CN105925728B - A kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and kit - Google Patents
A kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and kit Download PDFInfo
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Abstract
The invention belongs to technical field of biological, a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and kit are disclosed；First by designing and screening to obtain a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, the sequence of the upstream primer, downstream primer and probe is as shown in NO:1~3 SEQ ID；Using the primer and probe can specific amplification come out Sai Neijia paddy virus, real time fluorescent quantitative can acquire fluorescence data, identify SVV according to CT value by the combination situation of double-stranded DNA fluorescent dyestuff during real-time monitoring PCR and pcr amplification product；SVV can be identified after carrying out PCR amplification using the method, accuracy is high, specificity is good, reproducible, can accurately, fast and efficiently carry out identification SVV, be conducive to promote and apply in clinical practice.
Description
Technical field
The present invention relates to technical field of biological, quantitative more particularly, to a kind of Sai Neijia paddy virus real-time fluorescence
PCR detection primer and kit.
Background technique
Sai Neijia paddy viral (Seneca Valley virus SVV) is single strand plus RNA virus, is micro ribonucleic acid disease
The Typical Representative of malicious section Senecavirus Tobamovirus.It is initially considered as the pollutant in PER.C6 cell culture, then
It is separated in the swinery of America & Canada, occurs within 2007 blister Disease Clinical disease in the swinery in one slaughterhouse of the U.S.
There is cyllopodia in shape, about 80% pig, and it is feminine gender that PCR, which detects Schweineseuche, pig blisters, vesicular stomatitis and blister rash, and
SVV is but the positive, to be considered as primary blister disease.Recent pig Sai Neijia paddy viral (SVV) is in Brazilian swinery
Infection and outburst, it was demonstrated that pig Sai Neijia paddy virus is likely to be one of primary blister disease pathogen of pig.2015
There is SVV several times and infect swinery and serious clinical onset and dead symptom occur in year China and Brazil, cause serious warp
Ji loss.The bubble disease of SVV clinically caused clinical symptoms and some other pig is very much like, sick in clinical and tissue
It is difficult to distinguish in Neo-Confucianism, this just brings certain difficulty to making a definite diagnosis for the disease, and antidiastole usually need to be by laboratory diagnosis skill
Art, traditional detection method is time-consuming and laborious, and operating method is cumbersome, is unfavorable for the timely diagnoses and treatment of disease.
Summary of the invention
It is above-mentioned present in Sai Neijia paddy viral diagnosis in the prior art the technical problem to be solved by the present invention is to overcome
Defect provides a kind of primer of specific detection Sai Neijia paddy virus real-time fluorescence quantitative PCR.
A second object of the present invention is to provide the kits containing above-mentioned detection primer.
The purpose of the present invention is what is be achieved by the following technical programs:
A kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, the upstream primer, downstream primer and
The sequence of probe is as shown in NO:1~3 SEQ ID.
Preferably, 5 ' end labels of the probe is fluorescent reporter group；3 ' end labels are quenching groups.
It is highly preferred that the fluorescent reporter group is FAM, the quenching group is TAMAR.
The present invention also provides the reagents for containing the Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe
Box.
Preferably, the concentration of the upstream primer, downstream primer and probe is 10 μM.
Preferably, the kit further includes PCR reaction buffer, fluorescent dye, positive criteria product.
It is highly preferred that the preparation method of the positive criteria product be from SVV cell virus extract RNA and reverse transcription at
CDNA, then come out SVV-3C gene magnification through PCR, it obtains and target fragment amplified production of the same size.PCR product recycling
Afterwards, it connect with PMD19-T carrier, converted, expand the recombinant plasmid containing target fragment, identified through bacterium solution PCR and sequencing,
Confirmation has successfully constructed SVV recombinant plasmid standard items, is named as PMD19-T-3C, stand-by as positive criteria product.
Preferably, the kit carries out the reaction system of quantitative fluorescent PCR are as follows: Premix Ex Taq(Probe
QPCR) (2 ×) 10 μ L, 10 μM, 0.4 μ L upstream primer, 10 μM, 0.4 μ L downstream primer, ROX Reference Dye II
(50 ×) 0.2 μ L, 10 μM, 0.8 μ L probe, 2.0 μ L of DNA profiling, ddH2O 6.2μL。
Preferably, the kit carry out carry out quantitative fluorescent PCR reaction condition are as follows: 95 DEG C 30 seconds, 95 DEG C 5
Second, 60 DEG C 34 seconds, 40 circulation.
Compared with prior art, the invention has the following advantages:
The present invention is by designing and screening to obtain a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and spy
Needle, the sequence of the upstream primer, downstream primer and probe is as shown in NO:1~3 SEQ ID；It can be special using the primer and probe
Specific amplification comes out Sai Neijia paddy virus, real time fluorescent quantitative can by double-stranded DNA fluorescent dyestuff during real-time monitoring PCR with
The combination situation of pcr amplification product acquires fluorescence data, identifies SVV according to CT value；PCR amplification is carried out using the method
After can identify SVV, accuracy is high, specificity is good, reproducible, can accurately, fast and efficiently carry out identification SVV, favorably
In promoting and applying in clinical practice.
Detailed description of the invention
Fig. 1 is the PCR amplification result for recombinating plasmid standard PMD19-T-3C.
Fig. 2 is the standard curve of real-time fluorescence quantitative PCR.
Fig. 3 is that the sensitivity of real-time fluorescence quantitative PCR is tested；Wherein 1 is 1 × 109copies/μL；2 for 1 ×
108copies/μL；3 be 1 × 107copies/μL；4 be 1 × 106copies/μL；5 be 1 × 105copies/μL；6 for 1 ×
104copies/μL；7 be 1 × 103copies/μL；8 be 1 × 102copies/μL；9 be 1 × 101copies/μL；10 for 1 ×
100copies/μL；11 be negative control.
Fig. 4 is the specific test of real-time fluorescence quantitative PCR, wherein 1 is SVV；2 be FMDV；3 be SVDV；4 be VSV；5
For PRRSV；6 be PCV；7 be PRV；8 be PK-15 cell.
Specific embodiment
The contents of the present invention are further illustrated below in conjunction with Figure of description and specific embodiment, but should not be construed as pair
Limitation of the invention.In the case where without departing substantially from spirit of that invention and essence, modify made by the method for the present invention, step, condition
Or replacement, it all belongs to the scope of the present invention.Unless otherwise noted, experimental method used in embodiment is those skilled in the art
Conventional method and technology known to member, reagent or material are to be obtained by commercial sources.
The preparation of 1 standard positive template of embodiment
One, the extracting of virus total RNA
It is carried out by Invitrogen company's T RIZOL LS Reagent RNA extracts kit operation instructions.1.5
The resulting 750 μ L TRIZOL of virus liquid supernatant of cell proliferation that 250 μ L have been dispensed is added in mL eppendorf pipe,
It mixes well, is placed at room temperature for 10 min；The chloroform of 200 μ L is added, is aggressively shaken 15sec, is stored at room temperature 5min, 4 DEG C, 12000
Rpm is centrifuged 15 min；Supernatant is transferred in 1.5 new mL eppendorf pipes, 500 μ L isoamyl alcohol is added, mix well,
It is placed at room temperature for 10min, 4 DEG C, 12000 rpm are centrifuged 10 min；Liquid is discarded supernatant, ice-cold 70% ethyl alcohol, 1000 μ is precipitated
L is mixed gently, and washed once, and 4 DEG C, 12000 rpm are centrifuged 10 min；Liquid is discarded supernatant, is air-dried；It is handled with 20 μ L DEPC
Tri-distilled water dissolve RNA, -80 DEG C save or are directly used in reverse transcription.
Two, design of primers
According to SVV-001(DQ641257 in GenBank) 3C regional gene sequence, design PCR primer: upstream primer
P1:5 '-ATCCTTTTGCTGCCCATGTG-3 ', downstream primer P2:5 '-AGAACTCTACCCAACGCCTC-3 ', utilizes this
Primer amplification 3C gene, reaction system are as follows: 25 μ L of PremixTaq, 1 μ L of upstream primer P1,1 μ L of downstream primer P2, template
1 μ L of DNA, finally uses ddH2It is 50 μ L that O, which supplies reaction system total volume,.
Amplification program are as follows: (1) 94 DEG C of initial denaturation 5min, (2) 94 DEG C of denaturation 1min, 57 DEG C of 30 s of annealing, 72 DEG C extend 30
S, totally 32 recycle；(3) 72 DEG C of extension 10min.
PCR product (referring to the operation instructions of Omega company E.Z.N.A. Gel Extraction Kit) are recycled,
Agarose gel electrophoresis is carried out, correct target fragment is accredited as, is used for subsequent clone.
Three, the clone of target gene
(1) recovery product connects pMD19-T carrier
Referring to pMD 19-T Vector kit specification, coupled reaction system are as follows: 2 μ L of PCR product of purifying,
pMD19-T vector 0.5μL,Solution I 2.5μL；The total volume of final coupled reaction system is 5 μ L.It will be above-mentioned anti-
After answering system to mix and be slightly centrifuged, 4 DEG C of connections are overnight.
(2) connection product converts
The 5 μ L of connection product of (1) is lightly added in the competent escherichia coli cell of 50 μ L, ice bath 30min；42℃
Then heat shock 90s is quickly transferred in ice bath cooling 2min；It is added in 200 μ L LB liquid mediums, 37 DEG C, 160rpm vibration
Culture 45min is shaken, so that Escherichia coli is recovered, tolerant gene expression；The competent cell even spread Amp that will be recovered above+/LB
Solid medium plate, 37 DEG C of overnight incubations.
(3) the PCR identification and screening of recon
Three single colonies of picking from the plate that (2) are incubated overnight are inoculated in 1mL respectively and contain 100 μ g/mL Amp+'s
In LB liquid medium, 37 DEG C, 220rpm shaking culture 6h or more (OD600 value 0.3~0.4)；Bacterium solution is taken to carry out PCR identification,
Product is detected with 1% agarose gel electrophoresis；The bacterium solution for taking 20 μ L identification positive is inoculated into 2mL Amp with the ratio of 1:100+LB liquid medium in, 37 DEG C, 220rpm shake overnight incubation.
(4) extraction and identification of recombinant plasmid
Referring to the specification of Omega company E.Z.N.A. Plasmid Mini Kit I, carried out to bacterium solution is incubated overnight
Plasmid extraction detects plasmid extraction effect with 1% agarose gel electrophoresis.
1% agarose gel electrophoresis of recombinant plasmid extract product is detected, qualification result is shown in Fig. 1, the weight of the identified positive
Group pMD19-T plasmid send Beijing AudioCodes biotechnology Co., Ltd to carry out nucleotide sequencing.
The design and screening of 2 primer of embodiment and TaqMan probe
According to the 3C conservative fragments design special primer and TaqMan probe in SVV gene order, by Hua Da, company is synthesized,
Primer and probe sequence is shown in Table 1, and the fluorescent reporter group of 5 ' end labels of probe is FAM, and the quenching group of 3 ' end labels is
TAMRA。
By the primer and probe in table 1, PCR is carried out to SVV, as a result, it has been found that: other 3 pairs of primers occur non-specific expansion
Increasing or primer dimer do not meet requirement of experiment, therefore only primer 1 and primer 2 can be used as specific amplification detection SVV's
Primer.
3 quantitative fluorescent PCR reaction condition optimization of embodiment and standard curve making
Primer concentration, concentration and probe concentration and annealing temperature condition are optimized, 20 μ L reaction system of Real-Time PCR is
Premix Ex Taq(Probe qPCR) (2 ×) 10 μ L, each specificity upstream and downstream primer (10 μM) each 0.4 μ L, ROX
Reference Dye II(50 ×) 0.2 μ L, 0.8 μ L of fluorescence probe (10 μM), DNA profiling 2.0 μ L, ddH2O 6.2 μ
L.Optimum reaction condition are as follows: 95 DEG C of 30 s, 95 DEG C of 5 s, 60 DEG C of 34 s, 40 circulations.
The OD of measurement standard recombination positive plasmid260nmAnd OD280nmValue and its ratio calculate plasmid concentration, and are converted into and copy
Shellfish number is serially diluted into 7 gradients (10 with distilled water 10 again8~103Copies/ μ L).As template, 20 μ L reaction is established
System expands, gained standard curve is shown in figure according to the PCR reaction system after optimization on 7500 type fluorescence quantitative PCR instrument of ABI
2.As a result, the linear relationship between amplification curve and Ct value is close, related coefficient (R2) up to 0.999.
The sensitivity of 4 real-time fluorescence quantitative PCR of embodiment is tested
SVV positive criteria product 10 is serially diluted again carries out real-time fluorescence quantitative PCR (l × 10 as template0~l ×
109Copies/ μ L, totally 10 gradients, to determine their detection lower limit.As a result, using PMD19-T-3C standard items as template, it is glimmering
The detection lower limit of Fluorescent Quantitative PCR is 1 × 102Copies/ μ L(Fig. 3).
The specific test of 5 real-time fluorescence quantitative PCR of embodiment
Using the cDNA or DNA of SVV, FMDV, SVDV, VSV, PRRSV, PCV, PRV, PEDV and normal PK-15 cell as mould
Plate carries out real-time fluorescence quantitative PCR amplification using the reaction system and reaction condition that the embodiment 3 established optimizes, as a result sends out
Now the PCR amplification only has SVV to be positive (Fig. 4).
The repetitive test of 6 real-time fluorescence quantitative PCR of embodiment
The positive criteria product plasmid of SVV is carried out 109、107、105Totally 3 gradients are template to copies/ μ L, are carried out in group
The Repeatability checking between group, each gradient will be repeated 5 times, and do 3 secondary responses altogether.The method set up to SVV carries out
PMD19-T-3C standard items plasmid Repeatability checking.As a result shown in table 2: the coefficient of variation (CV) in group between group is respectively less than
0.96%。
The composition of 7 kit of embodiment
According to the following kit for forming and being formulated for detecting pig Sai Neijia paddy virus: 10 μM of primers, 1,10 μM of primer 2,
10 μM of probes, ROX Reference Dye II(50 ×), Premix Ex Taq(Probe qPCR) (2 ×).
A kind of reaction system of the kit can be with are as follows: Premix Ex Taq(Probe qPCR) (2 ×) 10 μ L, 10 μ
M, 0.4 1,10 μM of μ L primer, 0.4 μ L primer 2, ROX Reference Dye II(50 ×) 0.2 μ L, 10 μM, 0.8 μ L spy
Needle, 2.0 μ L of DNA profiling, ddH26.2 μ L of O, reaction total volume are 20 μ L.
Utilize the kit carry out real-time fluorescence quantitative PCR reaction condition are as follows: 95 DEG C 30 seconds, 95 DEG C 5 seconds, 60 DEG C
34 seconds, 40 circulations.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and kit
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>primer 1
<400> 1
gagcttcaat ctcctaga 18
<210> 2
<211> 19
<212> DNA
<213>primer 2
<400> 2
gtgtcatcat tctcgttag 19
<210> 3
<211> 23
<212> DNA
<213>probe 1
<400> 3
cagacattcg agccaagcaa caa 23
<210> 4
<211> 18
<212> DNA
<213>primer 3
<400> 4
ccctaacgag aatgatga 18
<210> 5
<211> 19
<212> DNA
<213>primer 4
<400> 5
cactgccaag attatcaag 19
<210> 6
<211> 20
<212> DNA
<213>probe 2
<400> 6
ccgcagctag agcaagacct 20
<210> 7
<211> 18
<212> DNA
<213>primer 5
<400> 7
ggctctgtag aactagag 18
<210> 8
<211> 18
<212> DNA
<213>primer 6
<400> 8
tagggctctg tagaacta 18
Claims (6)
1. a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, which is characterized in that upstream primer, downstream
The sequence of primer and probe is as shown in NO:1~3 SEQ ID；5 ' end labels of the probe are fluorescent reporter group FAM；3'
End label is quenching group TAMAR.
2. the kit containing Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe described in claim 1.
3. kit according to claim 2, which is characterized in that the concentration of the upstream primer, downstream primer and probe
It is 10 μM.
4. kit according to claim 3, which is characterized in that the kit further includes PCR reaction buffer, fluorescence
Dyestuff, positive criteria product.
5. kit according to claim 4, which is characterized in that the kit carries out the reactant of quantitative fluorescent PCR
System are as follows: 2 × Premix Ex Taq10 μ L, 10 μM of upstream primers 0.4 μ L, 10 μM of downstream primers 0.4 μ L, 50 × ROX
Reference Dye II0.2 μ L, 10 μM of 0.8 μ L of probe, DNA profiling 2.0 μ L, ddH2O 6.2μL。
6. kit according to claim 4, which is characterized in that the kit carries out the reaction item of quantitative fluorescent PCR
Part are as follows: 95 DEG C 30 seconds, 95 DEG C 5 seconds, 60 DEG C 34 seconds, 40 circulation.
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| CN107034313A (en) * | 2017-05-10 | 2017-08-11 | 广东温氏食品集团股份有限公司 | The RT PCR detection primers and RT PCR detection methods of a pair of Sai Nika paddy virus |
| CN107955840A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of swine foot-and-mouth disease virus and Sai Neijia paddy virus |
| CN107937617A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses |
| CN107937618A (en) * | 2017-12-29 | 2018-04-20 | 中国检验检疫科学研究院 | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application |
| CN110257554A (en) * | 2018-03-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit and its primer special and TaqMan probe of Seneca Valley virus |
| CN108384893A (en) * | 2018-05-03 | 2018-08-10 | 中国农业科学院兰州兽医研究所 | Real-time fluorescence quantitative RT-PCR kit for detecting foot and mouth disease virus and Sai Nika paddy viruses and its application |
| CN109097495A (en) * | 2018-08-16 | 2018-12-28 | 金宇保灵生物药品有限公司 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
| CN109234464A (en) * | 2018-11-23 | 2019-01-18 | 山东新希望六和集团有限公司 | For detecting primer and probe, the PCR kit for fluorescence quantitative and methods and applications of Seneca Valley virus |
| CN110093320B (en) * | 2019-03-27 | 2023-07-21 | 华南农业大学 | Swine sai-Ka virus GD-SVA-2018 strain and application thereof |
| CN110229933B (en) * | 2019-06-24 | 2022-06-07 | 派生特（福州）生物科技有限公司 | Primer group and kit for nested RT-PCR (reverse transcription-polymerase chain reaction) detection of porcine Saikovia virus and application of primer group and kit |
| CN110527746A (en) * | 2019-07-30 | 2019-12-03 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of pig Senecan paddy virus Yu 3 type of annulus virus |
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