CN105936944A - Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtype - Google Patents
Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtype Download PDFInfo
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Abstract
The invention aims to provide a triple fluorescent RT-PCR detection kit, primer and probe for classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus H1 (SIV H1) subtype. The first objective of the invention is to provide the triple fluorescent quantitative RT-PCR detection kit for the classical swine fever virus, the porcine reproductive and respiratory syndrome virus and the swine influenza virus H1 subtype; the second objective of the invention is to provide the detection primer and probe for the classical swine fever virus, the porcine reproductive and respiratory syndrome virus and the swine influenza virus H1 subtype; the third objective of the invention is to provide a triple fluorescent quantitative RT-PCR detection method for the classical swine fever virus, the porcine reproductive and respiratory syndrome virus and the swine influenza virus H1 subtype. Rapid and early identification diagnosis of the 3 pathogens in pigs is realized, and because the primer and the probe have high specificity and sensitivity, early identification detection efficiency is improved, and the detection method is simplified.
Description
Technical field
It is an object of the present invention to provide a kind of for swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1
The triple fluorescent RT-PCR detection kit of hypotype and primer, probe.
Background technology
Swine fever virus (Classical swine fever virus, CSFV) is ssRNA virus, flaviviridae Pestivirus
Belonging to, virion is rounded, and its RNA is single-stranded positive.Porcine reproductive and respiratory syndrome virus (Porcine reproductive
And respiratory syndrome virus, PRRSV) it is Arterivirus, virion is ball-type, is that one has capsule
The single strand plus RNA virus of film.Swine influenza virus (Swine influenza virus, SIV) H1 hypotype is orthomyxovirus section first
Type influenza virus, virion pleomorphism, there is cyst membrane, genome is segmented sub-thread strand RNA.Three of the above cause of disease is
RNA viruses, all can cause pig industry tremendous economic to lose, and the clinical symptoms caused has similar part, it could even be possible to mix simultaneously
Close and infect, but hazardness, popularity and prevention and control measure thereof that Different Kinds of Pathogens infection causes are completely different.Therefore, a kind of district is set up
The detection method dividing three kinds of cause of diseases is extremely important, and the sensitiveest detection method can effectively prevent transmission of disease, reduces economy
Loss.
The immunologys such as ELISA, RT-PCR and molecular biology for detection have been applied in the diagnosis of above-mentioned epidemic disease, but
Being currently used primarily in the detection of single cause of disease, the aspect such as its susceptiveness, specificity and simplicity all needs to be improved further, commonly
The judgement of RT-PCR testing result needs product is carried out gel electrophoresis analysis, complex operation.The present invention uses triple fluorescent RT-
PCR detection method can detect differentiation these three virus simultaneously, not only can improve susceptiveness, specificity and simplicity etc., also may be used
Save plenty of time and cost.
Above-mentioned 3 kinds of cause of diseases can be by conservative region design specific primer and probe, carrying out fluorescence RT-PCR inspection
Survey.Fluorescence real-time quantitative PCR (Real-time PCR) is the probe adding fluorophor labelling in PCR reaction system, utilizes
Whole PCR process is monitored in fluorescence signal accumulation in real time, is analyzed result finally by amplification curve.AllgloTMProbe is
The latest generation fluorescent quantitation probe that AlleLogic Biosciences company of the U.S. releases, it breaks traditions Taqman probe
Reporter group one end, one end essence go out the restriction of group, its sharpest edges are the fluorophors that probe two ends labelling is same, probe
The mutual cancellation of fluorophor before hydrolysis, is report luminophore after hydrolysis, be greatly improved fluorescence intensity and the spirit of probe
Quick property, AllgloTMContaining the special chemical group (BGB) that can improve Tm value above probe dye, probe can be greatly improved
Specificity.Multiple fluorescence quantitative PCR technology refers to expand multiple purpose base in same fluorescent quantitative PCR is tested simultaneously
Cause.At multiple quantitative application, AllgloTMThe problem that the TaqMan probe that avoids probe mutually suppresses, probe dye uses
It is easier the chemical group of enzyme action, a plurality of AllgloTMProbe interferes the least in a reaction system, make one glimmering
The effect of photoreaction detection multiple pathogenic microorganisms is the best.According to this principle design specificity multi-fluorescence probe, conventional
Fluorophor has MAR, JUP, URA etc., can detect multiple fluorescence signal by different fluorescence detection channel simultaneously.Due to this
Method introduces specificity amplification primer and fluorescent probe so that sensitivity and the specificity of detection are increased significantly
By force, thus avoid that other detection method specificitys are the highest and the problem of easy missing inspection.
Summary of the invention
In order to realize synchronous detecting swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype, this
First purpose of invention is to provide the triple of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Fluorescence quantitative RT-PCR detecting kit；Second object of the present invention is to provide swine fever virus, Porcine reproductive and respiratory syndrome
Virus and the detection primer of swine influenza virus H1 hypotype and probe；It is numerous that third object of the present invention is to provide swine fever virus, pig
Grow and breath syndrome virus and the triple fluorescent quantitative RT-PCR detection method of swine influenza virus H1 hypotype.The present invention realizes
In pig, the quick Early Identification to above-mentioned 3 kinds of cause of diseases diagnoses, and owing to primer and probe have the highest specificity and sensitivity, carries
High Early Identification detection efficiency, and simplify detection method.
In order to realize above-mentioned first purpose, the technical solution used in the present invention is:
The triple fluorescent RT-PCR detection of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Test kit, this test kit includes deoxynucleotide triphosphates mixture, reverse transcriptase M-MLV, MgCl2, archaeal dna polymerase, specificity
Primer and fluorescent probe, positive reference material, negative reference product, described specific primer and Allglo fluorescent probe sequence are such as
Under:
Swine fever-F:GGACTAGCCRTAGTGGCGAGC
Swine fever-R:GGCATGCCCTCGTCCAC
Swine fever-P:MAR-CCTGGGTGGTCTAAGTCCTGAGTACAGG-MAR；
Porcine reproductive and respiratory syndrome virus-F:TTGTTTGGCTTCACAATCGC
Porcine reproductive and respiratory syndrome virus-R:GTAGAGCGCGCACGGAGTA
Porcine reproductive and respiratory syndrome virus-P:JUP-TTGGCTGGTGGTCTTATGCATCAGACT-JUP；
Swine influenza virus H1 type-F:GTGCTATAAACACCAGCCTYCCA
Swine influenza virus H1 type-R:CGGGATATTCCTTAATCCTGTRGC
Swine influenza virus H1 type-P:URA CAGAATATACATCCRGTCACAATTGGARAA-URA.
For deoxynucleotide triphosphates mixture, MgCl2, archaeal dna polymerase etc., these components are for those skilled in the art
For
Belong to common knowledge, can select as required.
As preferably, the sequence of swine fever virus positive reference material:
AACGGAGGGACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGA
CGTGGGCAGAAGCCCACCTCGAGATGCTATGTGGACGAGGGCATGCCCAAGACACACC；
The sequence of Porcine reproductive and respiratory syndrome disease virus-positive reference material:
CTCAGGGTGAACGGTAGAGCGCGCACGGAGTACCGCGGAGCAAACCAGTCTGATGCATAAGACCACCAGCCAACCGG
CGATTGTGAAGCCAAACAAAATGGGC；
The sequence of swine influenza virus H1 type positive reference material:
ACCCAAGGGTGCTATAAACACCAGCCTCCCATTTCAGAATATACATCCAGTCACAATTGGAGAATGTCCAAAATATG
TCAAAAGCACAAAATTGAGAATGGCTACAGGACTAAGGAATATCCCGTCTATTCAAT。
In order to realize above-mentioned second purpose, the technical solution used in the present invention is:
The triple fluorescent RT-PCR detection of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
As follows with specific primer and Allglo fluorescent probe sequence, described specific primer and Allglo fluorescent probe sequence:
Swine fever-F:GGACTAGCCRTAGTGGCGAGC
Swine fever-R:GGCATGCCCTCGTCCAC
Swine fever-P:MAR-CCTGGGTGGTCTAAGTCCTGAGTACAGG-MAR；
Porcine reproductive and respiratory syndrome virus-F:TTGTTTGGCTTCACAATCGC
Porcine reproductive and respiratory syndrome virus-R:GTAGAGCGCGCACGGAGTA
Porcine reproductive and respiratory syndrome virus-P:JUP-TTGGCTGGTGGTCTTATGCATCAGACT-JUP；
Swine influenza virus H1 type-F:GTGCTATAAACACCAGCCTYCCA
Swine influenza virus H1 type-R:CGGGATATTCCTTAATCCTGTRGC
Swine influenza virus H1 type-P:URA CAGAATATACATCCRGTCACAATTGGARAA-URA.
In order to realize above-mentioned the 3rd purpose, the technical solution used in the present invention is:
The triple fluorescent RT-PCR detection of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Method, described method is as follows:
1) extracting testing sample RNA, sample RNA described in this step extracts and can carry out according to a conventional method, as used Shanghai brightness
The post method nucleic acid extraction test kit of farsighted bio tech ltd or other test kit description are extracted；
2) with testing sample RNA as template, RT-PCR buffer is HR One-step qPCR Master Mix buffering
Liquid；
Specific primer and fluorescent probe sequence are as follows:
Swine fever-F:GGACTAGCCRTAGTGGCGAGC
Swine fever-R:GGCATGCCCTCGTCCAC
Swine fever-P:MAR-CCTGGGTGGTCTAAGTCCTGAGTACAGG-MAR；
Porcine reproductive and respiratory syndrome virus-F:TTGTTTGGCTTCACAATCGC
Porcine reproductive and respiratory syndrome virus-R:GTAGAGCGCGCACGGAGTA
Porcine reproductive and respiratory syndrome virus-P:JUP-TTGGCTGGTGGTCTTATGCATCAGACT-JUP；
Swine influenza virus H1 type-F:GTGCTATAAACACCAGCCTYCCA
Swine influenza virus H1 type-R:CGGGATATTCCTTAATCCTGTRGC
Swine influenza virus H1 type-P:URA CAGAATATACATCCRGTCACAATTGGARAA-URA；
3) RT-PCR product is carried out fluoroscopic examination, sentence according to the minimum Ct value of fluoroscopic examination and high fluorescent
Disconnected result.
4) described fluorescence quantitative RT-RCR reaction condition is: 50 DEG C of 30min, 95 DEG C of 5min, then 95 DEG C of 10s, 55 DEG C
40s, carries out triple fluorescent detection at 55 DEG C, carries out 40 circulations altogether.
The present invention relates to swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 subtype specific primers
And probe.Based on having swine fever virus 5 ' the non-coding region gene sequence (gene order number: JX262391) of high degree of specificity, pig
Reproductive and respiratory syndrome virus gene ORF2 gene (gene order number: KP771746), swine influenza virus H1 hypotype
Hemagglutinin (HA) gene (gene order number: KR074351) separately designs respective primer and probe, and 3 spies
Different fluorescence signals is marked, under conditions of optimizing PCR reaction system, it is achieved triple fluorescent quantitative PCR synchronizes on pin
Detection swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype.Owing to the nucleic acid of these 3 kinds of pathogen is equal
For RNA, therefore, RNA simultaneous extraction not only can be realized, it is also possible to carry out fluorescence RT-PCR synchronous detecting, enormously simplify detection
Method.Owing to the method introduces high specific amplimer and the most fluorescently-labeled probe, it is ensured that this method can quickly,
Special and sensitive disposable discriminating detects swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype,
Early detective rate can be improved, effectively prevent transmission of disease, reduce economic loss.
Accompanying drawing explanation
Fig. 1 is the sensitivity map of kit measurement swine fever virus of the present invention.
Fig. 2 is the sensitivity map of kit measurement porcine reproductive and respiratory syndrome virus of the present invention.
Fig. 3 is the sensitivity map of kit measurement swine influenza virus H1 hypotype of the present invention.
Fig. 4 is to measure swine fever virus, porcine reproductive and respiratory syndrome virus, the mensuration of swine influenza virus H1 hypotype simultaneously
Figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
Embodiment 1: sample prepares and the extraction of viral RNA:
Aseptic collection pig Nasal swabs, brush,throat, blood and internal organs are (such as: spleen, trachea, lung, lymph node, flat
Fructus Persicae body, kidney, spleen etc.), it is placed in the PBS liquid containing antibiotics, airtight transportation, cryopreservation, standby.The extraction of viral RNA
Use the pillar nucleic acid extraction test kit of Hui Rui bio tech ltd, Shanghai, extract by test kit description.
Embodiment 2: primer and probe
Swine fever virus all over the world, porcine reproductive and respiratory syndrome virus, swine flue disease is downloaded from NCBI gene bank
Poison H1 subtype gene sequence, has carried out tetraploid rice to it.Based on the swine fever virus 5 ' noncoding region with high degree of specificity
Gene order (gene order number: JX262391), porcine reproductive and respiratory syndrome virus gene ORF2 gene (gene order number:
KP771746), swine influenza virus H1 hypotype hemagglutinin (HA) gene (gene order number: KR074351), believe with biology
Breath method is according to design of primers principle, and utilizes Primer Express design software, designs amplification said gene fragment
Specific primer and some groups of fluorescent probe.The primer that design is obtained and probe carry out one by one BLAST checking (http: //
Blast.ncbi.nlm.nih.gov/), to ensure the high specific of primer.And further with DNAStar software, checking is each
Whether primer dimer etc. can be produced between primed probe.Through above-mentioned checking, finally have selected primer as shown in table 1 and spy
Pin, and the fluorescence signal that labelling is different respectively on probe, to realize 3 Channel Synchronous detections.
Table 1 swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype detection primer and
Allglo probe
F: forward primer；R: reverse primer；P: probe.
Embodiment 3: the preparation of plasmid
The sequence of swine fever virus positive reference material:
AACGGAGGGACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAG
TAGTTCGACGTGGGCAGAAGCCCACCTCGAGATGCTATGTGGACGAGGGCATGCCCAAGACACACC
The sequence of porcine reproductive and respiratory syndrome virus positive reference material:
CTCAGGGTGAACGGTAGAGCGCGCACGGAGTACCGCGGAGCAAACCAGTCTGATGCATAAGACCACCAG
CCAACCGGCGATTGTGAAGCCAAACAAAATGGGC
The sequence of swine influenza virus H1 type positive reference material:
ACCCAAGGGTGCTATAAACACCAGCCTCCCATTTCAGAATATACATCCAGTCACAATTGGAGAATGTCC
AAAATATGTCAAAAGCACAAAATTGAGAATGGCTACAGGACTAAGGAATATCCCGTCTATTCAAT
Use the mode of synthetic to build the positive plasmid of corresponding virus by sequence above, build rear sequence verification sequence
The accuracy of row, so obtains the plasmid 3 kinds respectively containing a kind of purpose fragment.
Embodiment 4: fluorescence RT-PCR reaction system and the optimization of condition
Test kit: RT reaction buffer and enzyme mix selects the HR One-of Hui Rui bio tech ltd, Shanghai
Step qPCR Master Mix, Code:SJ-2106A, by specification operates, and reaction system is 25 μ l, wherein RT
Reaction buffer 7.5ul, enzyme mix 5ul, upstream and each 1 μ l of downstream primer (10 μm ol/L), probe (10 μm ol/L)
0.5 μ l, template ribonucleic acid 2-3 μ l, DEPC water supplies 25 μ l.Detecting with ABI7500 fluorescence detecting system, reaction condition is:
50 DEG C of 30min, 95 DEG C of 5min, then 95 DEG C of 10s, 55 DEG C of 40s, carry out triple fluorescent detection at 55 DEG C, carry out 40 circulations altogether.
Result judges: select fluoroscopic examination pattern MAR, JUP, URA, and the fluorescence signal that fluorescence baseline adjustment takes 3-15 circulation is average
Value, threshold value sets the peak being just above normal negative controls with threshold line, and sample is typical amplification curve, it is judged that for
Positive.Without typical amplification curve, it is judged that for feminine gender.The optimization Test of system, with same concentrations positive nucleic acid as template
In reaction system, primer concentration from 0.10～1.00 μM, concentration and probe concentration from 0.10～0.50 μM, use the preferred primer of matrix method and
The optium concentration of probe, selects optimal primer and concentration and probe concentration according to minimum Ct value and high fluorescent value added (Δ Rn).
Embodiment 5: substance fluorescent PCR sensitivity is verified
Respectively above-mentioned 3 kinds of cause of diseases are carried out the sensitivity checking of fluorescent PCR.Substance Fluorescence PCR system is as follows: wherein
RT reaction buffer 7.5ul, enzyme mix 5ul, upstream and each 1 μ l of downstream primer (10 μm ol/L), probe (10 μm ol/
L) 0.5 μ l, the plasmid in examples detailed above 3 takes 1ul (the plasmid content of each Concentraton gradient is as shown in table 2), and not enough volume is used
Water complements to 25ul.Detecting with ABI7500 fluorescence detecting system, reaction condition is: 50 DEG C of 30min, 95 DEG C of 5min, then
95 DEG C of 10s, 55 DEG C of 40s, carry out triple fluorescent detection at 55 DEG C, carries out 40 circulations altogether.The result of each substance PCR such as table 2
Shown in.When it is generally acknowledged Ct value less than or equal to 35, the positive findings obtained is reliable.Thus judge, CSFV, PRRSV, SIV
The detection sensitivity of primer and probe is 0.00025ng/ μ l.
The testing result of table 2 substance fluorescent PCR sensitivity checking
All experiments take the meansigma methods being repeated 3 times；Undet.:Ct value is less than detection limit.
Embodiment 6: multiple fluorescence PCR sensitivity is verified
When comparing triple fluorescent reaction, the detection sensitivity of each primed probe.Reaction system and response procedures such as example 5
Shown in, the most each primer concentration is identical, and each concentration and probe concentration is the most identical.When three kinds of plasmids (obtaining in example 3) concentration is 1:1:1,
Triple fluorescent PCR reaction is as shown in table 3 to three kinds of material detection sensitivities, i.e. the sensitivity to CSFV, PRRSV and SIV all reaches
To 0.0025ng/ μ l.
The testing result of table 3 triple fluorescent PCR sensitivity checking
All experiments take the meansigma methods being repeated 3 times；Undet:Ct value is less than detection limit.
Sequence table
<110>Zhejiang Entry-Exit Inspection and Quarantine Bureau
<120>the triple fluorescent RT-PCR inspection of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Test agent box and primer, probe
<160>17
<210>1
<211>21
<212>DNA
<213>artificial sequence
<400>1
GGACTAGCCR TAGTGGCGAGC 21
<210>2
<211>17
<212>DNA
<213>artificial sequence
<400>2
GGCATGCCCT CGTCCAC 17
<210>3
<211>28
<212>DNA
<213>artificial sequence
<400>3
CCTGGGTGGT CTAAGTCCTG AGTACAGG 28
<210>4
<211>20
<212>DNA
<213>artificial sequence
<400>4
TTGTTTGGCT TCACAATCGC 20
<210>5
<211>19
<212>DNA
<213>artificial sequence
<400>5
GTAGAGCGCG CACGGAGTA 19
<210>6
<211>27
<212>DNA
<213>artificial sequence
<400>6
TTGGCTGGTG GTCTTATGCA TCAGACT 27
<210>7
<211>23
<212>DNA
<213>artificial sequence
<400>7
GTGCTATAAA CACCAGCCTY CCA 23
<210>8
<211>24
<212>DNA
<213>artificial sequence
<400>8
CGGGATATTC CTTAATCCTG TRGC 24
<210>9
<211>30
<212>DNA
<213>artificial sequence
<400>9
CAGAATATAC ATCCRGTCAC AATTGGARAA 30
<210>10
<211>135
<212>RNA
<213>swine fever virus
<400>10
AACGGAGGGA CTAGCCGTAG TGGCGAGCTC CCTGGGTGGT CTAAGTCCTG AGTACAGGAC
AGTCGTCAGT AGTTCGACGT GGGCAGAAGC CCACCTCGAG ATGCTATGTG GACGAGGGCA TGCCCAAGAC
ACACC 135
<210>11
<211>103
<212>RNA
<213>Porcine reproductive and respiratory syndrome disease virus
<400>11
CTCAGGGTGA ACGGTAGAGC GCGCACGGAG TACCGCGGAG CAAACCAGTC TGATGCATAA
GACCACCAGC CAACCGGCGA TTGTGAAGCC AAACAAAATG GGC 103
<210>12
<211>134
<212>RNA
<213>swine influenza virus H1 type
<400>12
ACCCAAGGGT GCTATAAACA CCAGCCTCCC ATTTCAGAAT ATACATCCAG TCACAATTGG
AGAATGTCCA AAATATGTCA AAAGCACAAA ATTGAGAATG GCTACAGGAC TAAGGAATAT CCCGTCTATT
CAAT 134
Claims (2)
1. the triple fluorescent RT-PCR detection examination of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Agent box, it is characterised in that this test kit includes deoxynucleotide triphosphates mixture, reverse transcriptase M-MLV, MgCl2, DNA polymerization
Enzyme, specific primer and fluorescent probe, positive reference material, negative reference product, described specific primer and Allglo fluorescence are visited
Pin sequence is as follows:
Swine fever-F:GGACTAGCCRTAGTGGCGAGC
Swine fever-R:GGCATGCCCTCGTCCAC
Swine fever-P:MAR-CCTGGGTGGTCTAAGTCCTGAGTACAGG-MAR；
Porcine reproductive and respiratory syndrome virus-F:TTGTTTGGCTTCACAATCGC
Porcine reproductive and respiratory syndrome virus-R:GTAGAGCGCGCACGGAGTA
Porcine reproductive and respiratory syndrome virus-P:JUP-TTGGCTGGTGGTCTTATGCATCAGACT-JUP；
Swine influenza virus H1 type-F:GTGCTATAAACACCAGCCTYCCA
Swine influenza virus H1 type-R:CGGGATATTCCTTAATCCTGTRGC
Swine influenza virus H1 type-P:URA CAGAATATACATCCRGTCACAATTGGARAA-URA.
Swine fever virus the most according to claim 1, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype
Triple fluorescent RT-PCR detection kit, it is characterised in that the sequence of swine fever virus positive reference material:
AACGGAGGGACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGT
TCGACGTGGGCAGAAGCCCACCTCGAGATGCTATGTGGACGAGGGCATGCCCAAGACACACC；
The sequence of Porcine reproductive and respiratory syndrome disease virus-positive reference material:
CTCAGGGTGAACGGTAGAGCGCGCACGGAGTACCGCGGAGCAAACCAGTCTGATGCATAAGACCACCAGCCAA
CCGGCGATTGTGAAGCCAAACAAAATGGGC；
The sequence of swine influenza virus H1 type positive reference material:
ACCCAAGGGTGCTATAAACACCAGCCTCCCATTTCAGAATATACATCCAGTCACAATTGGAGAATGTCCAAAA
TATGTCAAAAGCACAAAATTGAGAATGGCTACAGGACTAAGGAATATCCCGTCTATTCAAT；
The triple fluorescent RT-PCR detection of swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 hypotype is special
Specific primer and Allglo fluorescent probe sequence, it is characterised in that described specific primer and Allglo fluorescent probe sequence are such as
Under:
Swine fever-F:GGACTAGCCRTAGTGGCGAGC
Swine fever-R:GGCATGCCCTCGTCCAC
Swine fever-P:MAR-CCTGGGTGGTCTAAGTCCTGAGTACAGG-MAR；
Porcine reproductive and respiratory syndrome virus-F:TTGTTTGGCTTCACAATCGC
Porcine reproductive and respiratory syndrome virus-R:GTAGAGCGCGCACGGAGTA
Porcine reproductive and respiratory syndrome virus-P:JUP-TTGGCTGGTGGTCTTATGCATCAGACT-JUP；
Swine influenza virus H1 type-F:GTGCTATAAACACCAGCCTYCCA
Swine influenza virus H1 type-R:CGGGATATTCCTTAATCCTGTRGC
Swine influenza virus H1 type-P:URA CAGAATATACATCCRGTCACAATTGGARAA-URA.
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|---|---|---|---|
| CN201610260124.2A CN105936944B (en) | 2016-04-22 | 2016-04-22 | Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtypes |
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|---|---|---|---|
| CN201610260124.2A CN105936944B (en) | 2016-04-22 | 2016-04-22 | Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtypes |
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|---|---|
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| CN105936944B CN105936944B (en) | 2019-12-20 |
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| CN (1) | CN105936944B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957924A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | It is a kind of simultaneously to detect and differentiate porcine reproductive respiratory syndrome and the detection kit and primer and probe of swine flu |
| CN115852049A (en) * | 2022-10-27 | 2023-03-28 | 广州海关技术中心 | Kit for detecting neocorona, respiratory syncytial, influenza and respiratory adenovirus and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816865A (en) * | 2011-10-31 | 2012-12-12 | 武汉大学 | H1N1, PRRSV and CSFV quadruple detection kit |
| CN102876661A (en) * | 2011-07-12 | 2013-01-16 | 爱科来株式会社 | Method and kit for amplifying and detecting polynucleotide |
| CN104195266A (en) * | 2014-08-30 | 2014-12-10 | 杭州市第一人民医院 | Quadruple fluorogenic quantitative PCR kit for detecting three pathogens of infantile pneumonia |
| CN104328218A (en) * | 2014-10-30 | 2015-02-04 | 广东出入境检验检疫局检验检疫技术中心 | Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof |
| CN104745731A (en) * | 2015-04-21 | 2015-07-01 | 天津出入境检验检疫局动植物与食品检测中心 | Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof |
| CN105349703A (en) * | 2015-12-09 | 2016-02-24 | 菲鹏生物股份有限公司 | Qualitative detection kit for influenza A virus subtype H1N1 nucleic acid |
-
2016
- 2016-04-22 CN CN201610260124.2A patent/CN105936944B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876661A (en) * | 2011-07-12 | 2013-01-16 | 爱科来株式会社 | Method and kit for amplifying and detecting polynucleotide |
| CN102816865A (en) * | 2011-10-31 | 2012-12-12 | 武汉大学 | H1N1, PRRSV and CSFV quadruple detection kit |
| CN104195266A (en) * | 2014-08-30 | 2014-12-10 | 杭州市第一人民医院 | Quadruple fluorogenic quantitative PCR kit for detecting three pathogens of infantile pneumonia |
| CN104328218A (en) * | 2014-10-30 | 2015-02-04 | 广东出入境检验检疫局检验检疫技术中心 | Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof |
| CN104745731A (en) * | 2015-04-21 | 2015-07-01 | 天津出入境检验检疫局动植物与食品检测中心 | Triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and preparation method and application thereof |
| CN105349703A (en) * | 2015-12-09 | 2016-02-24 | 菲鹏生物股份有限公司 | Qualitative detection kit for influenza A virus subtype H1N1 nucleic acid |
Non-Patent Citations (2)
| Title |
|---|
| 夏俊等: "复合RT-PCR检测猪繁殖与呼吸综合征和猪瘟及猪流感等3种病毒方法的建立", 《现代农业科技》 * |
| 梁晓艳等: "多重荧光RT-PCR技术检测猪流感病毒、猪乙型脑炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒方法的建立", 《中国动物检疫》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957924A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | It is a kind of simultaneously to detect and differentiate porcine reproductive respiratory syndrome and the detection kit and primer and probe of swine flu |
| CN115852049A (en) * | 2022-10-27 | 2023-03-28 | 广州海关技术中心 | Kit for detecting neocorona, respiratory syncytial, influenza and respiratory adenovirus and application thereof |
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