CN105112570A - Real-time RT-PCR (reverse transcription-polymerase chain reaction) specific primers, probe and method for detecting PSTVd (potato spindle tuber viroid) - Google Patents
Real-time RT-PCR (reverse transcription-polymerase chain reaction) specific primers, probe and method for detecting PSTVd (potato spindle tuber viroid) Download PDFInfo
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Abstract
The invention provides real-time RT-PCR (reverse transcription-polymerase chain reaction) specific primers, probe and method for detecting PSTVd (potato spindle tuber viroid) and relates to detection primers, detection probes and detection methods thereof. The sequences of the detection primers are shown in sequence tables Seq ID No: 1 and Seq ID No: 2, and the sequence of the probe is shown in a sequence table Seq ID No: 3. The detection method comprises the steps as follows: 1, RNA (ribonucleic acid) of to-be-detected potato tubers and test-tube plantlets is extracted and inversely transcribed into cDNA (complementary deoxyribonucleic acid) which is taken as a template; 2, the primers and the probe are adopted to calculate the content of PSTVd according to a standard curve and Cp value when the PCR efficiency reaches 90%-110%, the slope is -3.3 and negative control has no peak, analysis is performed through real-time RT-PCR, and a result is judged directly. The detection sensitivity is further improved by 100-1,000 times compared with RT-PCR.
Description
Technical field
The present invention relates to molecular detection primer, detection probes and detection method thereof.
Background technology
Potato is asexually propagated crop, infects and is bred by generation by stem tuber vegetative propagation, thus causing the degeneration of potato, had a strong impact on the yield and quality of potato due to virus and viroid.Most cause of disease disease can be peeled off by stem apex, tissue culture is eliminated and removed.But, potato spindle tuber viroid disease (Potatospindletuberviroid, PSTVd) uniquely can not/be difficult to be peeled off by stem apex, tissue culture eliminates and remove, and up to the present, also there is no specifics to prevent and treat, can only be eliminated by the potato seed strictly detecting production health and remove.Therefore, detection technique that is efficient, quick, sensitive, that be easy to promote is particularly important for control PSTVd.
At present, detect potato viroid and mainly contain the methods such as Biological Detection, electron microscope, round electrophoresis (R-PAGE), RT-PCR, NASH (nucleic acid spot hybridization) and real-timeRT-PCR, in these methods, real-timeRT-PCR is the highest method of sensitivity, PCR is combined with fluorescence detection method by the method, with its specificity high and highly sensitive (being about 1000 times of conventional RT-PCR), can quantitatively and level of automation advantages of higher, be used widely in medical field, microorganism and the quarantine of animals and plants disease.But, do not adopt this technology to be applied in PSTVd at present, in order to detect the report of PSTVd.
Summary of the invention
Real-timeRT-PCR technology is applied to and detects in PSTVd by the present invention first, and the PSTVdreal-timeRT-PCR detection system set up by the method substantially increases the sensitivity that PSTVd detects, for the prevention and control of PSTVd provide technical guarantee.
Detection potato spindle tuber viroid real-timeRT-PCR special primer of the present invention and probe, described primer sequence is as shown in sequence table SeqIDNo:1 and shown in SeqIDNo:2, and described probe sequence is as shown in sequence table SeqIDNo:3.
Potato spindle tuber viroid real-timeRT-PCR detection method of the present invention, it carries out according to following steps:
One, the foundation of typical curve:
(1) extract potato tuber to be detected and test-tube plantlet RNA, and reverse transcription is cDNA, as template;
(2) adopt the template of step (1), carry out pcr amplification, the object fragment after pcr amplification is reclaimed, be connected on pMD18-T carrier, be transformed in competent cell, extract plasmid DNA, obtain potato spindle tuber viroid detection standard plasmid;
(3) with the standard plasmid obtained for template, utilize real-timeRT-PCR special primer and the probe of claim 1, carry out quantitative fluorescent PCR, gather fluorescent signal, adopt BioEdit Software on Drawing to go out real time fluorescent quantitative typical curve;
Two, the standard of positive or negative is judged
When amplification efficiency reaches 90% ~ 110%, slope is-3.3, when negative control does not play peak, calculates the content of potato spindle tuber viroid, undertaken analyzing directly carry out result judgement by real-timeRT-PCR according to typical curve and Cp value.
Accurately calculate the content of PSTVd in the standard of judgement positive or negative of the present invention according to typical curve and Cp value, provide in the survey report that this result exports at instrument, can copy number that directly interpretation is concrete.Each examination criteria curve all needs again to increase.
The present invention comprises following beneficial effect:
The present invention passes through primer and the probe of design, successfully establish the real-timeRT-PCR detection system of PSTVd, the detection technique of China PSTVd has been enriched in the foundation of this system, and compared with the RT-PCR technology that sensitivity is relatively high, detection sensitivity improves again 100 ~ 1000 times.
The present invention, according to potato spindle tuber viroid (PSTVd) gene order, designs 3 pairs of Auele Specific Primers and 1 probe, and probe 5' holds and adopts FAM mark, and 3' end adopts TAMRA mark.Utilize NY/T1962-2010---the RT-PCR method amplification object fragment of regulation in " potato spindle tuber viroid detection ", reclaims, transforms, qualification, and check order, utilize this object fragment to be connected with pMD18-T, prepare the recombinant plasmid of PSTVd and pMD18-T.This plasmid is utilized to adopt serial dilution to prepare real-timeRT-PCR standard substance.Through screening, from 3 pairs of primers, filter out 1 to best primer, and establish the real-timeRT-PCR detection system of PSTVd accordingly.This system is through stability test, and the variation coefficient (CV) of Cp value is respectively 1.64%, 0.61%, 1.80%, 0.47%, 0.92%, 1.11%, 1.45% and 0.98%, shows that detection method has extraordinary stability.This system detection sensitivity reaches 31copies/ μ L, and carried out proof test with 22 known test-tube plantlets and dormancy stem tuber as sample, result is all coincide.The PSTVdreal-timeRT-PCR detection system that the present invention sets up substantially increases the sensitivity that PSTVd detects, for the prevention and control of PSTVd provide technical guarantee.
Real-timeRT-PCR technology is applied in PSTVd by the present invention first, the material that PSTVd content is extremely low can be detected, qualification for the valuable source PSTVd such as core seedling of preciousness is extremely important, loss can be reduced to minimum, early pinpoint the problems in pole, deal with problems, guarantee the production process of virus-free seed potato/seedling, ensure the quality of potato seed/seedling.In addition, this technical system foundation for PSTVd in pin main body copy and the field such as virulence research also significant.
Accompanying drawing explanation
Fig. 1 is the bacterium colony figure transformed;
Fig. 2 is that the competent escherichia coli cell of unconverted is directly coated with contrast figure;
Fig. 3 is positive bacterium colony PCR qualification result electrophorogram, and wherein, M is Marker, and size is each bacterium colony that 100bp, 1-18 swimming lane is respectively conversion, and size is 359bp;
Fig. 4 is take PSTV231F, PSTV296R as the real-timePCR typical curve of primer;
Fig. 5 is take PSTV234F, PSTV350R as the real-timePCR typical curve of primer;
Fig. 6 is take PSTV234F, PSTV356R as the real-timePCR typical curve of primer;
Fig. 7 is sensitivity test figure;
Fig. 8 is the amplification curve utilizing the real-timeRT-PCR system set up to detect PSTVd.
Embodiment
Content of the present invention is not limited only to the content of the respective embodiments described above, and the combination of one of them or several embodiment equally also can realize the object of inventing.
Embodiment one: the detection potato spindle tuber viroid real-timeRT-PCR special primer of present embodiment and probe, described primer sequence is as shown in sequence table SeqIDNo:1 and shown in SeqIDNo:2, and described probe sequence is as shown in sequence table SeqIDNo:3.
Embodiment two: the potato spindle tuber viroid real-timeRT-PCR detection method of present embodiment, it carries out according to following steps:
One, the foundation of typical curve:
(1) extract potato tuber to be detected and test-tube plantlet RNA, and reverse transcription is cDNA, as template;
(2) adopt the template of step (1), carry out pcr amplification, the object fragment after pcr amplification is reclaimed, be connected on pMD18-T carrier, be transformed in competent cell, extract plasmid DNA, obtain potato spindle tuber viroid detection standard plasmid;
(3) with the standard plasmid obtained for template, utilize real-timeRT-PCR special primer and the probe of claim 1, carry out quantitative fluorescent PCR, gather fluorescent signal, adopt BioEdit Software on Drawing to go out real time fluorescent quantitative typical curve;
Two, the standard of positive or negative is judged
When amplification efficiency reaches 90% ~ 110%, slope is-3.3, when negative control does not play peak, calculates the content of potato spindle tuber viroid, undertaken analyzing directly carry out result judgement by real-timeRT-PCR according to typical curve and Cp value.
Embodiment three: present embodiment and embodiment two unlike: described PCR in real time detector is LightCycler480 II real-timePCR instrument.Other is identical with embodiment two.
Embodiment four: present embodiment and embodiment two unlike: described PCR reaction system is as follows:
PCR response procedures is: 95 DEG C of 10min, 1 circulation, 95 DEG C of 10sec, 55 DEG C of 50sec, 72 DEG C of 1sec, 45 circulations.Other is identical with embodiment two.
Embodiment five: present embodiment and embodiment two unlike: the potato tuber to be detected described in step one and test-tube plantlet are PSTVd positive potato stem tuber and test-tube plantlet.Other is identical with embodiment two.
Embodiment six: present embodiment and embodiment two unlike: the primer carrying out pcr amplification in step one is PSTVd0054R and PSTVd0055-3F; Sequence is:
PSTVd0054R：5’-GGATCCCTGAAGCGCTCCTCCGAGCCG-3’；
PSTVd0055-3F：5’-CCGGGGAAACCTGGAGCGAACTGG-3’。Other is identical with embodiment two.
Embodiment seven: present embodiment and embodiment two unlike: reclaiming the object fragment after pcr amplification in step one is reclaimed by the centrifugal column type sepharose of Tiangen company that test kit reclaims amplified production, purifying.Other is identical with embodiment two.
Embodiment eight: present embodiment and embodiment two unlike: in step one, ligation system is 10 μ L, and wherein, pMD18-TVector is 0.8 μ L, and PCR purified product is 4 μ L, ddH
2o is 1.2 μ L; Solution I is 5 μ L; Reaction conditions is connect under 4 DEG C of conditions to spend the night.Other is identical with embodiment two.
Embodiment nine: present embodiment and embodiment two unlike: sensitivity test is carried out to the real-timeRT-PCR detection method of potato spindle tuber viroid.Other is identical with embodiment two.
Present embodiment carries out sensitivity test to the Real-timeRT-PCR system selected, and method is: the standard substance preparation standard curve utilizing concentration lower, as calculated, the concentration of each standard substance is respectively 3.11 × 10
7copies/ μ L ~ 3.11 × 10
-1copies/ μ L.
Embodiment ten: present embodiment and embodiment two unlike: stability test is carried out to the real-timeRT-PCR detection method of potato spindle tuber viroid.Other is identical with embodiment two.
Present embodiment utilizes Excel to calculate standard deviation and the average of Cp value, calculate the variation coefficient (Variationcoefficient, CV), method of calculation are: CV=standard deviation/mean number × 100%, verify the stability of detection system according to the variation coefficient (CV).
Beneficial effect of the present invention is verified by following examples:
Embodiment 1
1. test materials
1.1 for examination PSTVd cause of disease
The potato test-tube plantlet of infection PSTVd collected from all parts of the country and dormancy stem tuber and do not infect the Potatoes totally 30 of PSTVd, in table 1.
Table 1 potato samples is originated
1.2 reagent
RNA extracts test kit and DNA purifying reclaims test kit (centrifugal column type) for sky is with biological production, high purity plasmid in a small amount rapid extraction test kit and be the white biological production in Yuanping City without the water of DNA enzymatic, RNA enzyme, primer and probe are by the raw work biosynthesizing in Shanghai, Reverse Transcription box, PCR kit and pMD18-T carrier are TAKARA company and produce, competent cell is the bacillus coli DH 5 alpha that TranSGenBiotech produces, real-timePCR test kit is that Roche (Roche) is produced, and other conventional reagent are analytical pure level product.
1.31.3 plant and instrument
Trace ultraviolet spectrophotometer is ThermoNanodropND-1000, and PCR instrument is German BiometraTGradient, real-timePCR instrument is the LightCycler480 II that Roche (Roche) is produced.
2. test method
2.1RNA extract
Utilize Trizol (sky is with biological) reagent to extract potato tuber and test-tube plantlet RNA, finally use without enzyme water dissolution RNA, measure quality and the concentration of RNA solution with ND-1000 ,-20 DEG C save backup.
2.2 primers and probe design
With reference to the method for N.Boonhama, simultaneously online (
http:// www.ncbi.nlm.nih.gov/) search PSTVd sequence, after comparing, find out its conserved sequence, according to PSTVdAF483471 sequences Design Auele Specific Primer and probe, object fragment is at 100-150bp.
Table 2 primer and probe design
Wherein, PSTV234F primer sequence is as shown in SeqIDNo:1, and PSTV356R is as shown in SeqIDNo:2; PSTV251T probe sequence is as shown in SeqIDNo:3.
2.3 prepared by fluorescent quantitation standard substance
(1) object fragment reclaims:
Adopting agricultural industry criteria NY/T1962-2010---the RT-PCR method amplification object fragment of regulation in " potato spindle tuber viroid detection ", utilizes the centrifugal column type sepharose of Tiangen company to reclaim test kit and carries out recovery purifying to amplified production.Concrete operations following (or referring to specification sheets):
1) column equilibration: add 500 μ L balance liquid BL in adsorption column CA2,1,2000rpm is centrifugal, 1min, outwells waste liquid, and adsorption column is put back to collection tube;
2) amplified production is cut from sepharose, add triploid and amass PN sol solutions spin upside down centrifuge tube in centrifuge tube, blob of viscose is fully dissolved;
3) joined by sol solutions in CA1 adsorption column, the centrifugal 30s of 1,2000rpm, outwells waste liquid, and adsorption tube is put into new collection tube;
4) add the rinsing liquid PW of 600 μ L, the centrifugal 30s of 1,2000rpm, rinsing 3 times, removes rinsing liquid as far as possible repeatedly; Finally adsorption column room temperature is placed, dry;
5) add CA2, room temperature places 2min, and 1,2000rpm, 1min, remove waste liquid, puts back to collection tube; Add 600 μ L rinsing liquid PW, 12000rpm, 1min, waste liquid;
6) put back to collection tube, 1,2000rpm, 2min, room temperature places 5min;
7) CA2 is put into clean centrifuge tube, add 40 μ L elution buffer EB, room temperature places 2min, 1,2000rpm, 2min; On 1.5% sepharose, electrophoresis detection reclaims product.
(2) connection of object fragment
Ligation is carried out in 1.5mLEppendorf pipe, ligation system 10 μ L: carrier pMD18-TVector0.8 μ L; PCR purified product 4 μ L; ddH
2o1.2 μ L; Solution I 5 μ L, connects and spends the night under 4 DEG C of water bath condition.
(3) conversion of product is connected
The preparation of penbritin solution (100mg/mL):
In Bechtop, get 0.1g penbritin joins in 1mL sterilized water, with 0.22 μm of membrane filtration sterilizing, saves backup with latter-20 DEG C of sealed membrane sealing.
Other operations are carried out according to working instructions, specific as follows:
1) get the competent cell of 50 μ L thawed on ice, add 10 μ L target DNAs, mix gently, in ice bath, place 30min;
2) heat shock 45s in 42 DEG C of water-baths, transfers to 2min in ice bath then fast by pipe, and this process does not shake centrifuge tube;
3) in each centrifuge tube, add 500 μ LLB substratum (not containing microbiotic), mixing is placed on 37 DEG C, and 200rpm cultivates 1h, makes bacteria resuscitation;
4) draw the competent cell that 100-200 μ L has transformed, be added in the LB nutrient agar containing penbritin, cell is evenly spreadable, absorbed to liquid, be inverted dull and stereotyped, 37 DEG C of incubated overnight.With the competent escherichia coli cell of unconverted in contrast, as illustrated in fig. 1 and 2.
(4) positive bacterium colony PCR identifies
Observe the bacterium colony of incubated overnight, choose single bacterium colony and carry out coated plate cultivation, the bacterium colony obtained after cultivation is identified.
Authentication method still adopts NY/T1962-2010---and the RT-PCR method of regulation in " potato spindle tuber viroid detection ", is replaced by cDNA wherein and expands numerous single bacterium colony from flat board.3 PSTVd positive are chosen in this test, and the single bacterium colony coated plate of each sample picking 6 carries out bacterium colony PCR qualification after cultivating, and qualification result as shown in Figure 3.As seen from Figure 3, all bacterium colonies are the positive, show to transform successfully, all may be used for follow-up test preparation standard product.
(5) extraction of recombinant plasmid
Use " high purity plasmid is the rapid extraction test kit in a small amount " of Yuanpinghao (Tianjin) Biological Technology Co., Ltd. to extract being accredited as positive bacterium colony, concrete operations are as follows:
1) bacterium colony in Bechtop on picking flat board joins in the 5mLLB liquid nutrient medium containing penbritin and cultivates, 37 DEG C, 200rpm, overnight incubation;
2) get the positive bacterium colony nutrient solution of 2mL, the centrifugal 30s of 9000r/min, abandons supernatant as far as possible, collects thalline;
3) with the resuspended bacterial sediment of 250 μ LP1 solution, whirlpool concussion is to thoroughly suspending; If bacterium block suspends thorough, can cellular lysate be affected, thus cause extracted amount and purity on the low side;
4) add 250 μ LP2 solution, gentle upset makes the abundant cracking of thalline, until solution clarification, can not concuss, and avoid genomic dna to rupture;
5) add 400 μ LP3 solution, gentle upset makes the abundant cracking of thalline, leaves standstill 5min; 13000r/min, centrifugal 10min, collect supernatant;
6) be placed on collection tube by adsorption column, add 500 μ LP4 solution, 13000r/min, centrifugal 1min, abandons filtrate; Supernatant liquor proceeds in adsorption column AC that (adsorption column puts into collection tube, 13000r/min, and centrifugal 1min, abandons supernatant;
7) add 500 μ LPE protein liquid removals, 13000r/min, centrifugal 30 ~ 60s, abandons supernatant;
8) add the rinsing liquid WB of 500 μ L containing dehydrated alcohol, 13000r/min, centrifugal 30 ~ 60s, abandons supernatant;
9) repeat rinsing, 13000r/min, centrifugal 30 ~ 60s, abandons supernatant.13000r/min, centrifugal 2min; Leave standstill 3 ~ 5min, removing ethanol;
10) take out adsorption column AC, put into a clean centrifuge tube, 60 ~ 100 μ LEB elutriants are joined the central part of adsorption film, leave standstill 1min, 13000r/min, centrifugal 1min, eluted dna, measures plasmid concentration with ultraviolet spectrophotometer ND-1000 ,-20 DEG C of preservations.
(6) sequencing of recombinant plasmid
Checked order by Shanghai Sheng Gong Bioisystech Co., Ltd, sequential analysis adopts BioEdit software and internet blast to analyze.
The foundation of 2.4 real-time fluorescence quantitative PCR typical curves and the screening of primer with compare
2.4.1 the calculating of recombinant plasmid copy number
Plasmid copy number calculation formula:
(6.02 × 10
23copy number/mole) × (concentration ng/ μ L × 10
-9)/DNAlength × 660)=copies/mL;
Wherein 6.02 × 10
23avogadro constant number, dsDNA=base number × 660 dalton/base, uses PMD18-T carrier in this research, its base logarithm is 2964, the starting point concentration of the recombinant plasmid that this research uses is 101.17ng/ μ L, and therefore, the plasmid starting point concentration copy number used in this test is:
(6.02×10
23)×101.17ng/μl×10
-9/2964×660＝3.11×10
10copies/μL。
2.4.2 recombinant plasmid concentration gradient is diluted
Recombinant plasmid is done continuous gradient dilution preparation standard product, method is as follows:
Draw 27 μ L respectively without enzyme water in the PCR pipe of 11 200 μ L, be labeled as 1,2,3 respectively ... 11, drawing recombinant plasmid stoste 3 μ L joins in pipe 1, after abundant mixing, therefrom drawing 3 μ L again joins in pipe 2, by that analogy, dilute 11 gradients altogether, stoste and weaker concn represent with ng/ μ L and are respectively 101.17ng/ μ L ~ 1.0117ag/ μ L; Represent with copy number, be respectively 3.11 × 10
10~ 3.11 × 10
-1individual copy number (copies).
2.4.3 the comparison of primer and probe and screening
With stoste and front 5 dilution plasmid DNA solutions for template, use Roche
480 line fluorescent quantitative PCR apparatus are to three groups of primers (PSTV231F, PSTV296R; PSTV234F, PSTV350R; PSTV234F, PSTV356R) and probe PSTV251T carry out real-timePCR amplification, each sample 3 times repeats, and set up the typical curve of three individual system, reaction system is 20 μ L:
Response procedures is: 95 DEG C of 10min, 1 circulation, 95 DEG C of 10sec, 55 DEG C of 50sec, 72 DEG C of 1sec, 45 circulations.
Note: in this research, Tm value is 55 DEG C.
As shown in figures 4-6, in the standard substance in Fig. 4, plasmid concentration is respectively 3.11 × 10 to the selection result
10copies/ μ L, 3.11 × 10
9copies/ μ L, 3.11 × 10
8copies/ μ L, 3.11 × 10
7copies/ μ L, 3.11 × 10
6copies/ μ L, 3.11 × 10
5copies/ μ L; In Fig. 5 Plays product, plasmid concentration is respectively 3.11 × 10
10copies/ μ L, 3.11 × 10
9copies/ μ L, 3.11 × 10
8copies/ μ L, 3.11 × 10
7copies/ μ L, 3.11 × 10
6copies/ μ L, 3.11 × 10
5copies/ μ L; In Fig. 6 Plays product, plasmid concentration is respectively 3.11 × 10
10copies/ μ L, 3.11 × 10
9copies/ μ L, 3.11 × 10
8copies/ μ L, 3.11 × 10
7copies/ μ L, 3.11 × 10
6copies/ μ L, 3.11 × 10
5copies/ μ L.
The stability analysis of table 3PSTVdreal-timeRT-PCR detection method
2.5 sensitivity test
Carry out sensitivity test to the system selected in 2.5, method is: the standard substance preparation standard curve utilizing concentration lower, the concentration of each standard substance is respectively 3.11 × 10
7copies/ μ L ~ 3.11 × 10
-1copies/ μ L.
The relatively expanding effect of three groups of primers, have finally chosen the system that PSTV234F, PSTV356R and PSTV251T set up, and carried out sensitivity test to it, the concentration that the results are shown in Figure each standard substance in 7, Fig. 7 respectively is 3.11 × 10
7copies/ μ L ~ 3.11 × 10
-1copies/ μ L; Can see from this figure, plasmid concentration is 31.1copies/ μ L (101.17ag/ μ L), plasmid concentration and Logconcentration still keep good linear dependence, therefore the sensitivity that body series can detect is 31.1copies/ μ L (101.17ag/ μ L).
2.6real-timeRT-PCR system detects stability test
To 7 concentration gradients (3.11 × 10
7copies/ μ L ~ 3.11copies/ μ L) plasmid standard do 3 duplicate detection, Excel is utilized to calculate standard deviation and the average of Cp value, calculate the variation coefficient (Variationcoefficient, CV), method of calculation are: CV=standard deviation/mean number × 100%, verify the stability of detection system according to the variation coefficient (CV).
To 3.11 × 10
7the plasmid standard of ~ 3.11 copy number concentration does 3 duplicate detection, Cp value the results are shown in Table 2, the different plasmid concentrations detection Cp value variation coefficient (Variationcoefficient separately, CV) be respectively 1.64%, 0.61%, 1.80%, 0.47%, 0.92%, 1.11%, 1.45% and 0.98%, show that detection method has extraordinary stability.
To sum up, to prove successfully to establish real-timeRT-PCR system.
2.7 utilize the real-timeRT-PCR system set up to detect PSTVd sample
Utilize the system selected in 2.5 to carry out real-timeRT-PCR qualification to potato test-tube plantlet and dormancy stem tuber, each sample repeats for three times.
Detected result as shown in Figure 8, the LightCycler480 II real-timePCR instrument using Roche (Roche) to produce in Fig. 8, this instrument cannot show legend when 96 hole qualification results chart together, therefore, does not have legend to be shown as the amplification curve of test sample in this figure.As can be seen from Figure 8, positive amplification curve is typical S type, negative sample curve smoothing, without obviously sticking up tail, shows that detected result is reliable.
Claims (10)
1. detect potato spindle tuber viroid real-timeRT-PCR special primer and probe, it is characterized in that described primer sequence is as shown in sequence table SeqIDNo:1 and shown in SeqIDNo:2, described probe sequence is as shown in sequence table SeqIDNo:3.
2. potato spindle tuber viroid real-timeRT-PCR detection method, is characterized in that it carries out according to following steps:
One, the foundation of typical curve:
(1) extract potato tuber to be detected and test-tube plantlet RNA, and reverse transcription is cDNA, as template;
(2) adopt the template of step (1), carry out pcr amplification, the object fragment after pcr amplification is reclaimed, be connected on pMD18-T carrier, be transformed in competent cell, extract plasmid DNA, obtain potato spindle tuber viroid detection standard plasmid;
(3) with the standard plasmid obtained for template, utilize real-timeRT-PCR special primer and the probe of claim 1, carry out quantitative fluorescent PCR, gather fluorescent signal, adopt BioEdit Software on Drawing to go out real time fluorescent quantitative typical curve;
Two, the standard of positive or negative is judged
When amplification efficiency reaches 90% ~ 110%, slope is-3.3, when negative control does not play peak, calculates the content of potato spindle tuber viroid, undertaken analyzing directly carry out result judgement by real-timeRT-PCR according to typical curve and Cp value.
3. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that described PCR in real time detector is LightCycler480 II real-timePCR instrument.
4. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that described quantitative fluorescent PCR reaction system is as follows:
PCR response procedures is: 95 DEG C of 10min, 1 circulation, 95 DEG C of 10sec, 55 DEG C of 50sec, 72 DEG C of 1sec, 45 circulations.
5. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that the potato tuber to be detected described in step one and test-tube plantlet are PSTVd positive potato stem tuber and test-tube plantlet.
6. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, the primer that it is characterized in that carrying out in step one pcr amplification is PSTVd0054R and PSTVd0055-3F; Sequence is:
PSTVd0054R：5’-GGATCCCTGAAGCGCTCCTCCGAGCCG-3’；
PSTVd0055-3F：5’-CCGGGGAAACCTGGAGCGAACTGG-3’。
7. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that reclaiming the object fragment after pcr amplification in step one is reclaimed by the centrifugal column type sepharose of Tiangen company that test kit reclaims amplified production, purifying.
8. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, it is characterized in that in step one, ligation system is 10 μ L, wherein, pMD18-TVector is 0.8 μ L, and PCR purified product is 4 μ L, ddH
2o is 1.2 μ L; Solution I is 5 μ L; Reaction conditions is connect under 4 DEG C of conditions to spend the night.
9. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that carrying out sensitivity test to the real-timeRT-PCR detection method of potato spindle tuber viroid.
10. potato spindle tuber viroid real-timeRT-PCR detection method according to claim 2, is characterized in that the fluorescent quantitation typical curve to step one is drawn carries out stability test.
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| CN113774053A (en) * | 2021-08-19 | 2021-12-10 | 中国农业科学院植物保护研究所 | Nucleic acid probe and kit for simultaneously detecting multiple tomato viruses and application of nucleic acid probe and kit |
| CN114032338A (en) * | 2021-12-20 | 2022-02-11 | 黑龙江省农业科学院经济作物研究所 | Probe for detecting PSTVd by nucleic acid dot hybridization method, preparation method thereof, kit and detection method |
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