CN106868214A - Fluorescent quantitation RT PCR detect primer pair, probe, kit and the method for mouse rotavirus - Google Patents
Fluorescent quantitation RT PCR detect primer pair, probe, kit and the method for mouse rotavirus Download PDFInfo
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Abstract
The invention discloses a kind of fluorescent quantitation RT PCR detections primer pair of mouse rotavirus, probe, kit and method, the detection method be the cDNA with the brain tissue of mouse as template, SEQ ID No:1 and SEQ ID No:2 is primer, SEQ ID No:3 is probe, carries out fluorescent quantitation RT PCR amplifications, gathers fluorescence signal.Fluorescent quantitation RT PCR detection methods of the invention can realize it is quick, accurate, convenient and swift, specifically for mouse rotavirus nucleic acid detection in Mice Body, sensitivity, repeatability and specificity can ensure.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly it relates to a kind of fluorescence quantitative RT-RCR is detected
The primer pair of mouse rotavirus, probe, kit and method.
Background technology
Mouse rotavirus (Murine Rotavirus, MRV), is double-stranded rna virus, and the generally existing in mouse is caused
Epidemic Diarrhea of Laboratory (Epidemic Diarrhea of mice, EDIM), China opens this sick incidence in the mouse raised
Very high, serious harm opens the suckling mouse for raising each Strains of Mouse.EDIM Tobamovirus Reoviridae rotaviruses, are bifilar
RNA virus, have the specific antigen for being different from other rotavirus on its shell, MRV belongs to A group rotavirus, with baby children
The rotavirus of youngster has cross reaction, and does not have cross reaction with the B group rotavirus of small rat.So, MRV can only cause breast small
The morbidity of mouse.Mouse is unique natural reservoir (of bird flu viruses) of EDIM viruses.This disease, by alimentary canal and respiratory infectious, is that a kind of height connects
Touch sexually transmitted disease.The main suckling mouses within the age in days of the first tire 15 occurred frequently of EDIM.Suckling mouse can show diarrhoea, hypoevolutism, dehydration
It is dead once in a while etc. symptom.Adult mice is in subclinical infection and continuous outwards toxin expelling.
Mouse rotavirus is infected very generally in mouse group, and very big loss is all brought to production and scientific research.Due to
Clinically easily mutually obscure with the diarrhoea caused by other factors, this also brings certain difficulty to diagnosis.At present, people are not also right
This virus infection gives enough attention.Mouse is often examined after suffering from diarrhoea in terms of bacterium infection, feed, weather etc.
Consider.Therefore, people are often easy to ignore the seriousness of this subclinical infection, and potential danger is brought to productive group and population
Evil.At present, in the detection of experiment mice MRV, the method that the country currently uses is still limited to, Serologic detection MRV antibody,
Generally all it is to set up the ELISA method based on MRV structural proteins.Although this method can also carry out large sample detection, and inspection
Survey method is relatively easy, only need to detect serum sample, but the method needs to prepare specific antibody, and cannot be distinguished by being immunized
The antibody produced with infection.The wherein blood sampling of mouse, it is all especially cumbersome and time-consuming that serum is separated, particularly in large-scale experiment
Animal feeding mechanism, the detection of annual experimental mouse, conventional ELISA method is too cumbersome, and nowadays in the market has
Mouse rotavirus infection detect ELISA kit, major part be import reagent box, price all in 2000-3000 units or so, and
96 samples can be only detected, the deficiency such as traditional method has cumbersome, limitation, sensitivity is low, price is high.
So, the reliable detection method of MRV is set up, the health status of experimental mouse is found and monitored in time, reduce because of experiment
Mouse infection causes serious economic loss, and this research is proposed to found a kind of quick, easy, sensitive, special MRV quantitative fluorescent PCRs
Detection method, it is intended to supported for monitoring MRV provides method.
The content of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides a kind of detection of fluorescence quantitative RT-RCR
The primer pair of mouse rotavirus, probe, kit and method.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
A kind of fluorescence quantitative RT-RCR detects the primer pair of mouse rotavirus, and the primer pair has such as SEQ ID No:1
With SEQ ID No:Base sequence shown in 2.
Present invention also offers the probe that a kind of fluorescence quantitative RT-RCR detects mouse rotavirus, following technical side is taken
Case：
A kind of fluorescence quantitative RT-RCR detects the probe of mouse rotavirus, and the probe has such as SEQ ID No:Shown in 3
Base sequence.
Wherein in some embodiments, the 5 ' of the probe is combined with FAM, and the 3 ' of the probe is combined with BHQ1.
Present invention also offers the kit that a kind of fluorescence quantitative RT-RCR detects mouse rotavirus, following technology is taken
Scheme：
A kind of fluorescence quantitative RT-RCR detects the kit of mouse rotavirus, and the kit includes such as SEQ ID No:1
With SEQ ID No:Primer pair and SEQ ID No shown in 2:Probe shown in 3.
Present invention also offers a kind of method that fluorescence quantitative RT-RCR detects mouse rotavirus, following technical side is taken
Case：
A kind of method that fluorescence quantitative RT-RCR detects mouse rotavirus, comprises the following steps：With the brain tissue of mouse
CDNA is template, SEQ ID No:1 and SEQ ID No:2 is primer, SEQ ID No:3 is probe, carries out fluorescent quantitation RT-
PCR is expanded, and gathers fluorescence signal.
Wherein in some embodiments, the reaction system of the fluorescence quantitative RT-RCR amplification is：Premix Ex Taq
10 μ L, the μ L of 0.2 μ L, cDNA templates of ROX Reference Dye II 2, SEQ ID No:The μ L of 1 primer 0.4, SEQ ID No:2
The μ L of primer 0.4, SEQ ID No:The μ L of 3 probe 0.8, plus sterile purified water to 20 μ L.
Wherein in some embodiments, the reaction condition of the fluorescence quantitative RT-RCR amplification is：95 DEG C of predegenerations
30sec；95 DEG C of denaturation 5sec, 60 DEG C of annealing extend 34sec, 40 circulations.
Compared with prior art, the invention has the advantages that：
1st, the present invention optimizes the anti-of fluorescence quantitative RT-PCR detecting method by designing specific primer pair and probe
Condition is answered, so as to quickly, accurately, specifically for mouse rotavirus nucleic acid (MRV) in Mice Body be detected, not only can be with
Realize the large sample examination to experimental mouse, it is also possible to which exogenous factor detection is carried out to cell line and biomaterial；
2nd, fluorescence quantitative RT-PCR detecting method of the invention need to only collect the group of the cDNA that can extract micro in theory
Knit, MRV is mainly grown in intestinal epithelial cell, cause of disease precursor virus also can be largely contained in the excrement of infected mice, so
Fresh excreta can be collected, cDNA is extracted, and present invention only requires synthetic primer pair, probe, dye reagent box just can facilitate fast
Prompt thousands of samples of detection are convenient cheap；
3rd, the lowest detection of fluorescence quantitative RT-PCR detecting method of the invention is limited to 10 copy numbers/μ L, higher than common
100 times of PCR method, common Murine Virus strain does not find cross reaction, within-run and between-run analysis coefficient without specific amplification
Respectively less than 2%, sensitivity, repeatability and specificity can ensure.
Brief description of the drawings
For the testing result of clinical sample of the embodiment of the present invention 1, (X- axles represent PCR cycle number to Fig. 1, and Y- axles represent amplification
The fluorescent value of reaction)；
Fig. 2 is the canonical plotting of fluorescent quantitative PCR detection method in test example of the present invention 1；
Fig. 3 is the dynamic curve diagram of fluorescent quantitative PCR detection method in test example of the present invention 1；Wherein, 1-8 correspondences
Template concentrations are followed successively by：1×107、1×106、1×105、1×104、1×103、1×102、1×101、1×100Copy number/μ L；
9 is negative control (X- axles represent PCR cycle number, and Y- axles represent the fluorescent value of amplified reaction)；
Fig. 4 is the specific outcome figure of fluorescent quantitative PCR detection method in test example of the present invention 1.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
In the following example, the experimental technique of unreceipted actual conditions, generally routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(F.M. Ao Sibai, R.E. James Kingstons, J.G. Sai Deman etc. are edited, and Ma Xuejun, Su Yuelong's translates Beijing：Section
Learn publishing house, 2004) described in method carry out.
The fluorescence quantitative RT-RCR of embodiment 1 detects mouse rotavirus
1st, laboratory sample
246 parts of the intestinal tissue sample pathological material of disease of the mouse from Shanghai Laboratory Animal Research Institute.
2nd, cDNA synthesis
The intestinal tissue of some animals is taken, plus PBS adds steel ball to shake 1min or so in being put into oscillator, and 4000rpm is centrifuged,
2min, sucks supernatant, extracts RNA.Extracting method extracts box specification, the core of extraction with reference to TIANamp Virus RNA Kit
Sour RNA carries out reverse transcription synthesis cDNA.
Template is the viral RNA for extracting, and primer pair is OligdT18, carries out reverse transcription synthesis cDNA, reverse transcription system (40
μ L) be：The μ L of RNA templates 20, primer pair 2 μ L, 5 × reverse transcription Buffer 8 μ L, dNTP (10mmol/L) 4 μ L,
The μ L of 1 μ L, DEPC water of Ribonuclease Inhibitor (40U/ μ L) 1 μ L, Prime Script reverse transcriptase (200U) 4.42
DEG C water-bath 1h, 70 DEG C of water-bath 10min, ice bath 4min.- 20 DEG C save backup.
3rd, primer pair and probe are designed
According to mouse rotavirus P EC9 strains whole genome sequence (DQ288856.1) delivered on NCBI, in its conservative spy
At different region 919-1022nt, primer pair and probe are devised：
To qRT-MRV-F, its sequence is sense primer：
5’-AATTGCTTCTTCAGCCACATTC-3’(SEQ ID No:1)
To qRT-MRV-R, its sequence is anti-sense primer：
5’-CCTTCTCGGTCTGTGC TATTC-3’(SEQ ID No:2)
Taqman probe FAM-MRV, its sequence is：
5’-FAM-ACGTTAACCTGCTATCAGAGCAGCTG-BHQ1-3’(SEQ ID No:3)。
4th, TaqMan fluorescence quantitative PCR detections
Fluorescent quantitative PCR reaction total system is 20 μ L, wherein：
Fluorescent quantitative PCR reaction reaction condition be：95 DEG C of predegeneration 30sec；95 DEG C of denaturation 5sec, 60 DEG C of annealing
Extend 34sec, 40 circulations gather fluorescence.
5th, testing result
Testing result is as shown in Figure 1.Result shows do not occur mouse colyliform disease in the mouse of Shanghai Laboratory Animal Research Institute
The infection of malicious (MRV), the monitoring purpose for mouse rotavirus can be realized using the detection method of the present embodiment.
The drafting of the standard curve of the fluorescence quantitative RT-RCR of test example 1 and the determination of optimum linear scope
This test example have detected the specificity of the TaqMan probe fluorescent quantitative RT-PCR method of the MRV of the foundation of embodiment 1,
Sensitiveness and stability.
1st, the drafting of standard curve and kinetic curve
Artificial synthesized mouse rotavirus EB-C8/G16P [16] strain (Genbank：KJ477127.1) full-length genome
2421-2528nt DNA sequence dnas, send GENEWIZ companies to synthesize, and are connected with pUC57 carriers, build the plasmid control of pUC-MRV
Product.By 1 × 108Copy number/μ L are initial template concentration, with 10 doubling dilution number of times (respectively 1 × 107、1×106、1×
105、1×104、1×103、1×102、1×101、1×100Copy number/μ L) and corresponding Ct values draw out standard curve and dynamic
Force diagram, standard curve as shown in Fig. 2 as can be seen from Figure 2, when the ultimate density of MRV is respectively 1 × 107~1 × 100Copy
During number/μ L, its coefficient R2More than 0.99, standard curve all has good line relation, and each dilution factor correlation is good, by mistake
Difference is smaller；Amplification efficiency E=2.22, amplification efficiency is preferable, illustrates that reaction system and condition after optimization are fine.Kinetic curve
As shown in figure 3, the sensitivity of MRV can reach 10 copy/μ L, and kinetic enrichment exponent build phase and plateau
All complete, the result of quantitative fluorescent PCR is reliable.
2nd, sensitivity tests
The plasmid standard of pUC-MRV is done into 8 dilution factors from 1 × 10 respectively7~1 × 100Copy number/μ L is dilute with multiple proportions
Be interpreted as template carries out fluorescent quantitative PCR respectively, and Ct values are more than 35, are considered as feminine gender, while using primer pair SEQ ID No:1
With SEQ ID No:2 carry out conventional RT-PCR amplification.
Testing result such as Fig. 3, as a result shows, using the sensitivity of method of the present invention detection MRV standard plasmids up to 1 ×
101Copy number/μ L, and regular-PCR is minimum can detect 1 × 103Copy number/μ L, illustrates the method for the present invention higher than common
1000 times of PCR method.
3rd, specific test
By the detection method of the embodiment of the present invention 1, respectively to the virus of the usual infection of mouse, minute virus of mice
(MVM), mouse pneumonia virus (PVM), a59 virus strain (MHV-A59), sendai virus (SEV) are tested by Shanghai
Animal Research Center is provided.MVM extracts viral DNA, and PVM, MHV-A59 and SEV extract virus total RNA, then reverse transcription is DNA.
Adjust concentration after, respectively as template carry out EMV real-time fluorescence quantitative PCR reaction, detected, verification method it is special
Property.As a result result as shown in figure 4, show, in addition to the amplification curve that MRV standard items have characteristic, other viruses are without spy
The amplification curve of levying property, it was demonstrated that the fluorescent quantitative PCR detection method high specificity that the present invention sets up, common muroid disease with other
The equal no cross reaction of strain.In fig. 4,1：MVM；2：PVM；3：MHV-A59；4：SEV；5：Negative control.
4th, replica test
With 1 × 10 in MRV plasmid standards6With 1 × 104Copy number/dilution factors of μ L two as template, to this two
Individual dilution factor carries out 2 replications in different time sections, carries out 3 replications and setting 3 simultaneously to same template every time
Individual repeating hole, real-time fluorescence quantitative PCR is carried out according to the method described in the embodiment of the present invention 1.Wherein：The coefficient of variation (β)=mark
Quasi- deviation (SD)/average (X), the results are shown in Table 1.
The repeatability of the fluorescent quantitative PCR detection method of table 1.
The result of table 1 shows that within-run and between-run analysis coefficient is respectively less than 2%, illustrates the quantitative fluorescent PCR inspection of present invention foundation
Survey method has preferably repeatability, as a result reliable and stable.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
The ＞ China Agriculture Academe Shanghai Veterinary Institute of ＜ 110
The ＞ fluorescence quantitative RT-RCRs of ＜ 120 detect primer pair, probe, kit and the method for mouse rotavirus
The ＞ 3 of ＜ 130
The ＞ PatentIn version 3.1 of ＜ 170
The ＞ 1 of ＜ 210
The ＞ 22 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ artificial sequences of ＜ 213
The ＞ 1 of ＜ 400
aattgcttct tcagccacat tc 22
The ＞ 2 of ＜ 210
The ＞ 21 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ artificial sequences of ＜ 213
The ＞ 2 of ＜ 400
ccttctcggt ctgtgctatt c 21
The ＞ 3 of ＜ 210
The ＞ 26 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ artificial sequences of ＜ 213
The ＞ 3 of ＜ 400
acgttaacct gctatcagag cagctg 26
Claims (7)
1. a kind of fluorescence quantitative RT-RCR detects the primer pair of mouse rotavirus, it is characterised in that the primer pair has such as SEQ
ID No:1 and SEQ ID No:Base sequence shown in 2.
2. a kind of fluorescence quantitative RT-RCR detects the probe of mouse rotavirus, it is characterised in that the probe has such as SEQ ID
No:Base sequence shown in 3.
3. fluorescence quantitative RT-RCR according to claim 2 detects the probe of mouse rotavirus, it is characterised in that the spy
The 5 ' of pin are combined with FAM, and the 3 ' of the probe is combined with BHQ1.
4. a kind of fluorescence quantitative RT-RCR detects the kit of mouse rotavirus, it is characterised in that the kit is included as weighed
Profit requires the probe described in primer pair and Claims 2 or 3 described in 1.
5. a kind of method that fluorescence quantitative RT-RCR detects mouse rotavirus, it is characterised in that comprise the following steps：With the brain of mouse
The cDNA of tissue is template, SEQ ID No:1 and SEQ ID No:2 is primer, SEQ ID No:3 is probe, carries out fluorescence and determines
Amount RT-PCR amplifications, gather fluorescence signal.
6. the method that fluorescence quantitative RT-RCR according to claim 5 detects mouse rotavirus, it is characterised in that described glimmering
Light quantitative RT-PCR amplification reaction system be：Premix Ex Taq 10μL、ROX Reference Dye II 0.2μL、
The μ L of cDNA templates 2, SEQ ID No:The μ L of 1 primer 0.4, SEQ ID No:The μ L of 2 primer 0.4, SEQ ID No:The μ L of 3 probe 0.8, plus
Sterile purified water is to 20 μ L.
7. the method that fluorescence quantitative RT-RCR according to claim 5 detects mouse rotavirus, it is characterised in that described glimmering
Light quantitative RT-PCR amplification reaction condition be：95 DEG C of predegeneration 30sec；95 DEG C of denaturation 5sec, 60 DEG C of annealing extend 34sec,
40 circulations.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012032482A1 (en) * | 2010-09-07 | 2012-03-15 | Novartis Ag | Generic assays for detection of mammalian reovirus |
| CN102465137A (en) * | 2010-11-05 | 2012-05-23 | 上海生物制品研究所有限责任公司 | Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
-
2017
- 2017-03-22 CN CN201710173168.6A patent/CN106868214A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012032482A1 (en) * | 2010-09-07 | 2012-03-15 | Novartis Ag | Generic assays for detection of mammalian reovirus |
| CN102465137A (en) * | 2010-11-05 | 2012-05-23 | 上海生物制品研究所有限责任公司 | Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| F. ARNOLDI ET AL.,: ""Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2"", 《JOURNAL OF VIROLOGY》 * |
| 文心田: "《人兽共患病》", 30 September 2016, 中国农业大学出版社 * |
| 王云瑾等: ""TaqMan实时定量RT-PCR检测G3型轮状病毒VP7基因方法的建立"", 《中国新药杂质》 * |
| 陈艳君等: ""羊轮状病毒NT株VP1基因的测序和分子进化分析"", 《微生物学报》 * |
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Effective date of registration: 20171212 Address after: 200241 Minhang District purple Road, No. 518, Shanghai Applicant after: Shanghai Inst. of Animal Medicines, C.A.A.S Applicant after: Shanghai Lab. Animal Research Center Applicant after: Shanghai Bureau of Emigration &. Engression Examination &. Quarantine People's R Applicant after: Yangzhou University Address before: 200241 Minhang District purple Road, No. 518, Shanghai Applicant before: Shanghai Inst. of Animal Medicines, C.A.A.S |
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Application publication date: 20170620 |