CN110438259A - A kind of primer combination, detection method and the kit of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type - Google Patents
A kind of primer combination, detection method and the kit of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, disclose primer combination, detection method and the kit of a kind of LAMP of parting detection that can distinguish pig parvoviral 1-7 type.The present invention is utilized respectively the conservative section design primer combinations for covering LAMP primer, filtering out that specificity is good, and the parting that can distinguish pig parvoviral 1-7 type of high sensitivity detects of pig parvoviral NS1 gene and NP2 gene more.Primer combination can detect pig parvoviral 1-7 type respectively, and with the other viral no cross reactions in pig source.Fluorescent dye is added in LAMP reaction system by the present invention, it can be achieved that one-step method detection pig parvoviral 1-7 type, realizes whole real time monitoring in conjunction with fluorescent quantitation, can go out as a result, detection sensitivity can achieve 100 copies/μ L within an hour.Detection method of the present invention had both had easy, quick, sensitive, special advantage, in turn avoided error and caused environmental pollution of uncapping caused by artificially determining.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of can distinguish pig parvoviral 1-7 type parting detection
Primer combination, detection method and the kit of LAMP, distinguishes pig parvoviral 1-7 type for detecting.
Background technique
Pig parvoviral (porcine parvovirus) is one of the main pathogens for causing pregnant sow breeding difficulty,
It is characterized in that infected sow especially first farrowing sow shows as miscarriage, output stillborn foetus, mummy fetus and malformation fetus, or
Litter size is few, can also result in male and female pig infertility sometimes.Pig parvoviral has very high infectivity, susceptible healthy swinery one
Denier virus is incoming, almost can lead to the infection of swinery 100% in 3 months；The pig long period for infecting group keeps serological reaction
It is positive.Porcine parvovirus mostly occurs in spring, summer or sows farrowing and breeding season.When sow early pregnancy infects, embryo
Tire, the tire pig death rate may be up to 80%~100%.Sow 30~40 days most easy infections before period of pregnancy, pregnancy period different time
Infection will cause stillborn foetus, miscarriage, mummy, production weak pigle and sow respectively and match the different symptoms such as infertile long.Therefore, fast in early stage
Speed, precise Identification pig parvoviral are of great significance for control virus spread.
Mary and Mahnel had found pig parvoviral in 1966 for the first time, then the viral progression to multiple countries, the world
The area and.In the 1980s, multiple places all separation in China find porcine parvovirus.Classical porcine parvovirus
It is caused by 1 type of pig parvoviral (Porcine parvovirus type 1, PPV1) infection.Tiijikata in 2001 etc.
2 type of pig parvoviral (PPV2) is found from Swine serum when Burma carries out Hepatitis E investigation.2008, a kind of new pig was thin
Small virus, hokovirus (PHoV) is in Hong Kong confirmation, genome sequence and human parvovirus 4 (PARV4) and ox
Hokovirus (BHoP) is closely related, as 3 type of pig parvoviral (PPV3).PPV4 is 2005 from North Carolina, US
It infects in the swinery of porcine circovirus associated diseases (porcine circovirus associated disease, PCVAD) in state
It was found that, then China also has been reported that.2013, Xiao etc. had found a kind of novel PPV in U.S. swinery, was temporarily named as
PPV5, the virus have typical parvovirus genome structure.In December, 2014, Chinese scholar are drawn using sequence independence list
Object amplification method obtains a kind of parvovirus different from the past out of miscarriage pig fetus body, is temporarily named as PPV6.2016
Year is accredited PPV7 by macro gene order-checking method in U.S. swinery for the first time, and PPV7 is a kind of well differentiated novel fine
Small virus has also discovered the presence of PPV7 virus for 2017 in China.The NS1 of sequence alignment discovery PPV7 and other parvovirus
Sequence homology is no more than 17%.Due to constantly discovering for novel strain, the detection of pig parvoviral parting for viral genetic into
Change, Molecule Epidemiology Investigation monitoring, the prevention of pathological research and epidemic situation control are of great significance, and at present about pig
The detection research of parvovirus 1-7 type parting has not been reported.
Detection pig parvoviral common method has: Enzyme-linked Immunosorbent Assay reacts (ELISA), qualitative PCR technology, real-time fluorescence
Quantitative PCR technique and loop-mediated isothermal amplification technique (LAMP).ELISA sensitivity is higher, but testing result may include false sun
Property.Qualitative PCR technical costs is low, easily operated, but remolding sensitivity is lower, and since its amplified production need to pass through gel electrophoresis point
Analysis is primary, easily generates pollution.Though Real-Time Fluorescent Quantitative PCR Technique has the features such as highly sensitive, high specific, the experiment pair
Testing staff is more demanding and laboratory operating procedures need frequently to heat up and temperature-fall period, light detection time-consuming at least need
1h or more.Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) is to utilize one
Kind has the Bst archaeal dna polymerase of strand-displacement activity and waterfall type nucleic acid amplification function, carries out the change of nucleic acid under isothermal conditions
The strand displacement nucleic acid amplification reaction of property and automatic cycle.LAMP designs 4-6 special primer, benefit for 6 regions of target sequence
DNA amplification is constantly replicated at a constant temperature with the archaeal dna polymerase for having strand displacement function.It, can be in order to improve reaction efficiency
Two ring primers are added in reaction system, are allowed to respectively in conjunction with loop-stem structure, starting strand displacement synthesis, recursive copying.Therefore
The core of LAMP technology first is that design of primers, primer is the key factor for determining testing result sensitivity and specificity, is needed
Efficient, special, the sensitive LAMP primer group being adapted with reaction system can be just found by multiple primer screening and verifying
It closes.
And most LAMP reaction methods established need agarose gel electrophoresis or are added after needing to uncap aobvious at present
Toner be easy to cause laboratory pollution in this way or false positive results is caused to occur.What furthermore LAMP method was used to directly observe is aobvious
Most of color method is to uncap that fluorescent dye progress chromogenic reaction is added after reaction, has seen whether that colour developing carrys out interpretation test knot
Fruit had both caused Aerosol Pollution in this way and specific amplification and non-specific amplification cannot be distinguished, therefore increased false positive
The probability of diagnostic result；And weakly positive is reacted, it is likely to be mistaken for feminine gender by way of artificial naked eyes interpretation.
Summary of the invention
In view of this, it is an object of the invention to it is in the prior art there are aiming at the problem that, provide that a kind of speed is fast, spirit
Primer combination, detection method and the reagent of high, high specificity the parting detection LAMP that can distinguish pig parvoviral 1-7 type of sensitivity
Box.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
A kind of primer combination for the parting detection LAMP that can distinguish pig parvoviral 1-7 type, including primer sets 1-7；
The primer sets 1 include SEQ ID NO.1 and SEQ ID NO.2 shown in 2 outer primers, SEQ ID NO.3 and
2 ring primers shown in 2 inner primers and SEQ ID NO.5 and 6 shown in SEQ ID NO.4；
The primer sets 2 include SEQ ID NO.7 and SEQ ID NO.8 shown in 2 outer primers, SEQ ID NO.9 and
2 ring primers shown in 2 inner primers and SEQ ID NO.11 and 12 shown in SEQ ID NO.10；
The primer sets 3 include 2 outer primers, SEQ ID NO.15 shown in SEQ ID NO.13 and SEQ ID NO.14
With 2 ring primers shown in 2 inner primers shown in SEQ ID NO.16 and SEQ ID NO.17 and 18；
The primer sets 4 include 2 outer primers, SEQ ID NO.21 shown in SEQ ID NO.19 and SEQ ID NO.20
With 2 ring primers shown in 2 inner primers shown in SEQ ID NO.22 and SEQ ID NO.23 and 24；
The primer sets 5 include 2 outer primers, SEQ ID NO.27 shown in SEQ ID NO.25 and SEQ ID NO.26
With 2 ring primers shown in 2 inner primers shown in SEQ ID NO.28 and SEQ ID NO.29 and 30；
The primer sets 6 include 2 outer primers, SEQ ID NO.33 shown in SEQ ID NO.31 and SEQ ID NO.32
With 2 ring primers shown in 2 inner primers shown in SEQ ID NO.34 and SEQ ID NO.35 and 36；
The primer sets 7 include 2 outer primers, SEQ ID NO.39 shown in SEQ ID NO.37 and SEQ ID NO.38
With 2 ring primers shown in 2 inner primers shown in SEQ ID NO.40 and SEQ ID NO.41 and 42.
Wherein the primer sets 1 are the ring mediated isothermal amplification primer sets of 1 type of pig parvoviral, specific as follows (5 ' →
3 '):
Outer primer PPV1-F3:5 '-CTTGGTTGGTAAAGAAAGGT-3 ' (SEQ ID NO.1)
Outer primer PPV1-B3:5 '-AGCTAAATCCAGGTCCTC-3 ' (SEQ ID NO.2)
Inner primer PPV1-FIP:5 '-TGGGGTTTGCATTTTTGGCGGCTAGCTATATGC ATCATTG-3 ' (SEQ ID
NO.3)
Inner primer PPV1-BIP:5 '-TACACCAACAGACTCTCAGATTTCATTGGAGT TGCTGCGTA-3 ' (SEQ
ID NO.4)
Ring primer PPV1-LF:5 '-GACCAATCAGGTACATTTCC-3 ' (SEQ ID NO.5)
Ring primer PPV1-LB:5 '-ACATCAGTGAAAACTTCGC-3 ' (SEQ ID NO.6)；
The primer sets 2 are the ring mediated isothermal amplification primer sets of 2 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV2-F3:5 '-GCAAATGGAGCCCAGCAG-3 ' (SEQ ID NO.7)
Outer primer PPV2-B3:5 '-TGATCGCCTTCCCACCAG-3 ' (SEQ ID NO.8)
Inner primer PPV2-FIP:5 '-TTCAGCCACCCCCCCATCTCTTCACCTTCATCT CGCAGTG-3 ' (SEQ ID
NO.9)
Inner primer PPV2-BIP:5 '-ATGATAGAGCACAGGCCAGGCGAGCGGTCTT CCTCATCTCA-3 ' (SEQ
ID NO.10)
Ring primer PPV2-LF:5 '-CTCGGGCCTTTGTGTCG-3 ' (SEQ ID NO.11)
Ring primer PPV2-LB:5 '-GCCCCCCCCTTGTACTT-3 ' (SEQ ID NO.12).
The primer sets 1 are the ring mediated isothermal amplification primer sets of 3 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV3-F3:5 '-AGCAACTGGTTGAAGAGATGG-3 ' (SEQ ID N is O.13)
Outer primer PPV3-B3:5 '-GCAGCCTCAAATCTCTCCATG-3 ' (SEQ ID NO.14)
Inner primer PPV3-FIP:5 '-AGTCTCCCTTGTCTGGTCTTCCTCAACATTCCT AACTCGCCACA-3 '
(SEQ ID NO.15)
Inner primer PPV3-BIP:5 '-ATCAGTGCGACCTGACCTTTGTCAAGCATACC AGACATGCTCTA-3 '
(SEQ ID NO.16)
Ring primer PPV3-LF:5 '-GATATCCCACAGAATGCTCCAAC-3 ' (SEQ ID NO.17)
Ring primer PPV3-LB:5 '-AAGGTATCTACTGCCTAAAGTACCA-3 ' (SEQ ID NO.18).
The primer sets 1 are the ring mediated isothermal amplification primer sets of 4 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV4-F3:5 '-GACATTATGAACTGCCCTAT-3 ' (SEQ ID NO.19)
Outer primer PPV4-B3:5 '-AGGGATGAATTCAGTCTCAG-3 ' (SEQ ID NO.20)
Inner primer PPV4-FIP:5 '-TTCATTTCTTTTGGGGGTGGGTAAAAAAGGAC CTGCCAGT-3 ' (SEQ ID
NO.21)
Inner primer PPV4-BIP:5 '-AGAGCGGAACCAGATGAAATTCGTTTCTTCT TTCTCGGTGC-3 ' (SEQ
ID NO.22)
Ring primer PPV4-LF:5 '-TCTCTCCAACAGGAACTAAA-3 ' (SEQ ID NO.23)
Ring primer PPV4-LB:5 '-AATCCAGAAGAACTGGACC-3 ' (SEQ ID NO.24).
The primer sets 1 are the ring mediated isothermal amplification primer sets of 5 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV5-F3:5 '-GGAACATCACCAATCAAGA-3 ' (SEQ ID NO.25)
Outer primer PPV5-B3:5 '-GAGAGATCAATTTCATTGCG-3 ' (SEQ ID NO.26)
Inner primer PPV5-FIP:5 '-AGTTCTTTCTGTTCTCGGTGACCTATTAGAACA CGACTCAAGC-3 ' (SEQ
ID NO.27)
Inner primer PPV5-BIP:5 '-ATCAAACATGGAGCGGGAGATTTCCAGGACC TGTGTAG-3 ' (SEQ ID
NO.28)
Ring primer PPV5-LF:5 '-GCTTCTTCTGGTTGTTCTTC-3 ' (SEQ ID NO.29)
Ring primer PPV5-LB:5 '-CGGAACCGGTATCAACT-3 ' (SEQ ID NO.30).
The primer sets 1 are the ring mediated isothermal amplification primer sets of 6 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV6-F3:5 '-TCAAGATCAGGGAGTCATT-3 ' (SEQ ID NO.31)
Outer primer PPV6-B3:5 '-TAGTGCTCACAATACGCT-3 ' (SEQ ID NO.32)
Inner primer PPV6-FIP:5 '-GTAGTCACATCATTATCACACATCCACTGCTCC TACTTTTAAAGC-3 '
(SEQ ID NO.33)
Inner primer PPV6-BIP:5 '-CCTGTACTCTTTCCTTGCTACCGCAGCTCTAA GACACTGT-3 ' (SEQ ID
NO.34)
Ring primer PPV6-LF:5 '-CATTTTATCACCCGTAGCC-3 ' (SEQ ID NO.35)
Ring primer PPV6-LB:5 '-CCAGCTGGTACTCATTTG-3 ' (SEQ ID NO.36).
The primer sets 1 are the ring mediated isothermal amplification primer sets of 7 type of pig parvoviral, (5 ' → 3 ') specific as follows:
Outer primer PPV7-F3:5 '-GGTACCACAACTGGTACGAAG-3 ' (SEQ ID NO.37)
Outer primer PPV7-B3:5 '-TGGTGTTCCATGTCCATGTC-3 ' (SEQ ID NO.38)
Inner primer PPV7-FIP:5 '-CGGTGGTTGGTTTAGGGACGAGACACGCTCTG GAGAAGCT-3 ' (SEQ ID
NO.39)
Inner primer PPV7-BIP:5 '-ACCAAAGAAGGGGTCGGAAACAGGTTTCGG GTGCCGTGATG-3 ' (SEQ
ID NO.40)
Ring primer PPV7-LF:5 '-CCTTCTTTGTAGGCCACGT-3 ' (SEQ ID NO.41)
Ring primer PPV7-LB:5 '-TGGAGCAGACTTACACTCCC-3 ' (SEQ ID NO.42).
The present invention also provides the kits of the LAMP of parting detection that can distinguish pig parvoviral 1-7 type a kind of, including
Above-mentioned primer combination, wherein each equal independent packaging of primer in primer sets conjunction.
Preferably, kit of the present invention further includes the reaction solution containing fluorescent dye and/or sterilizing pure water.
In some embodiments, the reaction solution is the product of Capitalbio Corporation Co., Ltd., and catalog number is
CP.440020.It include fluorescent dye in the reaction solution.
Further, 2 outer primers in each primer sets in the combination of primer described in kit of the present invention
Reaction molal quantity is identical, and the reaction molal quantity of 2 inner primers is identical, and the reaction molal quantity of 2 ring primers is identical, in each primer sets
Outer primer: inner primer: the molar ratio of ring primer be (0.58-0.62): (4.6-5.0): (1.8-2.2).Such as primers F 3, primer
B3, primers F IP, primer BIP, primer LF and primer LB molar ratio be (0.29-0.31): (0.29-0.31): (2.3-2.5):
(2.3-2.5): (0.9-1.1): (0.9-1.1).
In some embodiments, the outer primer in each primer sets: inner primer: the molar ratio of ring primer is 0.6:4.8:2.
In certain embodiments, the primers F 3, primer B3, primers F IP, primer BIP, primer LF and primer LB
Molar ratio is 0.3:0.3:2.4:2.4:1:1.
The present invention also provides the detection methods of the LAMP of parting detection that can distinguish pig parvoviral 1-7 type a kind of, make
LAMP amplification is carried out with above-mentioned primer combination, starts the time occurred by observing real-time fluorescence PCR instrument " S " type amplification curve
Judge testing result.
Wherein, the outer primer that the reaction system of the amplification of LAMP described in detection method of the present invention is 0.3mM is each
0.12 μ L, each 0.96 μ L of inner primer of 2.4mM, 0.4 μ L of ring primer of 1mM, 2 × contain 10 μ L of reacted fluorogenic dye liquid, 2 μ L
Sample to be tested DNA adds sterilizing pure water to mend to 20 μ L.
In some embodiments, the reaction system is each 0.12 μ L of outer primer F3 and B3 of 0.3mM；2.4mM's is interior
Each 0.96 μ L of primers F IP and BIP；Each 0.4 μ L of ring primer LF and LB of 1mM；2 × reaction solution, 10 μ L；The sample to be tested DNA of 2 μ L,
Sterilizing pure water is added to mend to 20 μ L.
Preferably, it is 60 DEG C of -65 DEG C of constant temperature 50min that LAMP described in the detection method, which detects reaction condition,.It is more excellent
It is selected as, the LAMP detection reaction condition is 65 DEG C of constant temperature 50min.
As shown from the above technical solution, the present invention provides a kind of parting detections that can distinguish pig parvoviral 1-7 type
Primer combination, detection method and the kit of LAMP.The present invention is utilized respectively the guarantor of pig parvoviral NS1 gene and NP2 gene
The design of defending zone section covers LAMP primer, and it is specific good that finishing screen is selected, point that can distinguish pig parvoviral 1-7 type of high sensitivity
The primer combination of the LAMP of type detection.Primer combination can detect pig parvoviral 1-7 type respectively, and with the other diseases in pig source
(such as circovurus type 2 (PCV2), Pseudorabies virus (PRV), swine fever virus (CSFV), swine influenza virus (SIV), height cause a disease poison
Pig blue-ear disease poison (HP-PRRSV) etc.) no cross reaction.Fluorescent dye is added, it can be achieved that one in the present invention in LAMP reaction system
Footwork detects pig parvoviral 1-7 type, realizes whole real time monitoring in conjunction with fluorescent quantitation, can go out within an hour as a result, detection
Sensitivity can achieve 100 copies/μ L, provide strong tool for pig parvoviral parting research.Detection side of the present invention
Method had both had easy, quick, sensitive, special advantage, in turn avoided error and caused ring of uncapping caused by artificially determining
Border pollution.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1-3 is respectively the amplification curve of primer sets PPV1-1-PPV1-3；
Fig. 4-6 is respectively the amplification curve of primer sets PPV2-1-PPV2-3；
Fig. 7-9 is respectively the amplification curve of primer sets PPV3-1-PPV3-3；
Figure 10-12 is respectively the amplification curve of primer sets PPV4-1-PPV4-3；
Figure 13-15 is respectively the amplification curve of primer sets PPV5-1-PPV5-3；
Figure 16-18 is respectively the amplification curve of primer sets PPV6-1-PPV6-3；
Figure 19-21 is respectively the amplification curve of primer sets PPV7-1-PPV7-3；
Figure 22 is to use primer sets PPV1-1 in genome copy numbers for 104、103Amplification curve when detection；
Figure 23 is to use primer sets PPV1-2 in genome copy numbers for 104、103Amplification curve when detection；
Figure 24 is to use primer sets PPV2-1 in genome copy numbers for 104、103Amplification curve when detection；
Figure 25 is to use primer sets PPV3-1 in genome copy numbers for 104、103Amplification curve when detection；
Figure 26 is to use primer sets PPV4-2 in genome copy numbers for 104、103Amplification curve when detection；
Figure 27 is to use primer sets PPV4-3 in genome copy numbers for 104、103Amplification curve when detection；
Figure 28 is to use primer sets PPV5-3 in genome copy numbers for 104、103Amplification curve when detection；
Figure 29 is to use primer sets PPV6-2 in genome copy numbers for 104、103Amplification curve when detection；
Figure 30 is to use primer sets PPV7-1 in genome copy numbers for 104、103Amplification curve when detection；
Figure 31 is to use primer sets PPV7-2 in genome copy numbers for 104、103Amplification curve when detection；
Figure 32-34 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV1；
Figure 35-37 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV2；
Figure 38-40 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV3；
Figure 41-43 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV4；
Figure 44-46 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV5；
Figure 47-49 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV6；
Figure 50-52 is respectively in genome copy numbers 103、102、101The amplification curve of Shi Caiyong primer sets PPV7；
Figure 53-59 is respectively to use amplification curve of the primer sets PPV1-PPV7 in specificity verification；
Wherein, A-H is the corresponding fluorescence curve color of each reaction tank in figure, and E4 is to be in genome copy numbers in figure
104Amplification curve when detection, it is 10 that E3, which is in genome copy numbers,3Amplification curve when detection.
Specific embodiment
The invention discloses primer combination, the detections of a kind of LAMP of parting detection that can distinguish pig parvoviral 1-7 type
Method and detection kit.Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.Especially need
, it is noted that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as wrapping
It includes in the present invention.Method and product of the invention is described by preferred embodiment, and related personnel obviously can be not
Be detached from the content of present invention, method described herein be modified in spirit and scope or appropriate changes and combinations, realizing and
Using the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, material, reagent involved in the embodiment of the present invention are commercial product, can pass through quotient
Industry channel obtains.Experimental method in embodiment is unless otherwise specified conventional method.Wherein, containing the anti-of fluorescent dye
Answering liquid is the product of Capitalbio Corporation Co., Ltd., catalog number CP.440020.
The calculation method of DNA copy number is as follows:
50 μ g/ml of 1A260 absorbance value=dsDNA；
Nucleic acid concentration=(OD260) × (extension rate) × (50)=x ng/ μ l；
Average molecular weight (MW) representative gram/mol, unit dalton (dolton), i.e. 1dolton=1g/mol；
Mole=6.02 × 1023；
Average molecular weight (MW): dsDNA=(base number) × (660 dalton/base)；
Copy number calculation formula:
(6.02×1023Copies/ moles) × (l × 10 x ng/ μ-9)/(DNA length × 660)=copies/ μ l.
The screening preparation that embodiment 1, primer combine
One, the screening of primer combination
1, the primer sequence is synthesized by Sheng Gong company, designs multiple groups primer for pig parvoviral NS1 or VP2 gene
Group, each group primer sets sequence are as shown in table 1.
1 primer sets sequence of table
In above-mentioned primer combination, the respective independent packaging of each single stranded DNA.
In above-mentioned primer combination, primers F 3, primer B3, primers F IP, primer BIP, primer LF in each primer sets and draw
The molar ratio of object LB is 0.3:0.3:2.4:2.4:1:1.
2, using pig parvoviral 1-7 type detection gene plasmid DNA as template, the primer sets pair of step 1 preparation are respectively adopted
Template carries out ring mediated isothermal amplification detection.
1 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for U44978.1, L23427.1, KF742500.2, JN872448.1 compression conservative sequence
The DNA molecular of column, obtains recombinant plasmid, which is 1 type of pig parvoviral detection gene plasmid.
2 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for AB076669.1, KP765690.1, KX517759.1, GU938299.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 2 type of pig parvoviral detection gene plasmid.
3 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for KU167028.1, EU200677.1, JN990268.1, KX827774.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 3 type of pig parvoviral detection gene plasmid.
4 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for JQ236636.1, GQ387499.1, KC701339.1, GU978965.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 4 type of pig parvoviral detection gene plasmid.
5 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for JX896318.1, KF661535.1, KU745628.1, KX352456.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 5 type of pig parvoviral detection gene plasmid.
6 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for KF999681.1, KP245953.1, KR709262.1, KX384813.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 6 type of pig parvoviral detection gene plasmid.
7 type of pig parvoviral detects gene plasmid: in pUC57 plasmid (Sangon Biotech (Shanghai) Co., Ltd.)
The site SacI be inserted into No. Genebank for KY996756.1, KY996757.1, KY996758.1, KU563733.1 compression guarantor
The DNA molecular for keeping sequence, obtains recombinant plasmid, which is 7 type of pig parvoviral detection gene plasmid.
Reaction system (20 μ L): 10 μ L contain reaction solution (Capitalbio Corporation Co., Ltd.'s product, the product of fluorescent dye
Catalog number (Cat.No.): CP.440020), 2.96 μ L primer mixtures, 2 μ L template DNAs (5pg-50pg), mend sterilizing pure water to 20 μ L.Primer
The mixture of each primer composition in mixture, that is, primer combination.In reaction system, the outer primer F3 and B3 each 0.12 of 0.3mM
μL；Each 0.96 μ L of inner primer FIP and BIP of 2.4mM；Each 0.4 μ L of ring primer LF and LB of 1mM.
Reaction condition: 65 DEG C of constant temperature 50min.
In reaction process, fluorescence signal is detected using fluorescent PCR instrument.
3 repetitions are arranged in each reaction system.
If occurring positive amplification curve (i.e. amplification curve is typical " S type " amplification curve) in 45min, show anti-
Answer the corresponding gene group content in system that can be detected.If not occurring positive amplification curve in 45min (to expand
Increasing curve is typical " S type " amplification curve), show that the corresponding gene group content in reaction system cannot be detected.
1 type primer screening experimental result of pig parvoviral: the testing result of primer sets 1 such as Fig. 1, the detection knot of primer sets 2
Fruit such as Fig. 2, such as Fig. 3 of primer sets 3, the results show that primer sets 1 and 2 are preferable.
2 type primer screening experimental result of pig parvoviral: the testing result of primer sets 1 such as Fig. 4, the detection knot of primer sets 2
Fruit such as Fig. 5, such as Fig. 6 of primer sets 3, the results show that primer sets 1 are preferable.
3 type primer screening experimental result of pig parvoviral: the testing result of primer sets 1 such as Fig. 7, the detection knot of primer sets 2
Fruit such as Fig. 8, such as Fig. 9 of primer sets 3, the results show that primer sets 1 are preferable.
4 type primer screening experimental result of pig parvoviral: the testing result of primer sets 1 such as Figure 10, the detection knot of primer sets 2
Fruit such as Figure 11, primer sets 3 such as Figure 12.The results show that primer sets 2 and 3 are preferable.
5 type primer screening experimental result of pig parvoviral: the testing result of primer sets 1 such as Figure 13, the detection knot of primer sets 2
Fruit such as Figure 14, primer sets 3 such as Figure 15.The results show that primer sets 3 are preferable.
6 type primer screening experimental result of pig circular ring virus: the testing result of primer sets 1 such as Figure 16, the detection knot of primer sets 2
Fruit such as Figure 17, primer sets 3 such as Figure 18.The results show that primer sets 2 are preferable.
7 type primer screening experimental result of pig circular ring virus: the testing result of primer sets 1 such as Figure 19, the detection knot of primer sets 2
Fruit such as Figure 20, primer sets 3 such as Figure 21.The results show that primer sets 1 and 2 are preferable.
Embodiment 2, sensitivity primary dcreening operation
Sample to be tested: the plasmid of pig parvoviral 1-7 type prepared by embodiment 1.
1, the Plasmid DNA for extracting sample to be tested, carries out gradient dilution with sterile water, obtains each dilution.
2, for the dilution obtained using step 1 as template, the primer combination prepared using embodiment 1 carries out ring mediated isothermal expansion
Increase.
Reaction system (20 μ L): 10 μ L reaction solutions (Capitalbio Corporation Co., Ltd.'s product, catalog number:
CP.440020), (genome copy numbers contained in 1 μ L dilution are respectively for 2.96 μ L primer mixtures, 2 μ L template dilutions
104With 103), moisturizing to 20 μ L.The mixture of each primer composition in primer mixture, that is, primer combination.In reaction system,
Each 0.12 μ L of outer primer F3 and B3 of 0.3mM；Each 0.96 μ L of inner primer FIP and BIP of 2.4mM；Ring the primer LF and LB of 1mM is each
0.4μL。
Reaction condition: 65 DEG C of constant temperature 50min.
In reaction process, fluorescence signal is detected using fluorescent PCR instrument.
According to the difference of genome copy numbers in dilution, total following 2 reaction systems:
The genome copy numbers contained in reaction system 1:1 μ L dilution are 104；
The genome copy numbers contained in reaction system 2:1 μ L dilution are 103；
3 repetitions are arranged in each reaction system.
It is 10 that primer sets PPV1-1, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 22), 3
A detection is detectable out and reproducible, and primer sets PPV1-2 is 10 in gene copy number43 detections can when (Figure 23)
Complete appearance and repeatability is preferably, but gene copy number is 103When, 3 detections can not appearance and less reproducible completely, therefore
Primer sets PPV1-1 is selected to carry out sensitivity secondary screening.
It is 10 that primer sets PPV2-1, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 24), 3
A detection is detectable to be come out and reproducible
It is 10 that primer sets PPV3-1, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 25), 3
A detection is detectable out and reproducible.
It is 10 that primer sets PPV4-2, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 26), 3
A detection is detectable out and reproducible, and primer sets PPV4-3 is 10 in gene copy number43 detections can when (Figure 27)
Complete appearance and repeatability is preferably, but gene copy number is 103When, 3 detections can not appearance and less reproducible completely, therefore
Primer sets PPV4-2 is selected to carry out sensitivity secondary screening.
It is 10 that primer sets PPV5-3, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 28), 3
A detection is detectable out and reproducible.
It is 10 that primer sets PPV6-2, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 29), 3
A detection is detectable out and reproducible.
It is 10 that primer sets PPV7-1, which detects target gene genome copy numbers in 1 μ L dilution,4With 103When (Figure 30), 3
A detection is detectable out and reproducible, and primer sets PPV7-2 is 10 in gene copy number43 detections can when (Figure 31)
Complete appearance and repeatability is preferably, but gene copy number is 103When, 3 detections can not appearance and less reproducible completely, therefore
Primer sets PPV7-1 is selected to carry out sensitivity secondary screening.
The above result shows that some primers screened are after reducing template concentrations, expanding effect can be deteriorated, and we will
The primer of primary dcreening operation qualification carries out secondary screening, further test sensitivity.
Embodiment 3, sensitivity secondary screening
Sample to be tested: the plasmid of pig parvoviral 1-7 type prepared by embodiment 1.
1, the Plasmid DNA for extracting sample to be tested, carries out gradient dilution with sterile water, obtains each dilution.
2, the dilution obtained using step 1 carries out ring mediation etc. using the primer combination of Examples 1 and 2 preparation as template
Temperature amplification.
Reaction system (20 μ L): 10 μ L reaction solutions (Capitalbio Corporation Co., Ltd.'s product, catalog number:
CP.440020), (genome copy numbers contained in 1 μ L dilution are respectively for 2.96 μ L primer mixtures, 2 μ L template dilutions
103、102Or 101), moisturizing to 20 μ L.The mixture of each primer composition in primer mixture, that is, primer combination.Reaction system
In, each 0.12 μ L of outer primer F3 and B3 of 0.3mM；Each 0.96 μ L of inner primer FIP and BIP of 2.4mM；The ring primer LF of 1mM and
Each 0.4 μ L of LB.
Reaction condition: 65 DEG C of constant temperature 50min.
In reaction process, fluorescence signal is detected using fluorescent PCR instrument.
According to the difference of genome copy numbers in dilution, total following 3 reaction systems:
The genome copy numbers contained in reaction system 1:1 μ L dilution are 103；
The genome copy numbers contained in reaction system 2:1 μ L dilution are 102；
The genome copy numbers contained in reaction system 3:1 μ L dilution are 101；
20 repetitions are arranged in each reaction system.
If occurring positive amplification curve (i.e. amplification curve is typical " S type " amplification curve) in 45min, show anti-
Answer the corresponding gene group content in system that can be detected.If not occurring positive amplification curve in 45min (to expand
Increasing curve is typical " S type " amplification curve), show that the corresponding gene group content in reaction system cannot be detected.
It is 10 that primer sets PPV1-1, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 32) and 102(figure
33) 20 detections are detectable out and reproducible when, and 101When (Figure 34) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L
It is 10 that primer sets PPV2-1, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 35) and 102(figure
36) 20 detections are detectable out and reproducible when, and 101When (Figure 37) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L.
It is 10 that primer sets PPV3-1, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 38) and 102(figure
39) 20 detections are detectable out and reproducible when, and 101When (Figure 40) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L
It is 10 that primer sets PPV4-2, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 41) and 102(figure
42) 20 detections are detectable out and reproducible when, and 101When (Figure 43) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L.
It is 10 that primer sets PPV5-3, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 44) and 102(figure
45) 20 detections are detectable out and reproducible when, and 101When (Figure 46) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L.
It is 10 that primer sets PPV6-2, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 47) and 102(figure
48) 20 detections are detectable out and reproducible when, and 101When (Figure 49) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L.
It is 10 that primer sets PPV7-1, which detects target gene genome copy numbers in 1 μ L dilution,3(Figure 50) and 102(figure
51) 20 detections are detectable out and reproducible when, and 101When (Figure 52) 20 detection can not completely appearance and repeatability compared with
Difference, therefore the sensitivity of primer sets is 100 copy numbers/μ L.
Embodiment 4, specificity experiments
Classical Swine Fever Virus Shimen Strain AV1411 (04/08/87) and high pathogenic porcine reproductive and respiratory syndrome virus (PRRSV-
JXA1) purchased from China Veterinary Drugs Supervisory Inst., swine influenza virus (VR-333TM) it is purchased from ATCC, porcine pseudorabies virus
(BNCC 129649), pig parvoviral (BNCC124946) and porcine circovirus 2 type (BNCC128527) receive biology purchased from north
BNCC；Pig parvoviral 2-7 type plasmid is by giving birth to work building.
Each sample to be tested carries out following steps respectively:
1, the genomic DNA or RNA of sample to be tested are extracted.
2, using step 1 extract genomic nucleic acids as template, be respectively adopted embodiment 3 screening obtain each primer sets into
Row ring mediated isothermal amplification.
Reaction system (20 μ L): 10 μ L reaction solutions (Capitalbio Corporation Co., Ltd.'s product, catalog number:
CP.440020), 2.96 μ L primer mixtures, 2 μ L genomic templates (5pg-50pg), (template is 0.5 μ L AMV reverse transcriptase
It is added when RNA), RNase-free water moisturizing to 20 μ L.What each primer in primer mixture, that is, primer combination formed
Mixture.In reaction system, each 0.12 μ L of outer primer F3 and B3 of 0.3mM；Each 0.96 μ L of inner primer FIP and BIP of 2.4mM；
Each 0.4 μ L of ring primer LF and LB of 1mM.
Reaction condition: 65 DEG C of constant temperature 50min.
In reaction process, fluorescence signal is detected using fluorescent PCR instrument.
Figure 53 is shown in using the result of primer sets PPV1-1, only when the genomic DNA that sample to be tested is 1 type of pig parvoviral
Positive amplification curve is shown when (sample to be tested 1) (i.e. amplification curve is typical " S type " amplification curve).When sample to be tested is pig
Parvovirus 2-7 type Plasmid DNA or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), pig
Positive amplification curve is not shown when influenza virus (SIV), high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 54 is shown in using the result of primer sets PPV2-1, only when Plasmid DNA that sample to be tested is tiny sick 2 type of pig (to
Test sample sheet 2) when show positive amplification curve (i.e. amplification curve be typical " S type " amplification curve).When sample to be tested is other
Type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine influenza virus
(SIV), positive amplification curve is not shown when high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 55 is shown in using the result of primer sets PPV3-1, only when the Plasmid DNA that sample to be tested is 3 type of pig parvoviral
Positive amplification curve is shown when (sample to be tested 3) (i.e. amplification curve is typical " S type " amplification curve).When sample to be tested is it
Its type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine flu disease
Positive amplification curve is not shown when malicious (SIV), high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 56 is shown in using the result of primer sets PPV4-2, only when Plasmid DNA that sample to be tested is tiny sick 4 type of pig (to
Test sample sheet 4) when show positive amplification curve (i.e. amplification curve be typical " S type " amplification curve).When sample to be tested is other
Type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine influenza virus
(SIV), positive amplification curve is not shown when high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 57 is shown in using the result of primer sets PPV5-3, only when the Plasmid DNA that sample to be tested is 5 type of pig parvoviral
Positive amplification curve is shown when (sample to be tested 5) (i.e. amplification curve is typical " S type " amplification curve).When sample to be tested is it
Its type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine flu disease
Positive amplification curve is not shown when malicious (SIV), high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 58 is shown in using the result of primer sets PPV6-2, only when Plasmid DNA that sample to be tested is tiny sick 6 type of pig (to
Test sample sheet 6) when show positive amplification curve (i.e. amplification curve be typical " S type " amplification curve).When sample to be tested is other
Type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine influenza virus
(SIV), positive amplification curve is not shown when high pathogenic pig blue-ear disease malicious (HP-PRRSV).
Figure 59 is shown in using the result of primer sets PPV7-1, only when Plasmid DNA that sample to be tested is tiny sick 7 type of pig (to
Test sample sheet 7) when show positive amplification curve (i.e. amplification curve be typical " S type " amplification curve).When sample to be tested is other
Type pig parvoviral or porcine circovirus 2 type (PCV2), swine fever virus (CSFV), porcine pseudorabies virus (PRV), swine influenza virus
(SIV), positive amplification curve is not shown when high pathogenic pig blue-ear disease malicious (HP-PRRSV).
The above result shows that primer sets of the present invention have very high specificity to its target gene.
Applicant changes reaction system according to the method for above-described embodiment, and reaction system is in some embodiments
Each 0.12 μ L of outer primer F3 and B3 of 0.29mM；Each 0.96 μ L of inner primer FIP and BIP of 2.3mM；The ring primer LF of 0.9mM and
Each 0.4 μ L of LB.Reaction system is each 0.12 μ L of outer primer F3 and B3 of 0.31mM in further embodiments；Draw in 2.5mM
Each 0.96 μ L of object FIP and BIP；Each 0.4 μ L of ring primer LF and LB of 1.1mM.The results show that specificity and sensitivity and above-mentioned reality
It is similar to apply a result.
In conclusion being selected for 1 type NS1 GeneScreen of pig parvoviral optimal for detecting 1 type of pig parvoviral
Primer sets are following (5 ' → 3 '):
Outer primer PPV1-F3:5 '-cttggttggtaaagaaaggt-3 ' (SEQ ID NO.1)
Outer primer PPV1-B3:5 '-agctaaatccaggtcctc-3 ' (SEQ ID NO.2)
Inner primer PPV1-FIP:5 '-tggggtttgcatttttggcggctagctatatgcatcattg-3 ' (SEQ ID
NO.3)
Inner primer PPV1-BIP:5 '-tacaccaacagactctcagatttcattggagttgctgcgta-3 ' (SEQ ID
NO.4)
Ring primer PPV1-LF:5 '-gaccaatcaggtacatttcc-3 ' (SEQ ID NO.5)
Ring primer PPV1-LB:5 '-acatcagtgaaaacttcgc-3 ' (SEQ ID NO.6)
It is as follows that the optimal primer sets for detecting 2 type of pig parvoviral are selected for 2 type NS1 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV2-F3:5 '-gcaaatggagcccagcag-3 ' (SEQ ID NO.7)
Outer primer PPV2-B3:5 '-tgatcgccttcccaccag-3 ' (SEQ ID NO.8)
Inner primer PPV2-FIP:5 '-ttcagccacccccccatctcttcaccttcatctcgcagtg-3 ' (SEQ I D
NO.9)
Inner primer PPV2-BIP:5 '-atgatagagcacaggccaggcgagcggtcttcctcatctca-3 ' (SEQ ID
NO.10)
Ring primer PPV2-LF:5 '-ctcgggcctttgtgtcg-3 ' (SEQ ID NO.11)
Ring primer PPV2-LB:5 '-gccccccccttgtactt-3 ' (SEQ ID NO.12)
It is as follows that the optimal primer sets for detecting 3 type of pig parvoviral are selected for 3 type NS1 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV3-F3:5 '-agcaactggttgaagagatgg-3 ' (SEQ ID NO.13)
Outer primer PPV3-B3:5 '-gcagcctcaaatctctccatg-3 ' (SEQ ID NO.14)
Inner primer PPV3-FIP:5 '-agtctcccttgtctggtcttcctcaacattcctaactcgccaca-3 ' (SEQ
ID NO.15)
Inner primer PPV3-BIP:5 '-atcagtgcgacctgacctttgtcaagcataccagacatgctcta-3 ' (SE
Q ID NO.16)
Ring primer PPV3-LF:5 '-gatatcccacagaatgctccaac-3 ' (SEQ ID NO.17)
Ring primer PPV3-LB:5 '-aaggtatctactgcctaaagtacca-3 ' (SEQ ID NO.18)
It is as follows that the optimal primer sets for detecting 4 type of pig parvoviral are selected for 4 type NS1 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV4-F3:5 '-gacattatgaactgccctat-3 ' (SEQ ID NO.19)
Outer primer PPV4-B3:5 '-agggatgaattcagtctcag-3 ' (SEQ ID NO.20)
Inner primer PPV4-FIP:5 '-ttcatttcttttgggggtgggtaaaaaaggacctgccagt-3 ' (SEQ I D
NO.21)
Inner primer PPV4-BIP:5 '-agagcggaaccagatgaaattcgtttcttctttctcggtgc-3 ' (SEQ ID
NO.22)
Ring primer PPV4-LF:5 '-tctctccaacaggaactaaa-3 ' (SEQ ID NO.23)
Ring primer PPV4-LB:5 '-aatccagaagaactggacc-3 ' (SEQ ID NO.24)
It is as follows that the optimal primer sets for detecting 5 type of pig parvoviral are selected for 5 type VP2 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV5-F3:5 '-ggaacatcaccaatcaaga-3 ' (SEQ ID NO.25)
Outer primer PPV5-B3:5 '-gagagatcaatttcattgcg-3 ' (SEQ ID NO.26)
Inner primer PPV5-FIP:5 '-agttctttctgttctcggtgacctattagaacacgactcaagc-3 ' (SEQ
ID NO.27)
Inner primer PPV5-BIP:5 '-atcaaacatggagcgggagatttccaggacctgtgtag-3 ' (SEQ ID
NO.28)
Ring primer PPV5-LF:5 '-gcttcttctggttgttcttc-3 ' (SEQ ID NO.29)
Ring primer PPV5-LB:5 '-cggaaccggtatcaact-3 ' (SEQ ID NO.30)
It is as follows that the optimal primer sets for detecting 6 type of pig parvoviral are selected for 6 type NS1 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV6-F3:5 '-tcaagatcagggagtcatt-3 ' (SEQ ID NO.31)
Outer primer PPV6-B3:5 '-tagtgctcacaatacgct-3 ' (SEQ ID NO.32)
Inner primer PPV6-FIP:5 '-gtagtcacatcattatcacacatccactgctcctacttttaaagc-3 ' (SE
Q ID NO.33)
Inner primer PPV6-BIP:5 '-cctgtactctttccttgctaccgcagctctaagacactgt-3 ' (SEQ I D
NO.34)
Ring primer PPV6-LF:5 '-cattttatcacccgtagcc-3 ' (SEQ ID NO.35)
Ring primer PPV6-LB:5 '-ccagctggtactcatttg-3 ' (SEQ ID NO.36)
It is as follows that the optimal primer sets for detecting 7 type of pig parvoviral are selected for 7 type VP2 GeneScreen of pig parvoviral
(5 ' → 3 '):
Outer primer PPV7-F3:5 '-ggtaccacaactggtacgaag-3 ' (SEQ ID NO.37)
Outer primer PPV7-B3:5 '-tggtgttccatgtccatgtc-3 ' (SEQ ID NO.38)
Inner primer PPV7-FIP:5 '-cggtggttggtttagggacgagacacgctctggagaagct-3 ' (SEQ ID
NO.39)
Inner primer PPV7-BIP:5 '-accaaagaaggggtcggaaacaggtttcgggtgccgtgatg-3 ' (S EQ
ID NO.40)
Ring primer PPV7-LF:5 '-ccttctttgtaggccacgt-3 ' (SEQ ID NO.41)
Ring primer PPV7-LB:5 '-tggagcagacttacactccc-3 ' (SEQ ID NO.42).
Sequence table
<110>Capitalbio Corporation Co., Ltd.
Beijing animal epidemic prevention and control center
<120>a kind of primer combination, detection method and the reagent of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type
Box
<130> MP1905396
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cttggttggt aaagaaaggt 20
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agctaaatcc aggtcctc 18
<210> 3
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tggggtttgc atttttggcg gctagctata tgcatcattg 40
<210> 4
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tacaccaaca gactctcaga tttcattgga gttgctgcgt a 41
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
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gaccaatcag gtacatttcc 20
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
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acatcagtga aaacttcgc 19
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
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gcaaatggag cccagcag 18
<210> 8
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tgatcgcctt cccaccag 18
<210> 9
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ttcagccacc cccccatctc ttcaccttca tctcgcagtg 40
<210> 10
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgatagagc acaggccagg cgagcggtct tcctcatctc a 41
<210> 11
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ctcgggcctt tgtgtcg 17
<210> 12
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gcccccccct tgtactt 17
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
agcaactggt tgaagagatg g 21
<210> 14
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gcagcctcaa atctctccat g 21
<210> 15
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
agtctccctt gtctggtctt cctcaacatt cctaactcgc caca 44
<210> 16
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
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atcagtgcga cctgaccttt gtcaagcata ccagacatgc tcta 44
<210> 17
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gatatcccac agaatgctcc aac 23
<210> 18
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
aaggtatcta ctgcctaaag tacca 25
<210> 19
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gacattatga actgccctat 20
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
agggatgaat tcagtctcag 20
<210> 21
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ttcatttctt ttgggggtgg gtaaaaaagg acctgccagt 40
<210> 22
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
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<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tctctccaac aggaactaaa 20
<210> 24
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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aatccagaag aactggacc 19
<210> 25
<211> 19
<212> DNA
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ggaacatcac caatcaaga 19
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
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gagagatcaa tttcattgcg 20
<210> 27
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
agttctttct gttctcggtg acctattaga acacgactca agc 43
<210> 28
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
atcaaacatg gagcgggaga tttccaggac ctgtgtag 38
<210> 29
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gcttcttctg gttgttcttc 20
<210> 30
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cggaaccggt atcaact 17
<210> 31
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
tcaagatcag ggagtcatt 19
<210> 32
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tagtgctcac aatacgct 18
<210> 33
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gtagtcacat cattatcaca catccactgc tcctactttt aaagc 45
<210> 34
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
cctgtactct ttccttgcta ccgcagctct aagacactgt 40
<210> 35
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
cattttatca cccgtagcc 19
<210> 36
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ccagctggta ctcatttg 18
<210> 37
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ggtaccacaa ctggtacgaa g 21
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tggtgttcca tgtccatgtc 20
<210> 39
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
cggtggttgg tttagggacg agacacgctc tggagaagct 40
<210> 40
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
accaaagaag gggtcggaaa caggtttcgg gtgccgtgat g 41
<210> 41
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ccttctttgt aggccacgt 19
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
tggagcagac ttacactccc 20
Claims (9)
1. a kind of primer combination of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type, including primer sets 1-7；
The primer sets 1 include 2 outer primers, SEQ ID NO.3 and SEQ shown in SEQ ID NO.1 and SEQ ID NO.2
2 ring primers shown in 2 inner primers and SEQ ID NO.5 and 6 shown in ID NO.4；
The primer sets 2 include 2 outer primers, SEQ ID NO.9 and SEQ shown in SEQ ID NO.7 and SEQ ID NO.8
2 ring primers shown in 2 inner primers and SEQ ID NO.11 and 12 shown in ID NO.10；
The primer sets 3 include SEQ ID NO.13 and SEQ ID NO.14 shown in 2 outer primers, SEQ ID NO.15 and
2 ring primers shown in 2 inner primers and SEQ ID NO.17 and 18 shown in SEQ ID NO.16；
The primer sets 4 include SEQ ID NO.19 and SEQ ID NO.20 shown in 2 outer primers, SEQ ID NO.21 and
2 ring primers shown in 2 inner primers and SEQ ID NO.23 and 24 shown in SEQ ID NO.22；
The primer sets 5 include SEQ ID NO.25 and SEQ ID NO.26 shown in 2 outer primers, SEQ ID NO.27 and
2 ring primers shown in 2 inner primers and SEQ ID NO.29 and 30 shown in SEQ ID NO.28；
The primer sets 6 include SEQ ID NO.31 and SEQ ID NO.32 shown in 2 outer primers, SEQ ID NO.33 and
2 ring primers shown in 2 inner primers and SEQ ID NO.35 and 36 shown in SEQ ID NO.34；
The primer sets 7 include SEQ ID NO.37 and SEQ ID NO.38 shown in 2 outer primers, SEQ ID NO.39 and
2 ring primers shown in 2 inner primers and SEQ ID NO.41 and 42 shown in SEQ ID NO.40.
2. a kind of kit of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type, including described in claim 1 draw
Object combination, wherein each equal independent packaging of primer in primer sets conjunction.
3. kit according to claim 2 further includes the reaction solution containing fluorescent dye and/or sterilizing pure water.
4. kit according to claim 2, the molal quantity of 2 outer primers in each primer sets in the primer combination
Identical, the molal quantity of 2 inner primers is identical, and the molal quantity of 2 ring primers is identical, the outer primer in each primer sets: inner primer: ring
The molar ratio of primer is (0.58-0.62): (4.6-5.0): (1.8-2.2).
5. kit according to claim 2, outer primer in each primer sets: inner primer: the molar ratio of ring primer is
0.6:4.8:2。
6. a kind of detection method of the LAMP for the parting detection that can distinguish pig parvoviral 1-7 type, using described in claim 1
Primer combination carries out LAMP amplification, starts the time occurred judgement detection by observing real-time fluorescence PCR instrument " S " type amplification curve
As a result.
7. detection method according to claim 6, the outer primer each 0.12 that the reaction system of the LAMP amplification is 0.3mM
μ L, each 0.96 μ L of inner primer of 2.4mM, each 0.4 μ L of ring primer of 1mM, 2 × 10 μ L of reaction solution containing fluorescent dye, 2 μ L
Sample to be tested DNA adds sterilizing pure water to mend to 20 μ L.
8. detection method according to claim 6, the LAMP detects reaction condition are as follows: 60 DEG C of -65 DEG C of constant temperature 50min.
9. detection method according to claim 6, the LAMP detects reaction condition are as follows: 65 DEG C of constant temperature 50min.
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| CN105349702A (en) * | 2015-11-27 | 2016-02-24 | 广西壮族自治区兽医研究所 | PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof |
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| CN102002539A (en) * | 2010-11-23 | 2011-04-06 | 中国农业大学 | Porcine parvovirus assay kit and application thereof |
| CN104450976A (en) * | 2015-01-06 | 2015-03-25 | 福建农林大学 | Loop-mediated isothermal amplification reaction primer for detecting porcine parvovirus 5 |
| CN105349702A (en) * | 2015-11-27 | 2016-02-24 | 广西壮族自治区兽医研究所 | PPV (porcine parvovirus) LAMP (loop-mediated isothermal amplification) kit and application thereof |
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