, 1.29X 104Copy/. mu.L, 1.29X 103Copy/. mu.L, 1.29X 102Copy/. mu.L, 1.29X 101Copies/. mu.L. The amplification efficiency in the standard curve is 103.3%, and the correlation coefficient is 0.999, so that the amplification efficiency of the primer is very high, the correlation between the fluorescence curve and the concentration of the detected target gene is good, and the accuracy is high.
Eighthly, the kit for detecting and identifying the capripoxvirus virus is used for clinical sample tests, and 820 parts of clinical samples with known backgrounds are detected by adopting the kit for detecting and identifying the capripoxvirus virus. The test result shows that the established method can detect the positive nucleic acid samples of goat pox virus (GTPV), sheep pox virus (SPPV) and bovine sarcoidosis virus (LSDV). The established detection method has the advantages of high accuracy, reliability, practicability, low cost and simple and convenient operation.
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> kit for detecting and identifying capripoxvirus virus and use method thereof
<160> 5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence upstream primer (forward primer)
<400>
tccgccattt aagttcattt t 21
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence downstream primer (reverse primer)
<400>
ttggagtttt tatgtcatcg tca 23
<210> 3
<211> 160
<212> DNA
<213> goat pox virus (GTPV)
<400>
acgtaacaaa atgccaaatc gctattcgat gacatttttg ctctcacttt ctgagacttt 60
tttttcagaa ataccgtttt tgtgctcgca tgtattattt aaacaattcc atatatcatt 120
caatattaaa gtttttttaa ttggttcctc caaatccatt 160
<210> 4
<211> 160
<212> DNA
<213> sheep pox Virus (SPPV)
<400>
acgtaacaaa atgccaaatc gctattcgat gacatttttg ctatcacttt ctaaaacttt 60
tttttcagaa ataccgtttt tgtgatcaca tgtattattt aaacaattcc atatgttgtt 120
taatattaaa gtttttttaa ttggttcctc caaatccatt 160
<210> 5
<211> 160
<212> DNA
<213> bovine sarcoidosis virus (LSDV)
<400>
acgtaacaaa atgccaaatc gctattcgat gacatttttg ctctcacttt ctgaaacttt 60
tttttcagaa ataccgtttt tgtgctcgca tgtattattt aaacaattcc atatatcgtt 120
caatattaaa gtttttttaa ttggttcctc caaatccatt 160
Claims (5)
1. The kit for detecting and identifying the goat pox virus is characterized in that the kit comprises specific primers with sequences of SEQ ID No.1 and SEQ ID No. 2.
2. The kit for detecting and identifying the capripoxvirus virus of claim 1, wherein the kit further comprises GTPV nucleotide standard control having the sequence shown in SEQ ID NO.3, SPPV nucleotide standard control having the sequence shown in SEQ ID NO.4 and LSDV nucleotide standard control having the sequence shown in SEQ ID NO. 5.
3. The kit for detecting and identifying capripoxvirus virus of claim 1 or 2, wherein the kit further comprises a pre-mixed solution of saturated fluorescent dye and qPCR reaction.
4. The kit for detecting and identifying capripoxvirus virus according to claim 3, wherein the saturation dye in the kit is any one of Eva Green, LC Green Plus or SYTO 9.
5. Detection method for non-disease diagnostic purposes using any one of the kits of claims 1 to 4, characterized in that: extracting genome DNA of a sample to be detected, preparing a reaction system, carrying out qPCR amplification, collecting a fluorescence signal at a detection temperature, comparing a dissolution curve of the sample to be detected with a dissolution curve of a positive control, and detecting and identifying viruses and varieties in the sample to be detected.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831608A (en) * | 2021-03-12 | 2021-05-25 | 福建省农业科学院畜牧兽医研究所 | Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM detection reagent |
| CN113186358A (en) * | 2021-06-16 | 2021-07-30 | 中国动物卫生与流行病学中心 | Universal circulating fluorescent PCR (polymerase chain reaction) detection method for capripoxvirus viruses |
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| CN103276111A (en) * | 2013-05-24 | 2013-09-04 | 中国农业科学院兰州兽医研究所 | Kit used for detecting sheep pox virus and detection method thereof |
| CN104152584A (en) * | 2014-08-12 | 2014-11-19 | 重庆出入境检验检疫局检验检疫技术中心 | Capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method |
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| CN103276111A (en) * | 2013-05-24 | 2013-09-04 | 中国农业科学院兰州兽医研究所 | Kit used for detecting sheep pox virus and detection method thereof |
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| CN111118215A (en) * | 2020-01-16 | 2020-05-08 | 广东省农业科学院动物卫生研究所 | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus |
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| GELAYE等: "A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance", 《SCIENTIFIC REPORTS》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831608A (en) * | 2021-03-12 | 2021-05-25 | 福建省农业科学院畜牧兽医研究所 | Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM detection reagent |
| CN113186358A (en) * | 2021-06-16 | 2021-07-30 | 中国动物卫生与流行病学中心 | Universal circulating fluorescent PCR (polymerase chain reaction) detection method for capripoxvirus viruses |
| CN113186358B (en) * | 2021-06-16 | 2022-05-10 | 中国动物卫生与流行病学中心 | Universal cycle fluorescent PCR (polymerase chain reaction) detection method for capripoxvirus viruses |
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