CN110066888A - A kind of triple real time fluorescent quantitative RT-qPCR methods of synchronous detection TMV, CMV and PVY - Google Patents
A kind of triple real time fluorescent quantitative RT-qPCR methods of synchronous detection TMV, CMV and PVY Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses triple real time fluorescent quantitative RT-qPCR methods of a kind of synchronous detection TMV, CMV and PVY.The present invention designs to obtain one group of triple real time fluorescent quantitative RT-qPCR primers combination, including primer sets qTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets qPVY-F/qPVY-R as target sequence using the coat protein conserved sequence of TMV, CMV and PVY；Its nucleotide sequence is successively as shown in NO.1~6 SEQ ID.And further based on the primer sets, triple real time fluorescent quantitative RT-qPCR methods are established for the first time, quickly, accurately and efficiently can identify and detect TMV, CMV and PVY, and detection sensitivity is significantly higher than ELISA and RT-PCR both methods.In addition, this method is easy to operate, detection sensitivity is high, high specificity, stability are good, it is easy to base's detection unit and promotes the use of, there is wide development space and good practical application value.
Description
Technical field
The invention belongs to plant virus detection technique fields, more particularly, to a kind of synchronous detection TMV, CMV and PVY
Triple real time fluorescent quantitative RT-qPCR methods.
Background technique
Tobacco mosaic virus (TMV) (Tobacco mosaic virus, TMV), cucumber mosaic virus (Cucumber mosaic
Virus, CMV) and marmor upsilon (Potato virus Y, PVY) be to infect three kinds of tobacco main viruses.They cause
Virosis cigarette strain mainly there is soil band poison to pass poison the reason of virosis occurs for field, insect passes poison, seedling stage band poison etc.,
In, it is one of the main path that tobacco virus is propagated that vega soil band poison, which passes poison,.Cause the non-mediator of TMV, CMV and PVY soil
The reason of propagation, survival period in tobacco old complaint of mainly TMV, CMV and PVY was long, and they easily pass through root system
Wound invades cigarette strain, and root system can be made to generate many microtrauma mouths when field is transplanted seedlings, and the virus in soil invalid body will take advantage of the occasion to invade
Tobacco seedlings root.In recent years, the generation of tobacco virus is in rising trend, seriously affects the quality and yield of tobacco leaf, leads to tobacco
Industrial economy loss is huge, takes in tobacco grower and reduces, and reduces its enthusiasm for planting tobacco.Quickly and accurately detect on tobacco
Correlated virus be one of the important means of to produce upper prevention and control disease, reduce loss.
The common detection methods of virus have Electronic Speculum detection, enzyme linked immunosorbent assay (ELISA) (Enzyme-Linked
Immunosorbent Assay, ELISA) and polymerase chain reaction (polymerase chain reaction, PCR) is (such as
Fruit test object is RNA virus, then is RT-PCR) technology.ELISA is easy to operate, low in cost.But ELISA testing result
There is the interference of non-specific color reaction in the chromogenic reaction of substrate solution, in fact it could happen that false positive phenomenon, to influence the party
The accuracy and sensitivity of method.RT-PCR has many advantages, such as time saving, economical, efficient.Compared with ELISA, it has higher spirit
Sensitivity and faster detection speed, can save the plenty of time.But when RT-PCR response procedures are arranged it is noted that annealing temperature
The selection of degree, inappropriate annealing temperature may cause non-specific amplification or no band, to influence experimental result
Judgement.Therefore, need to establish it is a kind of for soil take it is viruliferous it is simple, quickly, high sensitivity, high specificity, stability it is good
Detection method.
Forefathers are less to the research of cause of disease analyte detection in vega soil, report.It avenges equality in week and refers to object method and ELISA
Method has detected the CMV in soil, it was demonstrated that CMV can carry out non-Vector transmission by soil.The comparative analysis such as Liu Min virus
Biological characteristis, DAS-ELISA, in 4 kinds of methods detection soil of colloidal gold strip detection and RT-PCR etc. the sensitivity of TMV and
Specificity.Ren Xiyi etc. combines real-time fluorescence RT-PCR and test tube to capture RT-PCR method, is changed to real-time fluorescence RT-PCR method
Into, and the multiple test tube capture real-time fluorescence RT-PCR detection body that can detect TMV and PVY simultaneously is successfully constructed on this basis
System.The method that Sun Jie etc. establishes CMV in detection banana with real-time fluorescence quantitative PCR.
These three viruses of TMV, CMV and PVY can seriously infect tobacco and cause tobacco virus, influence the yield and product of tobacco
Matter, but yet there are no both at home and abroad at present it is legal and quantitative by real time fluorescent quantitative RT-qPCR at the same detect TMV, CMV and
The report of these three viruses of PVY.Therefore, to avoid more huge economic loss, needing one kind can simultaneously quickly, efficiently, accurately
The molecular biology method of identification and detection TMV, CMV and PVY.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of synchronous detection
Triple real time fluorescent quantitative RT-qPCR methods of TMV, CMV and PVY.The main innovative point of the present invention is on tobacco
TMV, CMV and PVY, develop it is a kind of can quickly, efficiently, precise Identification and the above-mentioned three kinds of viral triple real-time fluorescences of detection it is fixed
RT-qPCR method is measured, in favor of promoting the use of in base's detection unit.
The first purpose of the invention is to provide triple real time fluorescent quantitative RT- of a kind of synchronous detection TMV, CMV and PVY
The combination of qPCR primer.
A second object of the present invention is to provide the combination of above-mentioned primer as or synchronous detection TMV, the CMV of preparation and/or
The reagent of PVY or the application in kit.
Third object of the present invention is to provide triple real time fluorescent quantitative RT- of a kind of synchronous detection TMV, CMV and PVY
QPCR method.
Fourth object of the present invention is to provide triple real time fluorescent quantitative RT- of a kind of synchronous detection TMV, CMV and PVY
QPCR kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention provides triple real time fluorescent quantitative RT-qPCR primer sets of a kind of synchronous detection TMV, CMV and PVY
It closes, including primer sets qTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets qPVY-F/qPVY-R；Its nucleosides
Acid sequence is successively as shown in NO.1~6 SEQ ID.
The sequence of the primer combination is successively as follows:
Primer qTMV-F (as shown in SEQ ID NO.1): 5 '-GATTTCCTGGCGATGTTT-3 ',
Primer qTMV-R (as shown in SEQ ID NO.2): 5 '-GTCGGACTCTGCTGGTTT-3 ',
Primer qCMV-F (as shown in SEQ ID NO.3): 5 '-TTCGTGGGTAGTGAAAGC-3 ',
Primer qCMV-R (as shown in SEQ ID NO.4): 5 '-GTCCGTGACTGAATCTGG-3 ',
Primer qPVY-F (as shown in SEQ ID NO.5): 5 '-GAAACTGAAATGCC AACT-3 ',
Primer qPVY-R (as shown in SEQ ID NO.6): 5 '-CATCATAACCCAAACTCC-3 '.
High sensitivity, high specificity, the stability of triple real time fluorescent quantitative RT-qPCR primer combinations are good, in synchronization
Had a good application prospect in detection TMV, CMV and/or PVY, therefore, the combination of above-mentioned primer synchronous detection TMV, CMV and/
Or the application in PVY, and as or the reagent or kit of synchronous detection TMV, CMV and/or the PVY of preparation in application,
It also all should be within protection scope of the present invention.
It is combined based on above-mentioned triple real time fluorescent quantitative RT-qPCR primers, the present invention also constructs a kind of synchronous detection
Triple real time fluorescent quantitative RT-qPCR methods of TMV, CMV and PVY, the method are utilized using sample to be tested RNA as template
Primer described in claim 1 combination carry out real time fluorescent quantitative RT-qPCR reaction, judged according to reaction result be in sample to be tested
It is no to contain TMV, CMV and/or PVY.
Preferably, the reaction system of the real time fluorescent quantitative RT-qPCR reaction are as follows: 2 μ L, 2 × One Step of RNA template
12.5 μ L, One Step Enzyme Mix of buffer 1 μ L, 0.5 μ L of upstream primer, downstream primer each 0.5 μ L, RNase
free ddH2O supplies 25 μ L.
Preferably, the response procedures of the real time fluorescent quantitative RT-qPCR reaction are as follows: 42 DEG C of reverse transcription 5min, 95 DEG C pre-
It is denaturalized 10s；95 DEG C of denaturation 5s, 60 DEG C~62 DEG C annealing 30s, totally 40 recycle.
It is highly preferred that the response procedures of real time fluorescent quantitative RT-qPCR reaction are as follows: 42 DEG C of reverse transcription 5min, 95 DEG C
Initial denaturation 10s；95 DEG C of denaturation 5s, 62 DEG C of annealing 30s, totally 40 recycle.
Preferably, the sample to be tested is vega soil.
Specifically, the result judgement method of the real time fluorescent quantitative RT-qPCR reaction are as follows: as utilization primer sets qTMV-
When the Ct value < 35 of F/qTMV-R amplification, then determine to contain TMV in sample to be tested；Expand when using primer sets qCMV-F/qCMV-R
When the Ct value < 35 of increasing, then determine to contain CMV in sample to be tested；As the Ct value < using primer sets qPVY-F/qPVY-R amplification
When 35, then determine to contain PVY in sample to be tested.
In addition, a kind of synchronous detection TMV, CMV comprising above-mentioned triple real time fluorescent quantitative RT-qPCR primers combination and
Triple real time fluorescent quantitative RT-qPCR kits of PVY, also should be within protection scope of the present invention.
The application method of the kit is identical as above-mentioned real time fluorescent quantitative RT-qPCR method.
Preferably, the kit includes the plasmid control respectively containing TMV, CMV and PVY viral gene target sequence
Product, every kind of virus particle standard items set 6 concentration gradients.
Preferably, the kit further includes virus RNA extraction reagent.
Above-mentioned triple real time fluorescent quantitative RT-qPCR methods or mentioned reagent box are in synchronous detection TMV, CMV and/or PVY
In application, also should be within protection scope of the present invention.
Compared with prior art, the invention has the following advantages:
The present invention by largely study and explore, obtained it is a kind of it is synchronous detection TMV, CMV and PVY it is triple in real time it is glimmering
Light quantifies the combination of RT-qPCR primer, including primer sets qTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets
qPVY-F/qPVY-R.The primer is used cooperatively, establish for the first time it is a kind of it is synchronous detection TMV, CMV and PVY it is triple in real time it is glimmering
Light quantifies RT-qPCR method, and this method quickly, accurately and efficiently can not only identify and detect target viral, and sensitivity is aobvious
It writes and is higher than ELISA and RT-PCR both methods.In addition, this method has easy to operate, high sensitivity, high specificity, stabilization
Property it is good, easy to spread the advantages that, be highly suitable for base's detection unit to three kinds of viral detections, there is good practical application
Value.
Detailed description of the invention
Fig. 1 is the amplification curve of pMD-TMV；Wherein, 1~6: concentration is respectively 1.42 × 107copies/μL、1.42×
106copies/μL、1.42×105copies/μL、1.42×104copies/μL、1.42×103copies/μL、1.42×
102The positive plasmid DNA, 7:ddH of copies/ μ L2O。
Fig. 2 is the standard curve (R of pMD-TMV2=0.999, E=106.1%).
Fig. 3 is the melting curve of pMD-TMV.
Fig. 4 is the amplification curve of pMD-CMV；Wherein, 1~6: concentration is respectively 1.31 × 108copies/μL、1.31×
107copies/μL、1.31×106copies/μL、1.31×105copies/μL、1.31×104copies/μL、1.31×
103The positive plasmid DNA, 7:ddH of copies/ μ L2O。
Fig. 5 is the standard curve (R of pMD-CMV2=0.986, E=107.9%).
Fig. 6 is the melting curve of pMD-CMV.
Fig. 7 is the amplification curve of pMD-PVY；Wherein, 1~6: concentration is respectively 1.1 × 108copies/μL、1.1×
107copies/μL、1.1×106copies/μL、1.1×105copies/μL、1.1×104copies/μL、1.1×
103The positive plasmid DNA, 7:ddH of copies/ μ L2O。
Fig. 8 is the standard curve (R of pMD-PVY2=0.986, E=108.2%).
Fig. 9 is the melting curve of pMD-PVY.
Figure 10 is the amplification curve of pedotheque TMV detection sensitivity；Wherein, 1~6: concentration be respectively 180ng/uL,
18ng/uL、1.8ng/uL、1.8×10-1ng/uL、1.8×10-2ng/uL、1.8×10-3Ng/uL, 7:ddH2O。
Figure 11 is the standard curve (R of pedotheque TMV detection sensitivity2=0.988, E=114.2%).
Figure 12 is the melting curve of pedotheque TMV detection sensitivity.
Figure 13 is the amplification curve of pedotheque CMV detection sensitivity；Wherein, 1~6: concentration be respectively 180ng/uL,
18ng/uL、1.8ng/uL、1.8×10-1ng/uL、1.8×10-2ng/uL、1.8×10-3Ng/uL, 7:ddH2O。
Figure 14 is the standard curve (R of pedotheque CMV detection sensitivity2=0.986, E=108.2%).
Figure 15 is the melting curve of pedotheque CMV detection sensitivity.
Figure 16 is the amplification curve of pedotheque PVY detection sensitivity；Wherein, 1~6 concentration be respectively as follows: 180ng/uL,
18ng/uL、1.8ng/uL、1.8×10-1ng/uL、1.8×10-2ng/uL、1.8×10-3Ng/uL, 7:ddH2O。
Figure 17 is the standard curve (R of pedotheque PVY detection sensitivity2=0.995, E=105.0%).
Figure 18 is the melting curve of pedotheque PVY detection sensitivity.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The design of 1 primer of embodiment and the extracting of pedotheque total serum IgE
1, design of primers
A kind of triple real time fluorescent quantitative RT-qPCR primers combination of synchronous detection TMV, CMV and PVY, including primer sets
QTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets qPVY-F/qPVY-R；The design method and sequence of primer
It arranges as follows: multiple groups primer is devised according to coat protein (CP) conserved sequence of TMV, CMV and PVY, by screening, obtain following
Primer combination: primer sets qTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets qPVY-F/qPVY-R, primer
Combined nucleotide sequence is as shown in table 1, and primer is synthesized in Shanghai Sheng Gong bioengineering Co., Ltd.
2, the extracting of pedotheque total serum IgE
With the total serum IgE of soil RNA rapidly extracting kit extracting vega soil sample, soil RNA rapidly extracting kit
It is purchased from Beijing Hua Yue ocean Biotechnology Co., Ltd.
The triple real time fluorescent quantitative RT-qPCR primer sets synkaryon nucleotide sequences of table 1
The foundation of embodiment 2 plasmid standard amplification curve, standard curve and melting curve
1, the foundation of amplification curve, standard curve and melting curve
By the recombination positive plasmid DNA standard items (offer of this laboratory) of TMV, CMV and PVY for having prepared, determined with absolute
Measure dilution Easy Dilution with 10 times concentration gradient serial dilution 5 times, as real time fluorescent quantitative RT-qPCR reaction
Template, while with RNase Free dH2O is arranged clear water and compares, and prepares reaction system, and response procedures are arranged, and carries out real-time fluorescence
3 parallel repetitions are done in quantitative RT-qPCR reaction, each standard items and clear water control respectively.According to pair of initial template copy number
The linear relationship of numerical value and the recurring number of reaction (i.e. Ct value), amplification curve, the standard curve of building detection TMV, CMV and PVY
And melting curve.Agents useful for same is purchased from TaRaKa company.
The reaction system of above-mentioned real time fluorescent quantitative RT-qPCR reaction are as follows: 2 μ L, 2 × One Step buffer of RNA template
12.5 μ L, One Step Enzyme Mix 1 μ L, 0.5 μ L of upstream primer, 0.5 μ L, RNase free ddH of downstream primer2O is mended
25 μ L of foot.
The response procedures of above-mentioned real time fluorescent quantitative RT-qPCR reaction are as follows: 42 DEG C of reverse transcriptions 5min, 95 DEG C of initial denaturation 10s；
95 DEG C of denaturation 5s, 62 DEG C of annealing 30s, totally 40 recycle；Melting curve: 95 DEG C of 15s, 60 DEG C of 30s, 95 DEG C of 15s.
2, the determination method of real time fluorescent quantitative RT-qPCR reaction result
When the Ct value < 35 of sample, judging result is the positive, value >=35 Ct of sample or when not expanding, then judging result is
It is negative.When using the method test sample of absolute quantitation, the Ct value of sample to be tested is substituted into calibration curve equation, the value acquired
The as starting copy number of sample to be tested.In order to guarantee the consistency of testing result, sample is measured using absolute quantification method every time
When product, it is necessary to draw standard curve, sample to be tested and standard items are put into fluorescence quantitative PCR instrument simultaneously and reacted, instead
After answering, the copy number of sample to be tested is calculated according to calibration curve equation.
3, result
The amplification curve of pMD-TMV, standard curve and melting curve are as shown in Figures 1 to 3, it can be seen that work as plasmid control
The concentration of product is 1.42 × 107~1.42 × 104When copies/ μ L, good linear relationship (Ct value < 35), explanation is presented
The primer and amplification system of design, can be very good amplification target gene, which can accurately quantify TMV；The standard of TMV
R in curve2=0.999, E=106.1%；Melting curve is in unimodal.Amplification curve, standard curve and the melting of pMD-CMV
Curve is as shown in figures 4-6, it can be seen that when the concentration of plasmid standard is 1.31 × 108~1.31 × 104When copies/ μ L,
Good linear relationship (Ct value < 35) is presented, illustrates the primer and amplification system of design, can be very good amplification target
Gene, the system can accurately quantify CMV；R in the standard curve of CMV2=0.986, E=107.9%；Melting curve is in single
Peak.Amplification curve, standard curve and the melting curve of pMD-PVY is as shown in figs. 7-9, it can be seen that dense when plasmid standard
Degree is 1.1 × 108~1.1 × 104When copies/ μ L, good linear relationship (Ct value < 35) is presented, illustrates drawing for design
Object and amplification system, can be very good amplification target gene, which can accurately quantify PVY；In the standard curve of PVY
R2=0.986, E=108.2%；Melting curve is in unimodal.
The evaluation of 3 detection method of embodiment
1, sensitivity test experience
(1) method
According to the method for extracting of 1 pedotheque total serum IgE of embodiment, the 1g soil to be measured containing TMV, CMV and PVY simultaneously is extracted
Earth sample total serum IgE, (this soil sample is detected by ELISA (enzyme linked immunosorbent assay (ELISA)), while containing there are three types of viruses), obtains
Total serum IgE by ultraviolet specrophotometer measure, learn RNA concentration be 180ng/uL.
With absolute quantitation dilution by total serum IgE with 10 times of gradient serial dilutions, 6 gradients, each sample sets 3 repetitions, into
Row real time fluorescent quantitative RT-qPCR reaction, reaction system and response procedures are same as Example 2.
(2) result
Pedotheque TMV detection sensitivity result is as shown in Figure 10~12, and pedotheque CMV detection sensitivity result is as schemed
Shown in 13~15, pedotheque PVY detection sensitivity result is as shown in Figure 16~18, from Figure 10~18 as can be seen that soil-like
The amplification curve of product TMV, CMV, PVY are smooth normal, and the interval between each curve is uniform, there is apparent baseline period, exponential increase
Phase, increased logarithmic phase and plateau；Each coefficient meets wanting for real time fluorescent quantitative RT-qPCR ideal standard curve in standard curve
It asks；Melting curve is unimodal.Real time fluorescent quantitative RT-qPCR method described above being capable of fast and accurately different detections
TMV, CMV, PVY, and detection sensitivity is high, the maximum sensitivity of detection TMV, CMV and PVY are successively 1g soil sample totals to be measured
RNA dilution 105Again, 103Times and 104Times, i.e. the maximum sensitivity of detection TMV is 1.8 × 10-3Ng/uL detects the highest spirit of CMV
Sensitivity is 1.8 × 10-1Ng/uL, the maximum sensitivity for detecting PVY is 1.8 × 10-2ng/uL；It is triple to illustrate that the present invention establishes
Real time fluorescent quantitative RT-qPCR method high sensitivity；Further illustrate the sensitive of real time fluorescent quantitative RT-qPCR primer combination
Degree is high.
2, specific detection is tested
(1) method
Take plant virus research department, Agricultural University Of South China save marmor erodens (Tobacco etch virus,
TEV), marmor solani (Potato virus A, PVA) and tomato spotted wilf virus (Tomato spotted wilt virus,
TSWV), reverse transcription is distinguished into cDNA as template, with 10 of the RNA simultaneously containing TMV, CMV and PVY2Times dilution is the positive
Control, with RNase Free ddH2O is clear water control, carries out real time fluorescent quantitative RT-qPCR, detects its specificity.
(2) result
The results are shown in Table 2 for the specific detection of real time fluorescent quantitative RT-qPCR, it can be seen that TMV, CMV and PVY are equal
Can accurately detected, and Ct value is between 17~22, and compare virus TEV, PVA, TSWV cannot detect it is any glimmering
Optical signal；Illustrate triple real time fluorescent quantitative RT-qPCR method high specificities that the present invention establishes；Further illustrate that this is glimmering in real time
Light quantifies the high specificity of RT-qPCR primer combination.
The triple real time fluorescent quantitative RT-qPCR of table 2 detect the specificity analysis of a variety of viruses
| Virus | Specific (Ct value) |
| TMV | 17.04 |
| CMV | 21.39 |
| PVY | 19.94 |
| TEV | — |
| PVA | — |
| TSWV | — |
Note: when Ct value is less than 35, determine that reaction result is the positive；"-" is represented without Ct value, can't detect any fluorescence
Signal.
3, the coefficient of variation is analyzed
(1) method
With 10~106Diluted TMV, CMV and PVY standard items are template again, carry out repeated experiment, and each sample does 3
A parallel repetition (repeating in batch), experiment are repeated 3 times and (repeat between batch).Calculate the coefficient of variation of resulting value.
(2) result
It is as shown in table 3 that experimental result is repeated in real time fluorescent quantitative RT-qPCR batches, between real time fluorescent quantitative RT-qPCR batches
Repeat experimental result it is as shown in table 4, from table 1 and 2 as can be seen that positive template through batch in and batch between it is each 3 times repetition test, circulation
Threshold average is almost the same, and the range of the duplicate coefficient of variation is 0.77%~1.04% in batch, duplicate variation lines between batch
Several ranges is 0.65%~1.21%, and the duplicate coefficient of variation is respectively less than 2% between repeating and criticizing in batch；Illustrate the testing result
It is good with good repeatability and reproducibility, the i.e. stability of real time fluorescent quantitative RT-qPCR system.
Experimental result is repeated in the triple real time fluorescent quantitative RT-qPCR of table 3 batches
Experimental result is repeated between the triple real time fluorescent quantitative RT-qPCR of table 4 batches
The preferred embodiment that the above specific embodiment is of the invention for ease of understanding and illustrates, but the invention is not limited to
Above-described embodiment does not mean that the present invention must rely on above-described embodiment and could implement.Person of ordinary skill in the field
It is the addition of equivalence replacement and auxiliary element to raw material selected by the present invention, specific it will be clearly understood that any improvement in the present invention
The selection etc. of mode, all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of triple real time fluorescent quantitative RT-qPCR primers combination of synchronous detection TMV, CMV and PVY, which is characterized in that packet
Include primer sets qTMV-F/qTMV-R, primer sets qCMV-F/qCMV-R and primer sets qPVY-F/qPVY-R；Its nucleotides sequence
Leie time is as shown in NO.1~6 SEQ ID:
Primer qTMV-F (as shown in SEQ ID NO.1): 5 '-GATTTCCTGGCGATGTTT-3 ',
Primer qTMV-R (as shown in SEQ ID NO.2): 5 '-GTCGGACTCTGCTGGTTT-3 ',
Primer qCMV-F (as shown in SEQ ID NO.3): 5 '-TTCGTGGGTAGTGAAAGC-3 ',
Primer qCMV-R (as shown in SEQ ID NO.4): 5 '-GTCCGTGACTGAATCTGG-3 ',
Primer qPVY-F (as shown in SEQ ID NO.5): 5 '-GAAACTGAAATGCC AACT-3 ',
Primer qPVY-R (as shown in SEQ ID NO.6): 5 '-CATCATAACCCAAACTCC-3 '.
2. the combination of primer described in claim 1 as or synchronous detection TMV, CMV and/or the PVY of preparation reagent or kit
In application.
3. a kind of triple real time fluorescent quantitative RT-qPCR methods of synchronous detection TMV, CMV and PVY, which is characterized in that the side
Method is to be combined using primer described in claim 1 using sample to be tested RNA as template and carry out real time fluorescent quantitative RT-qPCR reaction,
Judge whether contain TMV, CMV and/or PVY in sample to be tested according to reaction result.
4. according to the method described in claim 3, it is characterized in that, the reactant of real time fluorescent quantitative RT-qPCR reaction
System are as follows: 2 μ L, 2 × One Step buffer of RNA template, 12.5 μ L, One Step Enzyme Mix, 1 μ L, upstream primer 0.5
μ L, 0.5 μ L, RNase free ddH of downstream primer2O supplies 25 μ L.
5. according to the method described in claim 3, it is characterized in that, the reaction interval of real time fluorescent quantitative RT-qPCR reaction
Sequence are as follows: 42 DEG C of reverse transcriptions 5min, 95 DEG C of initial denaturation 10s；95 DEG C of denaturation 5s, 60 DEG C~62 DEG C annealing 30s, totally 40 recycle.
6. according to the method described in claim 3, it is characterized in that, the sample to be tested is vega soil.
7. according to the method described in claim 3, it is characterized in that, the result of real time fluorescent quantitative RT-qPCR reaction is sentenced
Determine method are as follows: when using the Ct value < 35 of primer sets qTMV-F/qTMV-R amplification, then determine to contain TMV in sample to be tested；When
When the Ct value < 35 expanded using primer sets qCMV-F/qCMV-R, then determine to contain CMV in sample to be tested；When utilize primer sets
When the Ct value < 35 of qPVY-F/qPVY-R amplification, then determine to contain PVY in sample to be tested.
8. a kind of triple real time fluorescent quantitative RT-qPCR kits of synchronous detection TMV, CMV and PVY, which is characterized in that described
Kit is combined comprising primer described in claim 1.
9. kit according to claim 8, which is characterized in that including containing TMV, CMV and PVY viral gene mesh respectively
The plasmid standard of sequence is marked, every kind of virus particle standard items set 6 concentration gradients.
10. kit according to claim 8, which is characterized in that further include virus RNA extraction reagent.
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