CN104450975A - RT-HDA primer, kit and method for detecting tomato spotted wilt virus - Google Patents
RT-HDA primer, kit and method for detecting tomato spotted wilt virus Download PDFInfo
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Abstract
The invention discloses an RT-HDA primer, kit and method for detecting a tomato spotted wilt virus. The primer is an oligonucleotide sequence represented by SEQ ID NO:1 and SEQ ID NO:2 of a sequence table. The kit comprises the following components: RT-HDA amplified reaction liquid, deionized water without RNA enzyme, a positive control sample and a negative control sample, the deionized water is adopted as blank control, the positive control sample is an RNA template with the tomato spotted wilt virus, and the negative control sample is a healthy tomato tissue RNA template without the tomato spotted wilt virus. The detecting method comprises the following steps that (1) RNA extraction is carried out on a sample to be detected; (2) RT-HDA amplification is carried out on the RNA of the tomato spotted wilt virus; (3) an electrophoresis detecting result is described and judged. The HDA detecting method is faster, safe and reliable, and compared with a PCR, no PCR instrument is needed, the requirement for equipment is simple, the method is suitable for fast detecting and confirming the tomato spotted wilt virus, and the method can be widely applied to epidemic situation monitoring in agricultural production and environment and monitoring and detecting of this kind of viruses in import and export trade.
Description
Technical field
The present invention relates to a kind of RT-HDA primer detecting tomato spotted wilf virus, test kit and method, belong to virus detection techniques field.
Background technology
In recent years, tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) has been listed in the world with its host range and tremendous economic loss of causing widely and has endangered one of maximum ten kind of plant viruses.In 60 ~ eighties of 20th century, this virus was once very popular on America and Europe and African tobacco and tomato, and annual sickness rate is 20% ~ 50%, causes the loss up to several 1,000,000,000 dollars.In Hawaii, America, Brazil, Italy and South Africa, TSWV once caused the crop such as tomato, lettuce to be close to total crop failure at the popular of 80 ~ nineties of 20th century.In recent years, Tospovirus virus particularly TSWV, has become the important factor that the whole world causes diversified economy crop and the very big financial loss of ornamental plant.
The vegetable seed such as capsicum, onion, romaine lettuce that China plants at present much comes from all over the world, especially the basic dependence on import of tomato seeds, import country origin comprises the states such as the U.S., Japan, Holland, Thailand, these importers have generation and the report of tomato spotted wilf virus at present, and China port once intercepted and captured TSWV virus in 2012 in the seed entered the territory.And traditional TSWV detects and by biological host, morphology tests, confirmation method generally judges whether plant has plant virus, these methods are time-consuming, complex operation, can not meet the needs of disease control.
In existing nucleic acid detection method, conventional has polymerase chain reaction (Standard PCR and real-time fluorescence PCR), and the method is sensitive, special, but needs high-quality thermal cycler, and because temperature cycle causes longer.Other nucleic acid amplification methods newly developed, such as NASBA (nucleic acid sequence-based amplification), 3SR (self-sustainedsequence replication), SDA (strand displacement amplification) etc., in amplification cycles, there is respective innovative point.NASBA and 3SR uses a series of transcribing to circulate to avoid high-temperature denatured effect with transcriptive process,reversed, and SDA then uses the template of restriction enzyme and modified to carry out cyclic amplification.Although their susceptibility is all very high, can detect and increase be less than 10 copy sample of nucleic acid, they need the shortcoming overcome in addition separately.The aspects such as technical requirements, material requirements, the specific defects of technology own have seriously fettered applying of these technology.Therefore, be necessary to provide one more convenient, the method for the detection tomato spotted wilf virus that detection sensitivity is higher.
Rely on helicase constant temperature gene amplification technology (Helicase-Dependent Isothermal Amplification, HDA) be BioHelix in 2004 researchist's simulated animal body in DNA replicanism invention a kind of external isothermal amplification technology newly.This technology utilizes DNA double chain helicase to open double-stranded DNA, and simultaneously under the effect of single strand binding protein (SSB) archaeal dna polymerase, amplified target fragment, realizes the amplification in vitro of goal gene.HDA technology compares with other gene amplifications, has following advantage:
1, compared with round pcr, HDA technology-imitation nature DNA synthesis mode, unties double-strand and complete amplification, and normal PCR is realized by the repeatedly circulation of sex change-annealing-extension process by means of DNA helicase.Due to the difference of principle, make the advantage of HDA especially outstanding: 1. HDA reaction is carried out at the same temperature, does not thus need expensive PCR instrument, is conducive to applying at laboratories.2. owing to only making the HDA reaction times obviously shorten with a kind of temperature amplification, only about 1h is needed.3. HDA technology adopts and eliminates 5 '-3 ' the BstDNA polysaccharase of 5 prime excision enzyme activity, this enzyme has the features such as high thermal resistance, efficient amplification reactivity, fabulous band homogeneity and high accuracy concurrently, not easily cause non-specific amplification, make HDA technology have higher specificity and susceptibility.
2, compared with other isothermal amplification technologies, HDA is a kind of constant temperature gene amplification method truly.Such as: SDA, LAMP etc. all need thermally denature, and HDA does not need this portion to complete reaction.In addition, although the reaction system group of HDA is slightly aobvious complicated, simple to operate easy to learn.Especially design of primers is simple.SDA technology needs four primers, also need the dNTP of modified mistake to make substrate, and the complicacy of target sequence also limit its range of application simultaneously.LAMP technology has simply, fast, the advantage such as efficient, high specificity, but design of primers is complicated, difficulty is comparatively large, and higher to the requirement of target sequence: aim sequence length is limited in about 200bp, four to six the primer Tm mutual difference therefrom designed, and 6 different regions can be identified.And the design of primers of HDA technology is identical with conventional PCR method, only need two general primers.In a word, compared with similar novel round pcr, HDA technology is simpler, more effectively, has a good application prospect.
According to the latest news, existing HDA technology also comprises one and not only can to increase the ring HDA method (T and circular helicase-dependent amplification, cHDA) of macromole DNA fragmentation, the complete loop plasmid DNA that also can directly increase.Utilize the specific amplification cyclic DNA ability of cHDA, directly can detect the virus etc. containing cyclic DNA from pathogenic agent.HDA technology combines with fluorescent labelling techniques can also realize real-time detection.Research shows, HDA technology can increase microbe genome DNA, RNA, plasmid DNA and cDNA etc.HDA method amplification ability is strong, and used Uvrd system amplification can reach more than 100 ten thousand times, the target gene lower than 10 copies can be detected from genomic dna.In sum, HDA technology is a kind of constant temperature gene amplification method of incomparable real practicality, is expected to become a kind of important diagnostic tool.
At present, the research of plant virus is not also detected both at home and abroad about HDA.We will set up the HDA technology of rapid detection tomato spotted wilf virus first.This invention achievement will be expected to the disruptive technology becoming vegetable plague molecular diagnosis field, is improving further and development of epiphytology detection technique system.
Summary of the invention
Technical problem to be solved by this invention is: a kind of RT-HDA amplimer for detecting tomato spotted wilf virus and test kit, namely RT-HDA amplification method is utilized to detect primer and the test kit of tomato spotted wilf virus, overcome the limitation of other detection methods at Viral diagnosis, for the detection of tomato spotted wilf virus provides effective tool, can fast, the detection tomato spotted wilf virus of high specificity, highly sensitive and low cost.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Tomato spotted wilf virus RT-HDA provided by the invention detects primer sets, and comprise primer pair HD-P1 and HD-P2, their base sequence is respectively SEQ ID NO:1 ~ 2.
Test kit of the present invention, comprises following component:
1) RT-HDA amplification reaction solution:
Comprise 10 × RT-HDA damping fluid 5 μ L, MgSO4 (100mM) 2 μ L, NaCl (500mM) 4 μ L, dNTP (3.0mM) 3.5 μ L, enzyme mixation enzyme mixation (DNA helicase 0.4 μ g, AMV-Bst 40U and ThermoScript ThermoScript II 4U) 3.5 μ L, each 1 μ L of RNase-Free Water 20 μ L, HD-P1 and HD-P2.
Wherein 10 × RT-HDA damping fluid comprises 10mmol/L KCL, 20mmol/L Tris-Cl (Ph8.8,25 DEG C), and described primer HD-P1 and HD-P2 has the base sequence of sequence table SEQ ID NO:1 ~ 2 respectively;
2) positive control sample: containing tomato spotted wilf virus RNA template, as positive template;
3) negative control sample: not containing the healthy tomato tissue RNA template of tomato spotted wilf virus, as negative template;
4) blank control sample: be DEPC deionized water;
Use above test kit, detect the method for tomato spotted wilf virus in sample, comprise the following steps: successively
1) measuring samples RNA extracts
Trizol method or RNA is taked to extract test kit extracting RNA template,
2) tomato spotted wilf virus RT-HDA increases
A: add template ribonucleic acid 10 μ L to be checked and RT-HDA amplification reaction solution 40 μ L successively in reaction tubes, fully mix;
B: under 65 DEG C of isothermal reaction conditions, carries out RT-HDA amplified reaction 90min;
During amplification, 3 contrasts should be set up: positive control (tomato spotted wilf virus RNA template replaces sample RNA template), negative control (replacing sample RNA template with negative template ribonucleic acid), blank (DEPC water replaces sample RNA template)
3) electrophoresis detection
Get 1.5g agarose, heat, fully dissolve in 100mL electrophoretic buffer, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L ~ 6 μ LHDA amplified productions are mixed with appropriate sample loading buffer respectively, point sample; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.If amplified fragments is 131bp, illustrate that virus to be checked is TSWV, if there is not 131bp amplified fragments, then illustrate that virus to be checked is not TSWV.
The application of above-mentioned tomato spotted wilf virus RT-HDA detection method on tomato, capsicum.
The molecular biology method of rapid detection tomato spotted wilf virus provided by the invention, have the features such as highly sensitive, high specificity, compared with prior art, beneficial outcomes of the present invention is:
1, simple and easy to do: this detection method is by thermostat water bath or have the equipment of stable thermal source just can test, and gets final product result of determination by reaction product colour-change, eliminate expensive plant and instrument, loaded down with trivial details electrophoresis process;
2, detect high efficiency: this detection method detection time used was less than 1 hour, and the pcr amplification time is longer, generally need more than 2 hours, the efficiency of detection can be substantially increased like this;
3, highly sensitive: with tomato spotted wilf virus RNA for template, the Monitoring lower-cut of the method is 10fg/ μ L, is 100 times of Standard PCR;
4, accuracy is high: this does not need purifying RNA from sample, incidence tissue or invalid body directly can be utilized to extract RNA rapid detection, substantially increase the accuracy of detection;
5, high specificity: HDA technology adopts and eliminates 5 '-3 ' the BstDNA polysaccharase of 5 prime excision enzyme activity, this enzyme has the features such as high thermal resistance, efficient amplification reactivity, fabulous band homogeneity and high accuracy concurrently, not easily cause non-specific amplification, make HDA technology have higher specificity.
Accompanying drawing explanation
Fig. 1 is RT-HDA specificity experiments figure of the present invention.1: tomato spotted wilf virus; 2: positive control; 3: garden balsam necrotic spot virus; 4: Tomato mosaic virus; 5: tomato yellow leaf curl virus; 6: tomato black ring virus.
Fig. 2 is RT-HDA sensitivity experiments figure of the present invention.Wherein 1 ~ 7:1.56 × 10
-1, 1.56 × 10
-2, 1.56 × 10
-3, 1.56 × 10
-4, 1.56 × 10
-5, 1.56 × 10
-6, 1.56 × 10
-7the susceptible material total serum IgE of ng/ μ LTSWV.
Fig. 3 is RT-PCR sensitivity experiments figure of the present invention.Wherein 1 ~ 6:1.56 × 10
-1, 1.56 × 10
-2, 1.56 × 10
-3, 1.56 × 10
-4, 1.56 × 10
-5, 1.56 × 10
-6the susceptible material total serum IgE of ng/ μ L TSWV.
Embodiment
The present invention adopts RT-HDA technology, and devise corresponding RT-HDA detection primer sets according to the gene order of tomato spotted wilf virus N fragment, comprising primer pair: HD-P1 and HD-P2, having invented the RT-HDA detection method for detecting tomato spotted wilf virus and test kit thus.Apply detection method of the present invention and test kit, only need by carrying out RNA extraction to the suspected infection such as capsicum, tomato Plant samples and carry out RT-HDA amplification, can rapid detection go out whether to infect tomato spotted wilf virus, basic unit is facilitated to carry out disease diagnosis and treatment fast, accurately, easily, and then take suitable prophylactico-therapeutic measures, reduce the loss that disease is brought.
Embodiment 1 specificity experiments
(1) design of primers
The present invention according to the N full length gene sequence of TSWV in NCBI GenBank (
kC294570.1(Kunming isolate), HM581936.1 (Nanjing isolate), JF730744.1 (Korea S's isolate), HM180089 (Taiwan isolate), HQ406984.1 (U.S.'s isolate), KC494503.1 (New Zealand's isolate), KM379142.1 (Turkey's isolate), KF146703.1 (Venezuela's isolate)), by the high conservative region of comparative analysis TSWV N gene under the prerequisite of the degenerate and versatility that ensure amplification, design RT-HDA primer pair HD-P1 and HD-P2, after having designed, primer to be compared under the Primer-Blast module of database checking, finally choose following RT-HDA and detect primer sets:
Upstream primer HD-P1:GGCTTGAATCAGAGGGTGAGA (see sequence table SEQ ID NO:1),
Downstream primer HD-P2:TTGGAGCCACTGACATGACC (see sequence table SEQ ID NO:2).
(2) extraction of viral RNA
Get 0.1g tomato spotted wilf virus, tomato black ring virus, garden balsam necrotic spot virus, Tomato mosaic virus, the bent leaf sample of tomato yellowing, add liquid nitrogen to be ground into powder, abrasive material is moved into rapidly in 1.5mL centrifuge tube, add 1mL TrizolReagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2mL chloroform, with hand concuss (not vortex oscillation), 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
(3) five kinds of viral RT-HDA increase
A: add five kinds of viral template ribonucleic acid to be checked 10 μ L and RT-HDA amplification reaction solution 40 μ L respectively successively in reaction tubes, fully mix.
B: under 65 DEG C of isothermal reaction conditions, carries out RT-HDA isothermal amplification reactions 90min.
(4) electrophoresis detection
Get 1.5g agarose, heat, fully dissolve in 100mL electrophoretic buffer, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L ~ 6 μ LHDA amplified productions are mixed with appropriate sample loading buffer respectively, point sample; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.If amplified fragments is 131bp, illustrate that virus to be checked is TSWV, if there is not 131bp amplified fragments, then illustrate that virus to be checked is not TSWV.
This group primer confirms to have very high specificity through the result of online BLAST, can not with other viral cross reactions.Tomato spotted wilf virus is Tospovirus virus, and therefore research have selected and belongs to main virus common on allied species virus garden balsam necrotic spot virus and other three kinds of tomatoes together for test seed culture of viruses.Empirical tests, except amplification 131bp band appears in tomato spotted wilf virus RNA, all the other viruses, all without corresponding amplified band, conform to expection, prove the good specificity of this group primer, with the equal no cross reaction of above-mentioned strain (see Fig. 1).
Embodiment 2 sensitivity experiments
(1) with DEPC water, TSWV viral RNA template liquid is done 10 times of gradient dilutions downwards, be followed successively by 1.56 × 10
-1, 1.56 × 10
-2, 1.56 × 10
-3, 1.56 × 10
-4, 1.56 × 10
-5, 1.56 × 10
-6, 1.56 × 10
-7ng/ μ L, respectively getting 2 μ L is that template carries out RT-HDA and RT-PCR amplified reaction respectively.Amplimer group comprises:
Upstream primer HD-P1:GGCTTGAATCAGAGGGTGAGA (see sequence table SEQ ID NO:1),
Downstream primer HD-P2:TTGGAGCCACTGACATGACC (see sequence table SEQ ID NO:2).
(2) extraction of viral RNA
Get the sample of the different weaker concn of 0.1g, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5mL centrifuge tube, add 1mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
(3) the tomato spotted wilf virus RT-HDA of different concns increases
A: the template ribonucleic acid to be checked 10 μ L and the RT-HDA amplification reaction solution 40 μ L that add different dilution multiple proportions respectively in reaction tubes successively, fully mix.
B: under 65 DEG C of isothermal reaction conditions, carries out RT-HDA amplified reaction 60min.
(4) RT-PCR amplification and detection
RT-PCR amplification system illustrates configuration with reference to the Quant One step RT-PCR test kit of TIANGEN company, and reaction conditions is 50 DEG C of reverse transcription 30min; 94 DEG C of denaturation 2min; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, circulating reaction 35 times; 65 DEG C extend 10min.
(4) electrophoresis detection
Get 1.5g agarose, heat, fully dissolve in 100mL electrophoretic buffer, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L ~ 6 μ L amplified productions are mixed with appropriate sample loading buffer respectively, point sample; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.
(5) result judges
RT-HDA Product Identification: electrophoresis result shows, tomato spotted wilf virus uses RT-HDA method to obtain 10
-5the template (see Fig. 2) of extension rate, can reach fg level level, uses RT-PCR method to obtain 10
-3extension rate (Fig. 3), only has pg level level.This illustrates that the detection sensitivity of RT-HDA method of the present invention exceeds PCR method order of magnitude.
Embodiment 3: actual sample detects and contrast experiment
By from Yunnan, Shandong, Deng Di field, Sichuan gather the sick sample with typical TSWV symptom and laboratory sample retention, adopt RT-HDA, RT-PCR to detect respectively, compare the effect of two kinds of methods, to assess the reliability of RT-HDA method further.
1, actual sample RT-HDA detects
1) extraction of viral RNA
Get 0.1g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5mL centrifuge tube, add 1mL TrizolReagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
(2) the tomato spotted wilf virus RT-HDA of different sources sample increases
A: the template ribonucleic acid to be checked 2 μ L and the RT-HDA amplification reaction solution A23 μ L that add different dilution multiple proportions respectively in reaction tubes successively, fully mix.
B: under 65 DEG C of isothermal reaction conditions, carries out RT-HDA isothermal amplification reactions 60min.
3) RT-HDA Product Identification
Observe electrophoresis result and record under Ultraviolet Detector, electrophoresis result shows, and in 9 increment product, 3 increment product are positive, and other samples are negative.
2, pcr amplification
1) method: conventional RT-PCR reaction conditions is 50 DEG C of reverse transcription 30min; 94 DEG C of denaturation 2min; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, circulating reaction 35 times; 65 DEG C extend 10min.
2) agarose gel electrophoresis result: in 9 increment product, 3 parts is positive, and all the other are 6 parts.
Conclusion: utilize above-mentioned RT-HDA and PCR method simultaneously to detect actual sample, the coincidence rate 100% of two kinds of methods, but RT-HDA is lower to equipment requirements, and convenience is higher.
The detection method that the present invention sets up can detect tomato spotted wilf virus fast and accurately, for scientific research and production practice provide a kind of detection technique easy, quick, with low cost, also for the early warning of spotted wilt and rational use of drug provide theoretical basis and technical director, Social and economic benef@ecological to increase all has reality and profound significance.
SEQUENCE LISTING
<110> Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120> detects the RT-HDA primer of tomato spotted wilf virus, test kit and method
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> upstream primer HD-P1
<400> 1
GGCTTGAATCAGAGGGTGAGA 22
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> downstream primer HD-P2
<400> 2
TTGGAGCCACTGACATGACC 20
Claims (9)
1. one group is detected the primer of tomato spotted wilf virus, it is characterized in that, the sequence of described primer sets is respectively shown in sequence table SEQ IDNO:1 ~ 2, and wherein SEQ ID NO:1 is the primer HD-P1 detecting tomato spotted wilf virus, SEQ ID NO:2 is the primer HD-P2 detecting tomato spotted wilf virus.
2. primer sets according to claim 1 is detecting the application in tomato spotted wilf virus.
3. primer sets according to claim 1 detects the application in the test kit of tomato spotted wilf virus in preparation.
4. detect a RT-HDA test kit for tomato spotted wilf virus, it is characterized in that, described test kit comprises following component:
1) RT-HDA amplification reaction solution:
Comprise 10 × RT-HDA damping fluid 5 μ L, MgSO4 (100mM) 2 μ L, NaCl (500mM) 4 μ L, dNTP (3.0mM) 3.5 μ L, enzyme mixation 3.5 μ L, each 1 μ L of RNase-Free Water 20 μ L, HD-P1 and HD-P2;
Wherein 10 × RT-HDA damping fluid comprises 10mmol/L KCL, 20mmol/L Tris-Cl (pH8.8,25 DEG C), and described primer HD-P1 and HD-P2 has the base sequence of sequence table SEQ ID NO:1 ~ 2 respectively;
Described enzyme mixation comprises DNA helicase 0.4 μ g, AMV-Bst 40U and ThermoScript ThermoScript II 4U;
2) without the deionized water of RNA enzyme, as blank template;
3) positive control sample: containing tomato spotted wilf virus RNA template, as positive template;
4) negative control sample: not containing the healthy tomato tissue RNA template of tomato spotted wilf virus, as negative template.
5. application rights requires that the test kit described in 4 detects a method for tomato spotted wilf virus in sample, and it is characterized in that, described method comprises the following steps:
1) measuring samples RNA extracts
Trizol method or RNA is taked to extract test kit extracting RNA template,
2) tomato spotted wilf virus RNA carries out RT-HDA amplification;
3) electrophoresis detection result and description and judgement
1. electrophoresis detection
2. result describes and judges
1. quality control standard:
Positive control at 131bp place by specific amplification band;
Negative control is without specific amplification band;
Be discontented with as negative control and positive condition and be enough to upper condition, it is invalid that this time test is considered as;
2. result judges:
Positive: at 131bp place by specific amplification band, represent in sample containing tomato spotted wilf virus;
Negative: without specific amplification band, to show in sample without tomato spotted wilf virus.
6. method according to claim 5, is characterized in that, has increased rear to RT-HDA amplified production sequence verification.
7. method according to claim 5, is characterized in that, step 2) tomato spotted wilf virus RNA carry out RT-HDA amplification be specially:
A: add template ribonucleic acid 10 μ L to be checked and RT-HDA amplification reaction solution 40 μ L successively in reaction tubes, fully mix;
B: under 65 DEG C of isothermal reaction conditions, carries out RT-HDA amplified reaction 90min;
During amplification, set up 3 contrasts: positive control tomato spotted wilf virus RNA template replaces sample RNA template, negative control to replace sample RNA template, blank DEPC water replacement sample RNA template with negative template ribonucleic acid.
8. method according to claim 5, is characterized in that, step 3) in electrophoresis detection be specially:
Get 1.5g agarose, heat, fully dissolve in 100mL electrophoretic buffer, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L ~ 6 μ LHDA amplified productions are mixed with appropriate sample loading buffer respectively, point sample; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.
9. above-mentioned tomato spotted wilf virus RT-HDA detection method detects the application of tomato spotted wilf virus on tomato, capsicum.
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| CN201410854297.8A CN104450975A (en) | 2014-12-31 | 2014-12-31 | RT-HDA primer, kit and method for detecting tomato spotted wilt virus |
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| CN105018486A (en) * | 2015-08-05 | 2015-11-04 | 江西师范大学 | HDA kit for detecting bacterial leaf streak pathogens of rice and detecting method |
| CN105063193A (en) * | 2015-07-31 | 2015-11-18 | 江西师范大学 | HDA kit and detecting method for detecting xanthomonas oryzae of rice |
| CN114058737A (en) * | 2021-10-22 | 2022-02-18 | 中国农业科学院烟草研究所 | crDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus |
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