CN111118211A - Bovine sarcoidosis detection kit and method based on loop-mediated isothermal amplification technology - Google Patents
Bovine sarcoidosis detection kit and method based on loop-mediated isothermal amplification technology Download PDFInfo
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- CN111118211A CN111118211A CN201910861380.0A CN201910861380A CN111118211A CN 111118211 A CN111118211 A CN 111118211A CN 201910861380 A CN201910861380 A CN 201910861380A CN 111118211 A CN111118211 A CN 111118211A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a bovine sarcoidosis detection kit and a method based on a loop-mediated isothermal amplification technology, wherein two specific inner primers and two specific outer primers are designed according to six specific regions of a 16S rRNA conserved region of bovine sarcoidosis, so that bovine sarcoidosis viruses and other pathogenic viruses can be specifically distinguished, and the high specificity of the loop-mediated isothermal amplification and the reliability of a detection result are ensured. The invention can amplify target sequence efficiently and specifically under isothermal condition, has simple operation, does not need expensive instrument and reagent, can judge result by naked eyes by directly developing the amplified product with fluorescent dye, has low cost, and is especially suitable for small and medium-sized units and field detection application. Particularly, lentinan is added into a loop-mediated isothermal amplification system until the final concentration is 0.1-0.5 mol/L, so that expensive betaine in a common LAMP kit can be partially replaced, and the amplification of high-content GC fragments can be well promoted.
Description
Technical Field
The invention relates to a biological detection kit and a method, in particular to a bovine sarcoidosis detection kit and a method based on a loop-mediated isothermal amplification technology.
Background
Bovine sarcoidosis (LSD), also known as bovine sarcoidosis, bovine nodular dermatitis or bovine pimple skin disease, is a bovine pox disease. The etiological agent is bovine sarcoidosis virus (LSDV), belonging to the genus capripoxvirus of the subfamily Chordopoxvirinae of the family Poxviridae, different from vaccinia virus. The clinical features of the disease include fever, nodules in the skin, mucous membranes and internal organs, emaciation, swollen lymph nodes, edema of the skin, and death in some cases. The disease can also cause a temporary decrease in milk production, cause temporary or permanent infertility in bulls, damage to skin, and even death of the animal due to secondary bacterial infections, thereby causing serious economic impact. The disease is a notification disease of OIE (OIE 2014 edition catalogue), is a type of infectious disease in the ' Advance animal quarantine epidemic disease catalogue of the people's republic of China ', and is a notification disease of Australia (Australia 2013 edition catalogue), Canada (Canada 2014 edition catalogue), America (American national animal health notification system disease catalogue) and European Union (European Union animal epidemic notification system disease catalogue). Since 2010, it was confirmed that up to 43 countries had or were developing the disease during this period, distributed in non-asian eurotrichia, with the african epidemic being particularly severe. In 2019, 8 and 12 months, the disease of bovine sarcoidosis occurred in Ili of the autonomous region of Uygur autonomous region of Xinjiang is confirmed by the foreign animal epidemic disease research center in the China animal health and epidemiology center, which is the first diagnosis of the disease in China. The morbidity of the disease is between 5 and 45 percent, and the mortality rate of the disease is 10 percent. The disease does not infect people, and the harm is mainly caused by the rising of the fatality rate of cattle, the reduction of the quality of cattle hide, the reduction of milk yield and slow weight increase, sometimes causes temporary or permanent sterility of bulls, even causes the death of sick cattle due to secondary bacterial infection, and can also cause the export obstruction of cattle and cattle products and the huge epidemic disease control and elimination cost, thereby causing serious economic loss.
At present, methods for detecting bovine sarcoidosis (LSD) mainly include a separation culture identification method, a Polymerase Chain Reaction (PCR) method, a fluorescent quantitative PCR (FQ-PCR) method, immunofluorescence, agar immunodiffusion, ELISA, and the like. However, the isolation culture assay is complicated and time-consuming to operate, and the PCR method, FQ-PCR and other methods require expensive instruments and reagents, have high detection cost, and are not suitable for small and medium-sized units and field detection applications.
Compared with other nucleic acid amplification technologies, the loop-mediated isothermal amplification (LAMP) technology can rapidly, efficiently and specifically amplify a target sequence under an isothermal condition, is simple and convenient to operate, does not need expensive instruments and reagents, and has a wide development prospect in the field of pathogenic microorganism detection. However, no report on the specific detection of bovine nodular skin disease (LSD) based on loop-mediated isothermal amplification technology has been found so far.
Disclosure of Invention
In view of the above, in order to overcome the defects of the prior art, an object of the present invention is to provide a bovine sarcoidosis (LSD) detection kit based on a loop-mediated isothermal amplification technique; the second purpose is to provide a method for detecting bovine nodular skin disease (LSD) by using the loop-mediated isothermal amplification technology-based bovine nodular skin disease (LSD) detection kit; the sensitivity is high, the specificity is good, the operation is simple, convenient and quick, and the result is accurate and reliable; and the third purpose is to further reduce the conventional LAMP detection cost and be suitable for small and medium-sized units and field detection application.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a cattle nodular skin disease (LSD) detection kit based on a loop-mediated isothermal amplification technology, which comprises the following specific primers:
an upstream inner primer FIP:
5’-ATCATTTCCAAATACAAGTGAGG-gtttttgattcttgtggaaaggtcg-3’；
the downstream inner primer BIP:
5’-AACCTCCCTGGAGGAAAATGC-caacactatagtagtagtagactt-3’；
the upstream outer primer F3: 5'-CATGTTTTTGACAAAAGCTGTT-3', respectively;
downstream outer primer B3: 5'-ttcaacaaagccatctgtatca-3'
Further, one or more of the following reagents are also contained: bacillus stearothermophilus (Bst) DNA polymerase, sagittaria verrucosa polysaccharide, 10 Xthermopolymerization buffer, deoxynucleotide mix of triphosphate bases (dNTPs), magnesium sulfate, betaine, sagittaria verrucosa polysaccharide, and fluorescent dye Saybolt I (SYBR Green I); the 10 Xthermal polymerization buffer solution consists of trihydroxymethyl aminomethane-hydrochloric acid (Tris-HCl) with the concentration of 250mmol/L, pH being 8.8, potassium chloride with the concentration of 100mmol/L, ammonium sulfate with the concentration of 100mmol/L, magnesium sulfate with the concentration of 20mmol/L and Triton (Triton) X-100 with the mass fraction being 1%; the dNTPs are composed of adenine deoxynucleotide triphosphate (dATP), guanine deoxynucleotide triphosphate (dGTP), cytosine deoxynucleotide triphosphate (dCTP) and thymidylate triphosphate (dTTP). The reagents are conventional reagents, can be directly purchased from commercial sources, and are usually prepared in relevant laboratories, so the reagent kit of the invention can be used without the reagents, or only one or more of the reagents, or all the reagents, and provides various options for users to purchase.
The invention also provides a method for detecting bovine nodular skin disease (LSD) by using the loop-mediated isothermal amplification technology-based bovine nodular skin disease (LSD) detection kit, which comprises the following steps:
a. extracting virus DNA of a sample to be detected as template DNA, and controlling OD of template DNA aqueous solution260/OD280A value of 1.6 to 2.0, a concentration of 10 to100ng/μl；
b. Loop-mediated isothermal amplification of bovine sarcoidosis (LSD): preparing a loop-mediated isothermal amplification reaction system with the total volume of 25 mu l in a reaction tube, wherein the loop-mediated isothermal amplification reaction system comprises the following components: upstream inner primer FIP and downstream inner primer BIP with the concentration of 0.2 mu mol/L respectively, upstream outer primer F3 and downstream outer primer B3 with the concentration of 0.8 mu mol/L respectively, 8U Bacillus stearothermophilus DNA polymerase, 1 Xthermal polymerization buffer solution, 1mmol/L adenosine triphosphate deoxynucleotide, guanine triphosphate deoxynucleotide, cytosine triphosphate deoxynucleotide and thymine triphosphate deoxynucleotide, 3mmol/L magnesium sulfate, 1mol/L betaine, 0.1-0.5 mol/L lentinan, 1 mu L of template DNA aqueous solution to be detected and the balance of water; carrying out heat preservation reaction on the reaction tube in a water bath at the temperature of 60-65 ℃ for 30-90 minutes, and then carrying out heat preservation in a water bath at the temperature of 80-95 ℃ for 3-5 minutes to terminate the reaction;
c. and (3) color development detection: adding 1 mu l of fluorescent dye cybor green I water solution with the mass fraction of 10% into a reaction tube, observing the color change by naked eyes, and if the color is yellow, indicating that the sample to be detected does not contain bovine sarcoidosis (LSD); if the color is changed to green, the sample to be detected contains bovine sarcoidosis (LSD).
The invention has the beneficial effects that: firstly, the invention establishes a cattle nodular skin disease (LSD) detection kit and a method based on a loop-mediated isothermal amplification technology, wherein the kit designs two specific inner primers and two specific outer primers according to six specific regions of a conserved region of cattle nodular skin disease (LSD) conserved Sequence DNA (NCBI Reference Sequence: NC-003027.1), thereby ensuring the reliability of a detection result; the invention detects bovine nodular skin disease (LSD) based on the loop-mediated isothermal amplification technology, six specific regions of a target sequence are identified by four primers, and the specificity is strong; amplification under isothermal condition, no time loss caused by temperature change, short time consumption, and amplification of target sequence to 10 in 1 hr9Double and influenced by non-target sequencesSmall noise, higher sensitivity compared with a PCR method, and detection limit of only a few copies; in addition, the technology does not need special and expensive instruments and reagents, the amplification product does not need gel electrophoresis, the result can be judged by naked eyes by directly developing with fluorescent dye, the operation is simple, convenient and quick, and expensive experimental instruments are not needed.
Secondly, the lentinan is used for loop-mediated isothermal amplification for the first time, the thermal stability of enzyme can be obviously improved, when the using concentration is not more than 0.5M, the lentinan can partially replace (about half of) expensive enzyme and betaine in a common LAMP kit, the amplification of high-content GC fragments can be well promoted, the detection reaction time can be reduced to 20-30 minutes, the cost is saved, the detection period is shortened, and the method has important significance for the loop-mediated isothermal amplification.
The kit and the method are particularly suitable for small and medium-sized units and field detection.
Drawings
FIG. 1 is a schematic diagram of a color reaction according to an embodiment of the present invention;
FIG. 2 is a LAMP specificity assay according to one embodiment of the present invention;
FIG. 3 is a LAMP sensitivity test according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention are described in detail below.
Example 1
1. A cattle nodular skin disease (LSD) detection kit based on a loop-mediated isothermal amplification technology comprises the following reagents:
a. an upstream inner primer FIP aqueous solution and a downstream inner primer BIP aqueous solution with the concentrations of 5 mu mol/L respectively, an upstream outer primer F3 aqueous solution and a downstream outer primer B3 aqueous solution with the concentrations of 20 mu mol/L respectively: the primer sequences are as follows:
an upstream inner primer FIP:
5’-ATCATTTCCAAATACAAGTGAGG-gtttttgattcttgtggaaaggtcg-3’；
the downstream inner primer BIP:
5’-AACCTCCCTGGAGGAAAATGC-caacactatagtagtagtagactt-3’；
the upstream outer primer F3: 5'-CATGTTTTTGACAAAAGCTGTT-3', respectively;
downstream outer primer B3: 5'-ttcaacaaagccatctgtatca-3'
b. Bst DNA polymerase aqueous solution with concentration of 8U/. mu.l;
c. 10 × thermal polymerization buffer: the reagent consists of Tris-HCl with the concentration of 250mmol/L, pH of 8.8, potassium chloride with the concentration of 100mmol/L, ammonium sulfate with the concentration of 100mmol/L, magnesium sulfate with the concentration of 20mmol/L and Triton X-100 with the mass fraction of 1 percent;
d. an aqueous solution of dNTPs at a concentration of 10 mmol/L: composed of dATP, dGTP, dCTP and dTTP at concentrations of 10mmol/L each;
e. magnesium sulfate aqueous solution with the concentration of 100 mmol/L;
f. a betaine aqueous solution having a concentration of 2 mol/L;
g. 10% of fluorescent dye SYBR Green I aqueous solution;
h. lentinan with a concentration of 0.1-0.5 mol/L.
The invention also provides a method for detecting bovine nodular skin disease (LSD) by using the loop-mediated isothermal amplification technology-based bovine nodular skin disease (LSD) detection kit, which comprises the following steps:
a. extracting bacterial DNA of a sample to be detected as template DNA: in this example, an experimental group and a blank control group are simultaneously set, wherein the experimental group is bovine nodular skin disease (LSD) and is derived from clinical isolation (LSD positive by PCR); extracting the bacterial DNA of each group by using a DNA extraction kit (Tiangen Biochemical technology Co., Ltd.), and performing the operation according to the kit specification to obtain the OD of the bacterial DNA aqueous solution of the experimental group260/OD280The value was 1.8 and the concentration was 20 ng/. mu.l.
b. Loop-mediated isothermal amplification of bovine sarcoidosis (LSD): preparing a loop-mediated isothermal amplification reaction system with the total volume of 25 mul in a reaction tube: adding 1 mul of upstream inner primer FIP aqueous solution and downstream inner primer BIP aqueous solution which are 5 mul mol/L respectively, 1 mul of upstream outer primer F3 aqueous solution and downstream outer primer B3 aqueous solution which are 20 mul mol/L respectively, 0.5 mul of Bst DNA polymerase aqueous solution with the concentration of 8U/mul, 2.5 mul of 10 Xthermal polymerization buffer solution, 2.5 mul of dNTPs aqueous solution with the concentration of 10mmol/L, 0.75 mul of magnesium sulfate aqueous solution with the concentration of 100mmol/L, 7 mul of betaine aqueous solution with the concentration of 2mol/L, 6 mul of lentinan with the concentration of 0.1-0.5 mol/L, diluting to 24 mul by DNA enzyme-free water, adding 1 mul of template DNA aqueous solution, and mixing uniformly to obtain the lentinan; carrying out heat preservation reaction on the reaction tube in a water bath at the temperature of 60-65 ℃ for 30-60 minutes, and then carrying out heat preservation in a water bath at the temperature of 80 ℃ for 5 minutes to terminate the reaction;
c. and (3) color development detection: 1 mu l of fluorescent dye cybor green I water solution with the mass fraction of 10% is added into a reaction tube, the mixture is shaken uniformly, and the color change is observed by naked eyes.
As a result: the blank control group was yellow in color, indicating that bovine sarcoidosis (LSD) was not present; the color of the experimental group turned green, indicating that bovine sarcoidosis (LSD) was present, consistent with the expected results (see fig. 1, from left to right: 1, 2 for control groups; 3, 4, 5, 6 for positive experimental groups).
d. Specificity assay of LAMP
And establishing an LAMP detection method by using LSDV and genome DNA of the SPPV vaccine strain, the SPPV wild strain and the GTPV vaccine strain as well as genome DNA of the GTPV wild strain and vaccinia virus (cowpox virus) according to the reaction system and conditions, and carrying out specificity test. The LSDV genome DNA is set as a positive control, ultrapure water is set as a negative control, and the result shows that only the LSDV genome has positive reaction, and the rest of LSDV genome has negative reaction (see figure 2, from left to right: 1, SPPV vaccine strain, 2, SPPV wild strain, 3, GTPV vaccine strain, 4, GTPV wild strain, 5, vaccinia virus (cowpox virus), 6, blank control, 7, positive experimental group).
f. LAMP susceptibility test
After the LSDV genomic DNA is diluted by 10-fold gradient, a negative control (ultrapure water) is arranged, and the LAMP detection method is established according to the reaction system and conditions so as to determine the sensitivity of the detection method.
Referring to fig. 3, from right to left: 1-8 are respectively LSDV DNA diluted by 10 times in a gradient manner; 9. 1.1 copy/reaction; 10. negative control (ultrapure water). The results show that: the established LSDV LAMP detection method can detect 11 copies/reacted LSDV DNA in a lateral sample.
Compared with the conventional PCR and fluorescence PCR methods, the LAMP amplification method has the following advantages:
the specificity is strong: the existence of the target gene can be determined only by whether amplification is carried out or not, thereby completing the qualitative detection of the seedlings and the viruses.
The sensitivity is high: the sensitivity of the conventional PCR detection method is 100 pg/reaction (l00 copy number/reaction), and the detection side lower limit is 27 fg/reaction (11 copy number/reaction) by adopting the detection method of the invention.
The operation and the identification are simple and quick: the conventional PCR process can obtain a result within 2-4 hours, the fluorescent quantitative PCR can also take 1-1.5 hours, the detection method provided by the invention can obtain a positive result within 30-60 minutes, the operation is simple and convenient, the requirement on instruments is low, only a common water bath is needed, the result can be directly observed through a dye, the electrophoresis step is omitted, and the method has wide application prospect in the practice of primary detection of bovine sarcoidosis (LSD).
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (3)
1. A bovine sarcoidosis detection kit based on a loop-mediated isothermal amplification technology is characterized in that: the kit contains the following specific primers:
an upstream inner primer FIP:
5’-ATCATTTCCAAATACAAGTGAGG-gtttttgattcttgtggaaaggtcg-3’；
the downstream inner primer BIP:
5’-AACCTCCCTGGAGGAAAATGC-caacactatagtagtagtagactt-3’；
the upstream outer primer F3: 5'-CATGTTTTTGACAAAAGCTGTT-3', respectively;
downstream outer primer B3: 5'-ttcaacaaagccatctgtatca-3' are provided.
2. The bovine sarcoidosis detection kit based on the loop-mediated isothermal amplification technology according to claim 1, characterized in that: the kit also contains one or more of the following reagents:
bacillus stearothermophilus DNA polymerase, 10 Xthermal polymerization buffer solution, base triphosphate deoxynucleotide mixture, magnesium sulfate, betaine, houndsia polysaccharide and fluorescent dye Saybolt I;
the 10 Xthermal polymerization buffer solution consists of trihydroxymethyl aminomethane-hydrochloric acid with the concentration of 250mmol/L, pH of 8.8, potassium chloride with the concentration of 100mmol/L, ammonium sulfate with the concentration of 100mmol/L, magnesium sulfate with the concentration of 20mmol/L and triton X-100 with the mass fraction of 1%;
the base triphosphate deoxynucleotide mixture consists of adenine triphosphate deoxynucleotide, guanine triphosphate deoxynucleotide, cytosine triphosphate deoxynucleotide and thymine triphosphate deoxynucleotide.
3. A method for detecting bovine sarcoidosis using the detection kit of claim 1, characterized in that: the method comprises the following steps:
a. extracting bacterial DNA of a sample to be detected as template DNA, and controlling OD of template DNA aqueous solution260/OD280The value is 1.6-2.0, and the concentration is 10-100 ng/mul;
b. loop-mediated isothermal amplification of bovine sarcoidosis: preparing a loop-mediated isothermal amplification reaction system with the total volume of 25 mu l in a reaction tube, wherein the reaction system comprises the following components: upstream inner primer FIP and downstream inner primer BIP with the concentration of 0.2 mu mol/L respectively, upstream outer primer F3 and downstream outer primer B3 with the concentration of 0.8 mu mol/L respectively, 8U Bacillus stearothermophilus DNA polymerase, 1 Xthermal polymerization buffer solution, 1mmol/L adenosine triphosphate deoxynucleotide, guanine triphosphate deoxynucleotide, cytosine triphosphate deoxynucleotide and thymine triphosphate deoxynucleotide, 3mmol/L magnesium sulfate, 1mol/L betaine, 0.1-0.5 mol/L lentinan, 1 mu L of template DNA aqueous solution to be detected and the balance of water; carrying out heat preservation reaction on the reaction tube in a water bath at the temperature of 60-65 ℃ for 30-90 minutes, and then carrying out heat preservation in a water bath at the temperature of 80-95 ℃ for 3-5 minutes to terminate the reaction;
c. and (3) color development detection: adding 1 mu l of fluorescent dye cybor green I aqueous solution with the mass fraction of 10% into a reaction tube, observing the color change by naked eyes, and if the color is yellow, indicating that the sample to be detected does not contain the bovine sarcoidosis; if the color is changed to green, the bovine sarcoidosis is contained in the sample to be detected.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094946A (en) * | 2020-09-11 | 2020-12-18 | 中国检验检疫科学研究院 | LAMP (loop-mediated isothermal amplification) detection primer and kit for bovine sarcoidosis virus and application of LAMP detection primer and kit |
| CN112198311A (en) * | 2020-08-19 | 2021-01-08 | 中国农业科学院兰州兽医研究所 | Competitive ELISA antibody detection kit for bovine sarcoidosis and preparation method thereof |
| CN112646927A (en) * | 2020-12-23 | 2021-04-13 | 广东省农业科学院动物卫生研究所 | Fluorescent RAA kit for detecting bovine sarcoidosis virus, detection method and application thereof |
| CN112941239A (en) * | 2021-04-08 | 2021-06-11 | 广州悦洋生物技术有限公司 | Primer pair, probe and kit for rapidly detecting bovine nodular skin disease virus |
| CN113373263A (en) * | 2021-05-27 | 2021-09-10 | 华南农业大学 | Primer and kit for detecting LSDV (localized surface plasmon resonance) |
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2019
- 2019-09-12 CN CN201910861380.0A patent/CN111118211A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112198311A (en) * | 2020-08-19 | 2021-01-08 | 中国农业科学院兰州兽医研究所 | Competitive ELISA antibody detection kit for bovine sarcoidosis and preparation method thereof |
| CN112094946A (en) * | 2020-09-11 | 2020-12-18 | 中国检验检疫科学研究院 | LAMP (loop-mediated isothermal amplification) detection primer and kit for bovine sarcoidosis virus and application of LAMP detection primer and kit |
| CN112094946B (en) * | 2020-09-11 | 2022-04-08 | 中国检验检疫科学研究院 | LAMP (loop-mediated isothermal amplification) detection primer and kit for bovine sarcoidosis virus and application of LAMP detection primer and kit |
| CN112646927A (en) * | 2020-12-23 | 2021-04-13 | 广东省农业科学院动物卫生研究所 | Fluorescent RAA kit for detecting bovine sarcoidosis virus, detection method and application thereof |
| CN112646927B (en) * | 2020-12-23 | 2023-02-28 | 广东省农业科学院动物卫生研究所 | Fluorescent RAA kit for detecting bovine sarcoidosis virus, detection method and application thereof |
| CN112941239A (en) * | 2021-04-08 | 2021-06-11 | 广州悦洋生物技术有限公司 | Primer pair, probe and kit for rapidly detecting bovine nodular skin disease virus |
| CN113373263A (en) * | 2021-05-27 | 2021-09-10 | 华南农业大学 | Primer and kit for detecting LSDV (localized surface plasmon resonance) |
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