CN106701961A - Reverse transcription loop-mediated isothermal amplification primer, kit and detection method for visually detecting Hobi-like pest virus - Google Patents
Reverse transcription loop-mediated isothermal amplification primer, kit and detection method for visually detecting Hobi-like pest virus Download PDFInfo
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Abstract
The invention belongs to the technical field of virus detection, and particularly relates to a reverse transcription loop-mediated isothermal amplification (LAMP) primer, a reverse transcription LAMP kit and a reverse transcription LAMP detection method for visually detecting a Hobi-like pest virus. An upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3 of the specific LAMP primer are synthesized; the kit is prepared from the following components: a 10*Thermo Pol reaction buffer solution, dNTP, MgSO4, Bst DNA polymerase, AMV Reverse Transcriptase, a Ribonuclease Ihibitor, DEPC water, a developing reagent and the primer. The kit is high in specificity, high in sensitivity, quick and high in operability.
Description
Technical field
The invention belongs to virus detection techniques field, and in particular to a kind of Visual retrieval Hobi-like pestivirus it is anti-
Transcription loop-mediated isothermal amplification (LAMP) primer, kit and detection method.
Background technology
Hobi-like pestivirus is a kind of new pestivirus for from Brazilian hyclone separate in 2004, with bovine viral
The property type of diarrhea virus 1（BVDV-1）, the type of bovine viral diarrhea virus 2（BVDV-2）, CSFV（CSFV）With sheep border disease virus
(BDV) flaviviridae is belonged to.Afterwards again in succession in other countries, such as Italy, the Switzerland in Europe, the Oceanian big profit of Australia
Asia, the state such as the Mexico in America and the Japan in Asia, India and Bangladesh is found that the viral distribu-tion and presence.The virus infects
The symptom that Niu Houbiao causes is similar to the symptom that BVDV-1 and BVDV-2 infected cattles show, clinic be mainly shown as respiratory tract,
Alimentary canal and genital diseases, therefore some virological investigations person tried to be named as " BVDV-3 " type.
Chinese scholar 2012 is separated to the virus first from the MDBK cells of pollution, this laboratory in 2015 first
Hobi-like pestivirus, strain full genome accession number GenBank are separated to from the sick sheep body with breathing problem
KU563155, shows that the virus not only with natural infection ox, and can infect the small ruminants such as sheep.Current veterinary clinic
On to the virus understanding be also insufficient to, good detection method is not set up clinically yet；Only a small amount of laboratory establishes often
The RT-PCR detection method of rule, Nest RT-PCR method and quantitative fluorescent PCR；These methods often exist need precision instrument and
The limitation of special experiment condition, constrains and now applies.Therefore, a kind of strong detection technique of practical application is set up to be pushed away in basic unit
Wide application, with very strong clinical meaning.
Loop-mediated isothermal amplification technique（Loop-mediated isothermal amplification, LAMP）It is to work as
Before a kind of one of the new technique of nucleic acid rapid amplifying that is most widely used, the method has fast and easy, simple to operate, quick
The features such as perceptual height, strong applicability, accurate result.Mainly using the strand replacement reaction of automatic cycle, nucleic acid expands the method principle
Increasing can typically be completed in 60 min, and amplification efficiency is generally up to 109~1010The individual order of magnitude.Moreover, the method is to reality
Test instrument requirements used very low, a common water-bath can just complete all experimentss reaction.
Chinese patent application CN103924006A discloses a kind of for detecting HoBi sample pestivirus（HoBi-like
pestivirus）The primer sequence of nucleotide fragments, belongs to animal pathogenic technical field of microbial detection.Primer pair includes：
By sequence for the sense primer HoBi-F and sequence of TGGTTCGACGCATTAAGGAAT are
The primer pair of the anti-sense primer HoBi-R compositions of TCTCTGCAGCACCCTATCAG.The set primer sequence is according to GenBank
Upper-UTR the gene orders of HoBi samples pestivirus 5 ' design, with sensitivity and specificity higher.But, the patent primer
Targeted sequence can not completely match the sequence corresponding to some classical strainses, i.e. primer sequence can not be detected all
The typical strain of Hobi-like pestivirus, can cause missing inspection, and need expensive quantitative real time PCR Instrument, Clinical practicability
Difference.
It is reverse transcription loop-mediated that Chinese patent CN101696453B discloses a kind of method for detecting bovine viral diarrhea virus
The preparation of isothermal amplification reaction reagent box and its application method, it is related to the preparation of reaction kit and its application method.And
The kit needs professional equipment and professional to participate in detection and is unsuitable for the problem of Animal Husbandry in China state of development at this stage.
Method：Primer is designed first, reverse transcription loop-mediated isothermal amplification reaction solution is then prepared, that is, complete.Application method：Take containing ox
The RNA extracts of viral diarrhea virus sample, add reaction solution, reverse transcription loop-mediated isothermal amplification are carried out after mixing anti-
Should, then detection is centrifuged；Or 10000 × SYBR GREEN I are added, it is subsequently placed in detection under uviol lamp；Or carry out electrophoresis detection；
Or insulation reaction detection is carried out using Real-time PCR instrument.But, the patent kit can only detect BVDV-1 types poison
Strain, can not detect HoBi-like pestivirus, and use ultraviolet detection or electrophoresis detection, still directly can not visually see
Examine, running cost is high.
The content of the invention
To overcome drawbacks described above, it is an object of the invention to provide a kind of the anti-of Visual retrieval Hobi-like pestivirus
Transcription loop-mediated isothermal amplification (LAMP) primer, kit and detection method.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of reverse transcription loop-mediated isothermal amplification (LAMP) primer of Visual retrieval Hobi-like pestivirus, the isothermal duplication draws
Thing includes upstream inner primer FIP, downstream inner primer BIP, upstream outer primer F3 and downstream outer primer B3, and sequence is as follows respectively：
FIP：TACCCGGGGCTTCGGTAATCCGACGAGCATAATGGGGGAC；
BIP：GGTTCGACGCATCAACGAGTGGCCCGCTTGGGTTAAGACAT；
F3：GCGAAGGCCGAAATAGGTTA；
B3：GGCCACATAATCGACCATCT.
A kind of reverse transcription ring mediated isothermal of the Visual retrieval Hobi-like pestivirus containing above-mentioned isothermal duplication primer
Amplification kit, the kit includes following component：10 × Thermo Pol reaction buffers, dNTP, MgSO4、Bst DNA
Polymerase, AMV Reverse Transcriptase, Ribonuclease Ihibitor, DEPC water, colour reagent and above-mentioned
Primer.
Preferably, the kit includes following component：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Colour reagent 3mM
DEPC water complements to 24 μ L.
Preferably, described colour reagent is hydroxynaphthol blue.
A kind of reverse transcription ring mediated isothermal of the Visual retrieval Hobi-like pestivirus carried out using above-mentioned kit
Amplification detection method, comprises the following steps：
（1）Samples are taken, after preliminary treatment, sample gene group is extracted using viral RNA extracts kit；
（2）Step is added in detection solution in 24 μ L loop-mediated isothermal amplification kits（1）The μ L of sample gene group 1, in 61
DEG C isothermal reaction 1h；Meanwhile, to add Hobi-like seasonal febrile diseases in the detection solution in 24 μ L loop-mediated isothermal amplification kits
μ L are used as positive control for malicious RNA templates 1；
（3）Reaction is directly visually observed after terminating, and positive findings is shown as sky blue, and negative findings is shown as purple.
Preferably, described Hobi-like pestivirus RNA templates are the cell extracted using viral RNA extracts kit
Culture Hobi-like pestivirus liquid geneome RNAs.
Preferably, step（2）Described reacting on carry out in PCR instrument or thermostat water bath.
Positive beneficial effect of the invention：
1. the present invention uses two pairs of primers, and four identification regions, relatively existing detection method, specificity is higher.
2. the reverse transcription ring mediated isothermal kit of Visual retrieval Hobi-like pestivirus of the present invention has special
Property is strong：Expand bovine viral diarrhea virus 1 type of the detection kit to extraction using Hobi-like pestivirus ring mediated isothermal
（BVDV-1）, bovine herpes virus-1（BHV-1）, bovine rota（BRV）, the type of bovine parainfluenza virus 3（BPIV3）Viral base
Because of group, while carrying out LAMP amplifications, and positive control is set, amplification show using other viruses as during template without the positive
Amplification, LAMP method specificity of the present invention is high；
The reverse transcription ring mediated isothermal kit of Visual retrieval Hobi-like pestivirus of the present invention has sensitiveness high：Profit
Expand detection kit with Hobi-like pestivirus ring mediated isothermal and conventional RT-PCR detected to the sample of doubling dilution,
Result shows：LAMP method can detect 1 TCID50/ mL, and the method for standard PCR amplification is only able to detect 100 TCID50/
mL.As can be seen here, LAMP method of the present invention has sensitiveness higher；
The reverse transcription ring mediated isothermal kit of Visual retrieval Hobi-like pestivirus of the present invention has rapidity：Relatively
The reaction time of common PCR reaction a few hours and detection time, detection method complete whole anti-by only needing 1 hour
Should and result judgement；
The reverse transcription ring mediated isothermal kit of Visual retrieval Hobi-like pestivirus of the present invention has operability：Phase
For Standard PCR, Hobi-like pestivirus ring mediated isothermal expands the PCR instrument that detection kit need not be expensive, it is only necessary to one
Simple thermostat water bath can complete reaction；It is special without gel electrophoresis etc. and the detection of result directly can visually judge
Instrument, therefore kit of the present invention has stronger existing ground operability.
3. detection method is easily operated, with low cost, can extensive use.
Brief description of the drawings
Fig. 1 is reverse transcription ring mediated isothermal detection method susceptibility results schematic diagram of the present invention, wherein 1：104 TCID50/
mL；2：103 TCID50/mL；3：102 TCID50/mL；4：101 TCID50/mL；5：100 TCID50/mL；6：10-1 TCID50/
mL；7：Negative control；
Fig. 2 is conventional RT-PCR detection method sensitive gels electrophoretogram, wherein, 1：Negative control；2：10-1 TCID50/mL；
3：100 TCID50/mL；4：101 TCID50/mL；5：102 TCID50/mL；6：103 TCID50/mL；7：104 TCID50/mL；M：
DL2000 DNA Marker；
Fig. 3 is reverse transcription ring mediated isothermal detection method specific detection gel electrophoresis figure of the present invention, wherein 1：BVDV-1；2：
BHV-1；3：BRV；4：BPIV-3；5：Positive control.
Specific embodiment
With reference to some specific embodiments, the present invention is further described.
1st, kit associated materials
The type of bovine viral diarrhea virus 1（BVDV-1）And bovine herpes virus-1（BHV-1）Come from China Veterinery Drug Inspection Office；
Bovine rota（BRV）, the type of bovine parainfluenza virus 3（BPIV3）With Hobi-like pestivirus for laboratory separates preservation strain.
Virus genom DNA/RNA extracts kits（Beijing Quanshijin Biotechnology Co., Ltd）, Betaine（SIGMA companies）,
BstDNA polymerases and 10 × Thermo pol reaction buffers（Biolabs Inc companies）, dNTP（Clontech companies）,
AMV Reverse Transcriptase （Promega companies）、Ribonuclease Ihibitor（NEB companies）、Mg2+And
DL2000（TAKARA companies）.
2nd, the design of LAMP primer and synthesis
With reference to the Hobi-like pestivirus sequences delivered at present（Accession number GenBank KU563155）, for pestivirus
Conservative region 5 '-UTR region, devise relevant primer using LAMP primer Photographing On-line software PrimerExplorer, draw
Thing includes：Upstream inner primer FIP (SEQ ID NO:And downstream inner primer BIP (SEQ ID NO 1):2), upstream outer primer F3
(SEQ ID NO:And downstream outer primer B3 (SEQ ID NO 3):4), this 4 primer sequences are as follows：
FIP：TACCCGGGGCTTCGGTAATCCGACGAGCATAATGGGGGAC；
BIP：GGTTCGACGCATCAACGAGTGGCCCGCTTGGGTTAAGACAT；
F3：GCGAAGGCCGAAATAGGTTA；
B3：GGCCACATAATCGACCATCT；
The primer for completing is designed by the deep fast biosynthesis in Suzhou.
3rd, viral genome is extracted
Using the virus genom DNA/RNA extracts kits of Beijing Quanshijin Biotechnology Co., Ltd, cell culture is extracted
Hobi-like seasonal febrile diseases venom, the doubtful infected animal tissue sample for having Hobi-like pestivirus infections, Specificity control virus
The viral genome of sample.
4th, the foundation of Hobi-like pestivirus RT-LAMP reaction systems
The kit includes following component：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Hydroxynaphthol blue 3mM
DEPC water complements to 24 μ L.
Described reaction system includes following component as follows：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Hydroxynaphthol blue 3mM
The μ L of sample template RNA 1
DEPC water complements to 25 μ L；
It is positioned over after said components are mixed in PCR instrument or 61 DEG C of constant temperature is acted on 1 hour in water-bath；Reaction can be with after terminating
Carry out gel detection or visually observe to judge result, both result of determination are consistent；When visually observing, positive findings shows
Sky blue is shown as, negative findings is shown as purple.
5th, the optimization of Hobi-like pestivirus RT-LAMP detection reagent box condition
5.1 primer concentrations optimize
With pair of primers as variable in the reaction system of 25 μ L, other reaction substrate compositions are identical, successively to FIP and BIP
（0.4μM、0.6μM、0.8μM、1.0μM、1.2μM、1.4μM、1.6μM、1.8μM、2.0μM）, F3 and B3（0.05μM、0.1μM、
0.2μM、0.3μM、0.4μM、0.5μM、0.6μM）Two pairs of primers carry out concentration gradient optimization, and each concentration is repeated 3 times, reaction knot
After beam, 2% agarose gel electrophoresis detection is carried out.
5.2 proliferation times optimize
Configuration reaction system, the sample of each time conditions sets 3 repetitions, reacted in thermostat water bath respectively 30 min,
Taken out after 45 min, 60 min, 75 min, carry out 2% agarose gel electrophoresis detection.
The optimization of 5.3 amplification temperature
Configuration reaction system, each temperature conditionss sample sets 3 repetitions, respectively 60 DEG C of different temperatures, 61 DEG C, 62 DEG C, 63
DEG C, 64 DEG C, 65 DEG C reaction 1 hour after take out, carry out 2% agarose gel electrophoresis detection.
5.4 Mg²+Concentration optimization
To Mg2+Concentration set gradient be 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L, 1.2 mmol/L, 1.4
Mmol/L, each concentration gradient sets 3 repetitions.After the reaction of all concentration gradients terminates, 2% agarose gel electrophoresis is carried out
Detection.
5.5 Bst archaeal dna polymerase concentration optimizations
Concentration to Bst archaeal dna polymerases is optimized, and its gradient is set to 160 U/mL, 320 U/mL, 480 U/mL, 640
U/mL, 800 U/mL, each concentration gradient set 3 repetitions.After the reaction of all concentration gradients terminates, carry out 2% agarose and coagulate
Gel electrophoresis are detected.
5.6 Reverse Transcriptase optimize
Concentration to AMV Reverse Transcriptase is optimized, its gradient be set to 160 U/mL, 320 U/mL,
480 U/mL, 640 U/mL, 800 U/mL, each concentration gradient set 3 repetitions.After the reaction of all concentration gradients terminates,
Carry out 2% agarose gel electrophoresis detection.
6th, the optimum results of Hobi-like pestivirus RT-LAMP methods
6.1 optimization systems and condition
By the optimization of above-mentioned condition, Hobi-like pestivirus ring mediated isothermal expands the system of the last optimization of detection kit
For：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Hydroxynaphthol blue 3mM
The μ L of sample template RNA 1
DEPC water complements to 25 μ L
Reaction condition is 61 DEG C of effect 1h.
Kit described in 6.2 includes following component：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Hydroxynaphthol blue 3mM
DEPC water complements to 24 μ L.
7th, the specific test of RT-LAMP
With clinically common bovine viral disease：The type of bovine viral diarrhea virus 1（BVDV-1）, bovine herpes virus-1（BHV-
1）, bovine rota（BRV）, the type of bovine parainfluenza virus 3（BPIV3）Viral genome as detection object, extract virus
Genome, and carry out LAMP amplifications as template and verify the specificity of its reaction, and sets positive control, as a result referring to
Fig. 3.
Reaction system described in positive control includes following component：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Hydroxynaphthol blue 3mM
The μ L of Hobi-like pestivirus RNA templates 1
DEPC water complements to 25 μ L.
Described Hobi-like pestivirus RNA templates are the cell culture extracted using viral RNA extracts kit
Hobi-like pestivirus liquid geneome RNAs.
Amplification shows that disperse shape band occurs in only positive control, and other four kinds of viruses do not occur, illustrate with it
There is no positive amplification when its virus is as template, LAMP method specificity of the present invention is high.
8th, the sensitivity tests of RT-LAMP
Take known TCID50Hobi-like pestivirus venom, be then diluted with common DMEM nutrient solutions, distinguish it
Equivalent to containing 1 TCID50/mL、10 TCID50/mL、100 TCID50/mL、1000 TCID50/mL、10000 TCID50/mL、
100000 TCID50/ mL, then extracts RNA by virus liquid, and is divided into two parts, the LAMP method inspection after part optimization
Survey, directly visually observed after adding hydroxyl bromophenol blue, determine the sensitiveness of the method；Another part is examined with conventional RT-PCR method
Survey, both testing results are compared, referring to Fig. 1 and Fig. 2, to verify its sensitiveness reacted.
Result shows：LAMP method can detect 1 TCID50/ mL, and the method for standard PCR amplification is only able to detect 100
TCID50/mL.As can be seen here, LAMP method of the present invention has sensitiveness higher.
9th, the accordance of RT-LAMP practical applications
57 Nasal swabs for suffering from respiratory tract infected cattle of clinical acquisitions, extract sample gene group, are detected using the RT-LAMP for setting up
The RT-PCR and fluorescent quantitative RT-PCR method of method and routine are detected, by being detected in three kinds of method practical applications
Results contrast, verifies the accordance of RT-LAMP.
Result shows：Expand detection kit and conventional RT-PCR and glimmering using Hobi-like pestivirus ring mediated isothermal
Light quantitative RT-PCR to obtain 57 parts of samples detect, find RT-LAMP recall rate（18/57）It is significantly higher than traditional
RT-PCR method（10/57）, it is more close with fluorescent quantitative RT-PCR method extracting rate（17/57）, and traditional RT-PCR and glimmering
The positive of light quantitative RT-PCR detection can be detected by RT-LAMP methods.
SEQUENCE LISTING
<110>Nanyang Normal College
<120>The reverse transcription loop-mediated isothermal amplification (LAMP) primer of Visual retrieval Hobi-like pestivirus, kit and detection
Method
<130> /
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 40
<212> DNA
<213>Manually
<400> 1
tacccggggc ttcggtaatc cgacgagcat aatgggggac 40
<210> 2
<211> 41
<212> DNA
<213>Manually
<400> 2
ggttcgacgc atcaacgagt ggcccgcttg ggttaagaca t 41
<210> 3
<211> 20
<212> DNA
<213>Manually
<400> 3
gcgaaggccg aaataggtta 20
<210> 4
<211> 20
<212> DNA
<213>Manually
<400> 4
ggccacataa tcgaccatct 20
Claims (7)
1. a kind of reverse transcription loop-mediated isothermal amplification (LAMP) primer of Visual retrieval Hobi-like pestivirus, it is characterised in that described
Isothermal duplication primer includes upstream inner primer FIP, downstream inner primer BIP, upstream outer primer F3 and downstream outer primer B3, sequence point
It is not as follows：
FIP：TACCCGGGGCTTCGGTAATCCGACGAGCATAATGGGGGAC；
BIP：GGTTCGACGCATCAACGAGTGGCCCGCTTGGGTTAAGACAT；
F3：GCGAAGGCCGAAATAGGTTA；
B3：GGCCACATAATCGACCATCT.
2. a kind of reverse transcription ring of the Visual retrieval Hobi-like pestivirus containing isothermal duplication primer described in claim 1
Mediated isothermal amplification kit, it is characterised in that the kit includes following component：10 × Thermo Pol reaction bufferings
Liquid, dNTP, MgSO4, Bst archaeal dna polymerases, AMV Reverse Transcriptase, Ribonuclease Ihibitor,
Isothermal duplication primer described in DEPC water, colour reagent and claim 1.
3. the reverse transcription ring mediated isothermal amplification reagent of Visual retrieval Hobi-like pestivirus according to claim 2
Box, it is characterised in that the kit includes following component：
The μ L of 10 × Thermo Pol reaction buffers 2.5
FIP/BIP 1.2μM
F3/B3 0.3μM
MgSO4 0.8mM
dNTP 1.4nM
Bst archaeal dna polymerases 8U
Ribonuclease Ihibitor 10U
AMV ReverseTranscriptase 10U
Colour reagent 3mM
DEPC water complements to 24 μ L.
4. the reverse transcription ring mediated isothermal amplification of the Visual retrieval Hobi-like pestivirus according to claim 2 or 3
Kit, it is characterised in that described colour reagent is hydroxynaphthol blue.
5. a kind of reverse transcription ring of the Visual retrieval Hobi-like pestivirus that kit using described in claim 2 is carried out
Mediated isothermal amplification detection method, it is characterised in that comprise the following steps：
（1）Samples are taken, after preliminary treatment, sample gene group is extracted using viral RNA extracts kit；
（2）Step is added in detection solution in 24 μ L loop-mediated isothermal amplification kits（1）The μ L of sample gene group 1, in 61
DEG C isothermal reaction 1h；Meanwhile, to add Hobi-like seasonal febrile diseases in the detection solution in 24 μ L loop-mediated isothermal amplification kits
μ L are used as positive control for malicious RNA templates 1；
（3）Reaction is directly visually observed after terminating, and positive findings is shown as sky blue, and negative findings is shown as purple.
6. the reverse transcription ring mediated isothermal amplification detection of Visual retrieval Hobi-like pestivirus according to claim 5
Method, it is characterised in that described Hobi-like pestivirus RNA templates are the cell extracted using viral RNA extracts kit
Culture Hobi-like pestivirus liquid geneome RNAs.
7. the reverse transcription ring mediated isothermal amplification detection of Visual retrieval Hobi-like pestivirus according to claim 5
Method, it is characterised in that step（2）Described reacting on carry out in PCR instrument or thermostat water bath.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113278736A (en) * | 2021-05-28 | 2021-08-20 | 深圳市易瑞生物技术股份有限公司 | Reagent and method for qualitative detection of bovine herpes virus type I |
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| CN113278736A (en) * | 2021-05-28 | 2021-08-20 | 深圳市易瑞生物技术股份有限公司 | Reagent and method for qualitative detection of bovine herpes virus type I |
| WO2022247186A1 (en) * | 2021-05-28 | 2022-12-01 | 深圳市爱医生物科技有限公司 | Reagent and method for qualitative detection of bovine herpesvirus type i |
| CN113278736B (en) * | 2021-05-28 | 2023-05-05 | 深圳市爱医生物科技有限公司 | Reagents and methods for qualitative detection of bovine herpesvirus type I |
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