CN107236824A - A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection - Google Patents
A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection Download PDFInfo
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Abstract
The invention provides a kind of fluorescent quantitation RT PCR kits for porcine reproductive and respiratory syndrome virus specific detection and application, belong to kit technical field, the kit includes following components：The specific primer pair and probe of reaction mixture, water and specific amplification porcine reproductive and respiratory syndrome virus ORF6 genes.The present invention has good linear relationship in the range of the templates of 101~108copies μ L 1, and sensitivity reaches 101copies μ L 1；This method can in real time be monitored to its result；This method has good specificity；Rapidly and accurately PRRSV can be detected in 2 3h, qualitative detection, and energy accurate quantitative analysis can be carried out, and classical strainses, highly pathogenic mutant strain and NADC30 like strains can be detected simultaneously.
Description
Technical field
It is used for pig breeding the present invention relates to a kind of fluorescence quantitative RT-PCR detecting kit, more particularly to one kind and breathes comprehensive
The primer pair of simulator sickness virus-specific detection and probe, fluorescence quantitative RT-PCR kit and application.
Background technology
Porcine reproductive and respiratory syndrome（Porcine reproductive and respiratory syndrome,
PRRS）It is by porcine reproductive and respiratory syndrome virus（PRRSV）The highly contagious disease of a caused boar.It is sent out with sow
The respiratory symptom and high mortality of the breeding difficulty such as heat, apocleisis and miscarriage, stillbirth, the weak son of production and piglet are characterized.1987
Year, the disease was found in the U.S. first, then broke out and be popular in almost all of pig-raising countries, caused international extensive pass
Note.China in 1996 reports first there is PRRS, hereafter spreads rapidly in China, and China's outburst in 2006 is high to be caused
Sick PRRS epidemic situations, involve scope extensively, extremely heavy economic loss are caused to pig industry.Since the end of the year 2013, multiple provinces
Part it is found that with U.S. NADC30 strains homology very high NADC30-like strains, the strain there is 131 in nsp2 areas in succession
The discontinuous missing of individual amino acid.Epidemiological investigation shows that NADC30-like strains are in gradually in the multiple areas of China
Epidemic status.PRRS has turned into one of main epidemic disease of harm China pig industry.
Current PRRSV common detection methods include virus purification and identification, indirect immuno-fluorescence assay (IFA),
Serum neutralization test (SN), reverse transcription-polymerase chain reaction (RT-PCR) and sequencing etc..These conventional methods are used for
PRRSV detect when specificity, sensitiveness and it is ageing in terms of Shortcomings, particularly virus infection early diagnosis in body
Now it is particularly evident.Standard PCR is combined by real-time fluorescence quantitative PCR with fluorescence probe detection technique, with high specificity, spirit
Sensitivity is high, it is reproducible the advantages of, and easy to operate, the used time is short, and pollution is few, by analysis software can automatically quantitative analysis,
As a result it is more accurate and visual, it is adaptable to the qualitative and quantitative detection of large batch of virus, it has been increasingly becoming the weight of pathogen detection
Want method.
The content of the invention
It is fixed it is an object of the invention to provide a kind of fluorescence for porcine reproductive and respiratory syndrome virus specific detection
Measure RT-PCR kit and application.
For achieving the above object, the present invention is realized by following measure：It is comprehensive with breathing that one kind is used for pig breeding
The specific primer pair and probe of simulator sickness virus-specific detection, wherein, the nucleosides of two primers of specific primer centering
Acid sequence difference is as follows：
Sense primer：5' CCATGGAAACCTGGAGATTCA 3'
Anti-sense primer：5' GCGGCCTAGCAAGCACAA 3'；
The probe sequence is as follows：
5' FAM-CACCTCCAGATGCC-MGB 3'。
In addition, the invention provides the fluorescence quantitative RT-RCR for porcine reproductive and respiratory syndrome virus specific detection
Kit, wherein, the kit includes following components：Reaction mixture, water and specific amplification porcine reproductive and respiratory syndrome
The specific primer pair and probe of virus O RF6 genes；
The nucleotide sequence difference of described two primers of specific primer centering is as follows：
Sense primer：5' CCATGGAAACCTGGAGATTCA 3'
Anti-sense primer：5' GCGGCCTAGCAAGCACAA 3'
The probe sequence is as follows：
5' FAM-CACCTCCAGATGCC-MGB 3'。
Wherein, the reaction mixture is the Premix Ex Taq purchased from TaKaRa companies（Probe qPCR）.
Wherein, the water is the RNase-free Water that water is available from TaKaRa companies.
In addition, the invention provides the described fluorescent quantitation for porcine reproductive and respiratory syndrome virus specific detection
Application of the RT-PCR kit in porcine reproductive and respiratory syndrome virus detection reagent is prepared.
Wherein, this kit answering in PRRSV classical strainses, highly pathogenic mutant strain and NADC30-like strains
With.
Beneficial effects of the present invention are：According in GenBank, porcine reproductive and respiratory syndrome virus（PRRSV）Sequence is set
1 pair of meter is directed to the primer and 1 specificity T aqMan-MGB probe of ORF6 genes, establishes a kind of quick detection PRRSV viruses
The specificity T aqMan-MGB fluorescent quantitative RT-PCR methods of carrying capacity.Pass through the optimization to reaction condition and reaction system so that
This method has good linear relationship in the range of 101~108copies μ L-1 templates, and sensitivity reaches 101copies μ
L-1, is 100 times of RT-PCR method.And compared with conventional RT-PCR method, this method can in real time be supervised to its result
Control, without further carrying out gel electrophoresis analysis.Experiment shows, this method and the viral no cross reaction of other swine diseases, with good
Good specificity.Measuring samples are extracted after RNA reverse transcriptions, quantitative fluorescent PCR reaction is carried out, utilizes set up fluorescent quantitation
RT-PCR method can rapidly and accurately be detected in 2-3h to PRRSV, can be carried out qualitative detection, can accurately be determined again
Amount, and classical strainses, highly pathogenic mutant strain and NADC30-like strains can be detected simultaneously.
Brief description of the drawings
Fig. 1 is the PCR amplification figures of purpose gene.M：DL2000 Marker；1：PRRSV classical strainses；2：It is highly pathogenic to become
Different strain；3：NADC30-like strains；4：Negative control.
Fig. 2 is the dynamic curve diagram of fluorescence quantitative RT-RCR.1-8 is respectively 108 ~ 101copies μ L-1 standard items
Fig. 3 is the canonical plotting of fluorescence quantitative RT-RCR.
Fig. 4 is the specific test figure of fluorescence quantitative RT-RCR.1：Standard items；2：Highly pathogenic mutant strain；3：It is classical
Strain；4：NADC30-like strains；5-11 is respectively CSFV, PRV, PEDV, TGEV, PCV2, RV and negative control.
Fig. 5 is the sensitivity tests figure of conventional RT-PCR.M：DL2000 Marker；1-7：Dilution factor be 1.2 × 107 ~
1.2 × 101copies μ L-1 template DNA；8：Negative control.
Fig. 6 is the replica test figure of fluorescence quantitative RT-RCR.
Embodiment
For the technical characterstic for illustrating this programme can be understood, below by embodiment, this programme is illustrated.
Embodiment 1 is used for the fluorescence quantitative RT-PCR kit of porcine reproductive and respiratory syndrome virus specific detection
The kit includes following components：Reaction mixture, water and specific amplification porcine reproductive and respiratory syndrome virus ORF6
The specific primer pair and probe of gene；
The nucleotide sequence difference of described two primers of specific primer centering is as follows：
Sense primer：5' CCATGGAAACCTGGAGATTCA 3'
Anti-sense primer：5' GCGGCCTAGCAAGCACAA 3'
The probe sequence is as follows：
5' FAM-CACCTCCAGATGCC-MGB 3'。
Wherein, the reaction mixture is the Premix Ex Taq purchased from TaKaRa companies（Probe qPCR）.
Wherein, the water is the RNase-free Water that water is available from TaKaRa companies.
In addition, fixed present invention also offers the described fluorescence for porcine reproductive and respiratory syndrome virus specific detection
Measure application of the RT-PCR kit in porcine reproductive and respiratory syndrome virus detection reagent is prepared.
Application of the kit of the present invention in detection porcine reproductive and respiratory syndrome virus
1 materials and methods
1.1 bacterial strains and strain
E.coli DH5 α are purchased from TaKaRa companies, PRRSV classical strainses VR2332（GenBank accession number AY150564.1）With
CH1-α（GenBank accession number AY032626.1）；Highly pathogenic strain HuN4（GenBank accession number EF635006.1）And
NADC30-like strains are preserved by this laboratory.
1.2 instruments and reagent
CFX96 quantitative real time PCR Instruments are purchased from Bio-Rad companies, and ultraviolet specrophotometer is purchased from Thermo companies；Plasmid extraction is tried
Agent box is purchased from AxyGen companies；Extraction reagent kit is purchased from invitrogen companies to DNA/RNA nucleic acid altogether；Premix Ex Taq
（Probe qPCR）Purchased from TaKaRa companies；5 × All-In-One RT MasterMix are purchased from abm companies.
The design and synthesis of 1.3 probes
Prepared according to the method for embodiment 1.
Using MegAlign softwares to PRRSV the classical strainses VR2332 and CH-1a, highly pathogenic that are announced in GenBank
The ORF6 gene orders of the strains such as variation strain's HuN4 and NADC30-like strain are compared analysis, in its conservative region
Design specific primer and TaqMan-MGB fluorescence probes.Upstream primer sequence is Primer F: 5'
CCATGGAAACCTGGAGATTCA 3', downstream primer sequence is Primer R: 5' GCGGCCTAGCAAGCACAA 3'.Visit
Pin sequence is Probe: 5'FAM- CACCTCCAGATGCC-MGB 3'.The fluorescent reporter group of probe 5' ends mark is FAM,
3' ends mark fluorescent quenching group is Non-fluorescent quencher and Minor Groove Binder (MGB).
The extraction and cDNA synthesis of 1.4 viral RNAs
With DNA/RNA nucleic acid, extraction reagent kit extracts viscera tissue homogenate, viral cultures and blood respectively by its operational manual altogether
Clear total serum IgE, is dissolved in the water that 20 μ L are free of RNase, for synthesizing cDNA.The μ L of total serum IgE 5.5 are taken to add 20 μ L reverse transcription systems
In, wherein being free of RNase water comprising 4 μ L 5 × All-In-One RT MasterMix and 10.5 μ L, mix after 25 DEG C
10min, 42 DEG C of 30min, gained cDNA can be directly used for quantitative fluorescent PCR or put -20 DEG C saving backup.
The preparation of 1.5 plasmid standards
By primer of Primer F/R by standard PCR amplification PRRSV ORF6 target gene, the recovered kits of product
PMD19-T carriers are connected afterwards, and conversion to E.coli DH5 α Escherichia coli is by sequencing acquisition positive colony plasmid
Plasmid standard.Determine after DNA concentration, be converted into copy number, and be diluted to 1010copies μ L-1, -20 DEG C of preservations, before
Dilution is used as plasmid standard.
The optimization of 1.6 quantitative fluorescent PCR reaction conditions
Using plasmid standard as template, Standard PCR reaction is carried out under different annealing temperature, amplified production passes through Ago-Gel
Electrophoresis is analyzed, it is determined that optimal primer annealing temperature.Application matrix method is entered to the primer and probe concentration of quantitative fluorescent PCR
Row optimization, to obtain optimal reaction system and reaction condition.
The foundation of 1.7 quantitative fluorescent PCR standard curves
It is 101-108copies μ L by 10 times of doubling dilutions of plasmid standard to concentration range-1.Each dilution factor sets 3 weights
It is multiple, fluorescence quantitative PCR detection is carried out, standard curve is drawn.
1.8 specific test
Using the fluorescence quantifying PCR method of foundation to CSFV（CSFV）, Pseudorabies virus（PRV）, Porcine Epidemic Diarrhea
Poison（PEDV）, transmissible gastro-enteritis virus（TGEV）, rotavirus（RV）And porcine circovirus 2 type（PCV2）Deng virus
DNA is detected；Simultaneously using set up method to PRRSV classical strainses, highly pathogenic mutant strain and in recent years I
Emerging NADC30-like strains carry out the specificity of detection checking this method in state swinery.
1.9 sensitivity tests
It is 10 every microlitre by 10 times of doubling dilutions to least concentration by positive DNA to copy, carries out quantitative fluorescent PCR reaction.Together
When using the DNA as template, carry out Standard PCR reaction, upstream and downstream primer be respectively 5'CCAGCCAGTCAATCARCTGTG 3' and
5' GCGAATCAGRCGCACWGTATG 3'.The μ L of amplified production 10 are taken, are analyzed with 1.5% agarose gel electrophoresis, than
Compared with the difference of the two sensitiveness.
1.10 replica test
Carry out the replica test in 4 batches between batch respectively with the standard items plasmid of 5 kinds of concentration and counted according to Ct values
Calculate the coefficient of variation.
The detection of 1.11 pairs of clinical samples
By new hope six and dynamic guarantor's suspicious 120 parts of PRRSV clinical samples of central collection, fluorescence quantitative RT-RCR detection is carried out respectively
With conventional RT-PCR detection, comparative analysis result.
2 results
The preparation of 2.1 plasmid standards
Pass through standard PCR amplification PRRSV ORF6 target gene by primer of Primer F/R（Fig. 1）, the recovered reagent of product
Box connects pMD19-T carriers after purification, and conversion to E.coli DH5 α Escherichia coli obtains positive colony matter by sequencing
Grain is plasmid standard.Determine after DNA concentration, be converted into copy number, and be diluted to 1010copies μ L-1, -20 DEG C of guarantors
Deposit, plasmid standard is used as with preceding dilution.
The optimization of 2.2 quantitative fluorescent PCR reaction conditions
By the optimization to annealing temperature and primer and concentration and probe concentration, the peak optimization reaction of fluorescence quantifying PCR method is finally determined
Condition.Quantitative fluorescent PCR reaction system is altogether 25 μ L, containing 12.5 μ L 2 × Premix Ex Taq（Probe qPCR）, 10 μ L
Water, 0.5 μ L Probe（10μM）, 0.5 μ L Primer F（10μM）, 0.5 μ L Primer R（10μM）, the μ L of plasmid standard 1.
Reaction condition is： 95℃5min；95 DEG C of 15sec, 60 DEG C of 45sec, 40 circulations.
The foundation of 2.3 quantitative fluorescent PCR standard curves
It is 101-108copies μ L to take concentration-1Standard items plasmid carry out fluorescent quantitative PCR for template and set up mark
Directrix curve.From Fig. 2 and Fig. 3, linear relationship is good, and standard curve is successfully established, available for clinical detection.
2.4 specific test
Testing result is equal to be detected to CSFV, PRV, PEDV, TGEV, RV and PCV2 with the fluorescence quantifying PCR method set up
For feminine gender, these viral amplification curves are horizontal line, not up to detect threshold value, and PRRSV classical strainses, highly pathogenic
Emerging NADC30-like strains have good amplification in variation strain and in recent years China swinery（Fig. 4）, experiment knot
Fruit shows that this method has good specificity.
2.5 sensitivity tests
The DNA profiling least concentration that fluorescence quantifying PCR method can be detected is 101copies μ L-1（Fig. 2）, and Standard PCR energy
The template least concentration of detection is 1.2 × 103copies μ L-1（Fig. 5）, show set up fluorescent quantitative PCR detection method
Sensitiveness be 100 times of conventional RT-PCR.
2.6 replica test
Carry out the replica test in batch between batch respectively with the standard items plasmid of 5 kinds of concentration.Test result indicates that, batch
The coefficient of variation of the interior replica test between batch is respectively less than 2.5%（Table 1 and Fig. 6）, show that this method has good repeatability
With stability.
The PRRSV TaqMan-MGB quantitative fluorescent PCR replica test results of table 1
2.7 clinical sample testing results
To 120 parts of the suspicious PRRSV clinical samples of collection, fluorescence quantitative RT-RCR detection and conventional RT-PCR detection are carried out respectively
（Table 2）.Testing result to clinical sample shows, fluorescent quantitative RT-PCR method positive rate（59.2%）Than conventional RT-
PCR method（46.7%）It is higher.In addition, there are 3 parts of sample Standard PCR testing results to be the positive in clinical sample detection process, and it is glimmering
The quantitative testing result of light is feminine gender, finally determines that this 3 parts of samples are false positive with sequencing by rechecking, this shows that fluorescence is determined
Amount method has higher specificity and sensitiveness, is more suitable for PRRSV clinical detection, especially infects early stage viral level
When relatively low.
The clinical sample testing result of table 2
Technical characteristic of the invention without description can be realized by or using prior art, will not be repeated here, certainly, above-mentioned
Illustrate it is not limitation of the present invention, the present invention is also not limited to the example above, those skilled in the art
The variations, modifications, additions or substitutions made in the essential scope of the present invention, should also belong to protection scope of the present invention.
Claims (6)
1. specific primer pair and probe for porcine reproductive and respiratory syndrome virus specific detection, it is characterised in that institute
The nucleotide sequence difference for stating two primers of specific primer centering is as follows：
Sense primer：5' CCATGGAAACCTGGAGATTCA 3'
Anti-sense primer：5' GCGGCCTAGCAAGCACAA 3'；
The probe sequence is as follows：
5' FAM-CACCTCCAGATGCC-MGB 3'。
2. the fluorescence quantitative RT-PCR kit for porcine reproductive and respiratory syndrome virus specific detection, it is characterised in that
The kit includes following components：Reaction mixture, water and specific amplification porcine reproductive and respiratory syndrome virus ORF6 genes
Specific primer pair and probe；
The nucleotide sequence difference of described two primers of specific primer centering is as follows：
Sense primer：5' CCATGGAAACCTGGAGATTCA 3'
Anti-sense primer：5' GCGGCCTAGCAAGCACAA 3'
The probe sequence is as follows：
5' FAM-CACCTCCAGATGCC-MGB 3'。
3. fluorescence quantitative RT-PCR kit according to claim 2, it is characterised in that the reaction mixture be purchased from
The Premix Ex Taq of TaKaRa companies（Probe qPCR）.
4. fluorescence quantitative RT-PCR kit according to claim 2, it is characterised in that the water is available from for water
The RNase-free Water of TaKaRa companies.
5. the fluorescence quantitative RT-RCR according to claim 2 for porcine reproductive and respiratory syndrome virus specific detection
Application of the kit in porcine reproductive and respiratory syndrome virus detection reagent is prepared.
6. application according to claim 5, it is characterised in that this kit is in PRRSV classical strainses, highly pathogenic mutant
The application of strain and NADC30-like strains.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611439A (en) * | 2018-04-19 | 2018-10-02 | 河北农业大学 | Kit for identifying and detecting porcine reproductive and respiratory syndrome virus and application thereof |
| CN110453015A (en) * | 2019-08-30 | 2019-11-15 | 苏州西山生物技术有限公司 | PRRS virus real-time fluorescence quantitative PCR detection kit and primer |
| CN114107570A (en) * | 2021-12-30 | 2022-03-01 | 青岛嘉智生物技术有限公司 | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method |
| CN115820929A (en) * | 2022-09-14 | 2023-03-21 | 温氏食品集团股份有限公司 | Fluorescent quantitative PCR (polymerase chain reaction) detection kit for NADC34-like porcine reproductive and respiratory syndrome virus |
| CN116064961A (en) * | 2022-11-14 | 2023-05-05 | 盐城师范学院 | Products and methods for fluorescent RT-RAA detection of genotype 2 porcine reproductive and respiratory syndrome virus |
| CN117844988A (en) * | 2024-03-06 | 2024-04-09 | 赛锐思生物技术(吉林)有限公司 | RT-qPCR detection primer and kit for porcine reproductive and respiratory syndrome virus and application of RT-qPCR detection primer and kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443076A (en) * | 2000-05-24 | 2003-09-17 | 梅瑞尔公司 | Porcine reproductive and respiratory syndrome virus (PRRSV) recombinant avipoxvirus vaccine |
| CN101343669A (en) * | 2008-08-25 | 2009-01-14 | 湖南省兽医总站 | Fast detecting reagent kit for porcine reproductive and respiratory syndrome virus |
| CN102140551A (en) * | 2011-03-25 | 2011-08-03 | 中国动物疫病预防控制中心 | General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus |
| CN102586477A (en) * | 2012-02-15 | 2012-07-18 | 山东省动物疫病预防与控制中心 | Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same |
| CN103305637A (en) * | 2013-06-29 | 2013-09-18 | 中国农业科学院兰州兽医研究所 | Taqman Real-time RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting PRRSV (porcine reproductive and respiratory syndrome virus) |
| CN103627816A (en) * | 2013-08-26 | 2014-03-12 | 中国农业科学院兰州兽医研究所 | Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof |
| CN105603127A (en) * | 2016-03-30 | 2016-05-25 | 天津市畜牧兽医研究所 | PCR detection kit for identifying NADC30-like PRRSV |
-
2017
- 2017-06-29 CN CN201710516979.1A patent/CN107236824A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443076A (en) * | 2000-05-24 | 2003-09-17 | 梅瑞尔公司 | Porcine reproductive and respiratory syndrome virus (PRRSV) recombinant avipoxvirus vaccine |
| CN101343669A (en) * | 2008-08-25 | 2009-01-14 | 湖南省兽医总站 | Fast detecting reagent kit for porcine reproductive and respiratory syndrome virus |
| CN102140551A (en) * | 2011-03-25 | 2011-08-03 | 中国动物疫病预防控制中心 | General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus |
| CN102586477A (en) * | 2012-02-15 | 2012-07-18 | 山东省动物疫病预防与控制中心 | Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same |
| CN103305637A (en) * | 2013-06-29 | 2013-09-18 | 中国农业科学院兰州兽医研究所 | Taqman Real-time RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting PRRSV (porcine reproductive and respiratory syndrome virus) |
| CN103627816A (en) * | 2013-08-26 | 2014-03-12 | 中国农业科学院兰州兽医研究所 | Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof |
| CN105603127A (en) * | 2016-03-30 | 2016-05-25 | 天津市畜牧兽医研究所 | PCR detection kit for identifying NADC30-like PRRSV |
Non-Patent Citations (4)
| Title |
|---|
| J. ZIEBUHR等: "Virus-encoded proteinases and proteolytic processing in the Nidovirales", 《JOURNAL OF GENERAL VIROLOGY》 * |
| R. ALLENDE等: "North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions", 《JOURNAL OF GENERAL VIROLOGY》 * |
| 刘涛等: "河北猪繁殖与呼吸综合征病毒地方株HB-4(hs)株ORF5-7基因变异分析", 《华北农学报》 * |
| 郝晓芳等: "高致病性猪繁殖与呼吸综合征病毒RT-PCR鉴别诊断方法的建立", 《中国预防兽医学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611439A (en) * | 2018-04-19 | 2018-10-02 | 河北农业大学 | Kit for identifying and detecting porcine reproductive and respiratory syndrome virus and application thereof |
| CN110453015A (en) * | 2019-08-30 | 2019-11-15 | 苏州西山生物技术有限公司 | PRRS virus real-time fluorescence quantitative PCR detection kit and primer |
| CN114107570A (en) * | 2021-12-30 | 2022-03-01 | 青岛嘉智生物技术有限公司 | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method |
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| CN116064961A (en) * | 2022-11-14 | 2023-05-05 | 盐城师范学院 | Products and methods for fluorescent RT-RAA detection of genotype 2 porcine reproductive and respiratory syndrome virus |
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Application publication date: 20171010 |