CN101812540A - Real-time fluorescent quantitative RT-PCR method for detecting garden balsam necrotic spot virus, primer, probe and kit thereof - Google Patents
Real-time fluorescent quantitative RT-PCR method for detecting garden balsam necrotic spot virus, primer, probe and kit thereof Download PDFInfo
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Abstract
The invention provides a real-time fluorescent quantitative RT-PCR method for detecting garden balsam necrotic spot virus, a primer, a probe and a kit thereof, which belong to the technical field of molecular biological detection and identification of plant virus. The sequence of an upstream primer is shown in SEQ ID NO:1; the sequence of a downstream primer is shown in SEQ ID NO:2; the fragment length of a target product is 182 bp; and the sequence of a TaqMan probe is shown in SEQ ID NO:3. The kit and the detection method comprise the primers and the probe. The invention has the advantages that the invention is good in reliability and repeatability, ensures that the difference value of Ct values obtained by repeating three samples is between 0.01 and 1.7, has good stability, ensures that the difference value of Ct average values detected on different hosts is between 0.25 and 0.34, can detect the INSV virus content of 6.99 copy mu l-1, has the effect of trace detection, is strong in detection specificity, and only allows INSV to have fluorescent signals.
Description
Technical field
The present invention relates to plant virus molecular Biological Detection and authenticate technology field, be specifically related at the TaqMan fluorescence probe quantitative RT-PCR detection method of garden balsam necrotic spot virus and used primer and probe.
Background technology
(Impatiens necrotic spot virus is one of the important disease of harm ornamental plant INSV) to garden balsam necrotic spot virus, also is one of the most scabrous problem in the external production of flowers and plants.INSV was listed in A2 class quarantine harmful organisms by Europe and Mediterranean Sea plant protection tissue in 1998.
(Impatiens necrotic spot virus INSV) belongs to bunyaviridae (Bunyaviridae) tomato spotted wilf virus and belongs to (Tospovirus) garden balsam necrotic spot virus.This virus particle is spherical, diameter 80-120nm, and virus has coating, and its genome is made of 3 single stranded RNA molecules.Garden balsam necrotic spot virus the earliest from the Symptomatic Flower of Garden Balsam isolation identification come out, can infect 50 sections, 648 kind of plant, wherein 39 genus of ornamental plant, 6 genus of vegetables.The symptom that INSV causes is various, with the host with infect different variation the in period, mainly show as spot, stem browning (ulcer), the broken look flower of the brown of dwarfing, ring spot, blade or stem to purple, when seriously taking place, disease can cause the dwarfing of many ornamental plants, even plant death, have a strong impact on the economic worth of ornamental plant.
Traditional biological mensuration, modern electron microscopy, serological technique, conventional RT-PCR technology etc. have been widely used in the diagnosis of INSV virus at present.It is by observing the differential host at postvaccinal symptomatic reaction that yet traditional biological is measured, and other biological is learned the standard that index is identified as virus; Modern electron microscopy is to determine by the feature of aspects such as making plant virion form, size, structure, inclusion body composition and substructure whether virus exists; Conventional PCR (polymerase chain reaction, Polymerase Chain Reaction is called for short PCR) can in vitro be expanded to 100,000 and even 1,000,000 times with goal gene or a certain dna fragmentation that will study at one in a few hours, make naked eyes energy direct viewing and judgement.Chinese patent application number is that 200810058612.0 " method of the garden balsam necrotic spot virus on the rapid detection oncidiumLuridum " (its publication number is CN101307366A, open day is on November 19th, 2008) set up the RT-PCR and the nest-type PRC technology of garden balsam necrotic spot virus, its specificity and sensitivity are all superior than ordinary method, but it only can the qualitative detection garden balsam necrotic spot virus, can not realize the detection by quantitative to viral sample.
Quantitative detection method has converting phosphor immunochromatography technique, real-time fluorescence quantitative PCR technology etc. in the biology at present, real-time fluorescence quantitative PCR (Real-time fluorescence quantitative PCR wherein, RTFQPCR) technology use in present quantitative measurement technology is the most extensive, and tolerance range is the highest.The regular-PCR technology can be carried out exponential amplification to the specific nucleotide segment.After amplified reaction finishes, can analyze qualitatively amplified production by the method for gel electrophoresis, also can carry out quantitative analysis by the densitometric scan that radionuclide mixes behind the mark.No matter qualitative still quantitative analysis, analysis all be the PCR end product.But in many cases, people interested be starting template amount before amplifying without the PCR signal, for example, if wonder genetically modified copy number of a certain genetically modified animals and plants or the expression amount of a certain specific gene in particular organization, the present invention has made up real-time fluorescence quantitative RT-PCR (RT-PCR has been a reverse transcriptional PCR under this demand, reverse transcription Polymerase Chain Reaction is called for short RT-PCR)), construct the suitable primer special that detects garden balsam necrotic spot virus, probe and detection optimal conditions etc., it is on conventional PCR basis FRET (fluorescence resonance energy transfer) to be combined with fluorescence labeling probe, dexterously nucleic acid amplification, hybridization, the creative technology that spectroscopic analysis and real-time detection technique merge, up to now, do not find that publish and the patent of invention similar detection garden balsam necrotic spot virus of the present invention.
Summary of the invention
The technical problem to be solved in the present invention is to overcome prior art in the defective aspect the garden balsam necrotic spot virus detection by quantitative, its objective is the real-time fluorescent quantitative RT-PCR method that a kind of detection garden balsam necrotic spot virus based on the TaqMan probe is provided, simultaneously, the present invention also is provided for garden balsam necrotic spot virus is carried out the primer that real-time fluorescence quantitative RT-PCR detects, and garden balsam necrotic spot virus is carried out TaqMan probe and test kit and the application that real-time fluorescence quantitative RT-PCR detects.
For solving the problems of the technologies described above, the present invention has following technical proposal:
1, a kind of being used for carried out the primer that real-time fluorescence quantitative RT-PCR detects to garden balsam necrotic spot virus, described primer is made up of upstream primer and downstream primer, the base sequence of described upstream primer is shown in SEQ ID NO:1 in the sequence table, the base sequence of described downstream primer is shown in SEQ ID NO:2 in the sequence table, and the target product fragment length is 182bp.
2, a kind of being used for carried out the TaqMan probe that real-time fluorescence quantitative RT-PCR detects to garden balsam necrotic spot virus, the base sequence of described TaqMan probe is shown in SEQ ID NO:3 in the sequence table, be marked with the report fluorophor at 5 of described TaqMan probe ' end, 3 ' end is marked with the cancellation fluorophor, and described TaqMan probe is between the upstream primer and downstream primer of the described primer of above-mentioned the 1st technical scheme.
Described report fluorophor can adopt some report fluorophors commonly used, and as FAM, described cancellation fluorophor can adopt some cancellation fluorophors commonly used, as TAMRA.
3, a kind of test kit that garden balsam necrotic spot virus is carried out the real-time fluorescence quantitative RT-PCR detection comprises primer and TaqMan probe; Described primer is the described primer of above-mentioned the 1st technical scheme, and described TaqMan probe is the described TaqMan probe of above-mentioned the 2nd technical scheme.
The mentioned reagent box also comprises the positive criteria product, it is from positive colony bacterium liquid and extracts plasmid, with the absorbancy of UV-light spectrophotometric determination D260nm and 280nm, and calculates the plasmid content that extracts, plasmid with the gradient dilution of ultrapure water with 10 times, is diluted to 10 at last
1, 10
2, 10
3, 10
4, 10
5Times positive criteria product that plasmid is made, each usage quantity respectively is the every reactions of 2 μ l/.
The mentioned reagent box also comprises the garden balsam necrotic spot virus immunity test strip; the M-MLV ThermoScript II; 5x M-MLV Buffer; 10Mm dNTP mixture; 50 μ l random primers, six aggressiveness; 40U/ μ l RNA inhibitor; PCR quality water; Illustra RNAspin Mini RNA Isolation Kit test kit; the PCR product of hundred Tyke biotech firms reclaims purification kit; competent cell DH5@; SOC substratum and X-Gal; IPTG; the blue hickie substratum of the LB of Amp; described competent cell DH5@; SOC substratum and X-Gal; IPTG; the blue hickie substratum of the LB of Amp is according to X-Gal in the common agents preparation of TaKaRa company; IPTG; the configuration of the blue hickie culture medium preparation of the LB of Amp method forms; plasmid reclaims purification kit (purchasing in hundred Tyke Bioisystech Co., Ltd), super mixed liquid of the Premix Ex TaqTM of TaKaRa company and PMD19 simple-T Vector, sterilization ultrapure water.
4, a kind of real-time fluorescent quantitative RT-PCR method that detects garden balsam necrotic spot virus, comprise the extraction of viral RNA to be measured and synthesizing of cDNA thereof, TaqMan fluorescence quantitative RT-RCR reaction system, carry out the reaction of TaqMan fluorescence quantitative RT-RCR in TaqMan fluorescence quantitative RT-RCR reaction system, by the preparation of standard substance and the foundation of typical curve, calculate detected result, the primer that uses in synthetic and described reaction is the described primer of above-mentioned the 1st technical scheme, and the TaqMan probe that uses in synthetic and described reaction is the described TaqMan probe of above-mentioned the 2nd technical scheme.
TaqMan fluorescence quantitative RT-RCR reaction system described in above-mentioned a kind of real-time fluorescent quantitative RT-PCR method that detects garden balsam necrotic spot virus can also be according to the specification sheets of the Premix Ex TaqTM of TaKaRa company, consumption to reagent is allocated, make it to reach better effect, the TaqMan fluorescence quantitative RT-RCR reaction system after allocating be: adopt 25 μ l reaction systems, Premix Ex TaqTM (2x) 12.5 μ l wherein, each 0.5 μ l of upstream primer and downstream primer, TaqMan probe 0.25 μ l, dH
2O (aqua sterilisa) 9.25 μ l, cDNA 2 μ l; All reaction reagents are all in preparation on ice; The real-time fluorescence quantitative PCR response procedures is 94 ℃ of pre-denaturation temperatures, 5min; 58 ℃ of annealing temperatures, 30s; Gather fluorescent signal 15s for 58 ℃.
5, with primer of the present invention and TaqMan probe of the present invention, with test kit of the present invention or with the application of real-time fluorescent quantitative RT-PCR method in detecting garden balsam necrotic spot virus of detection garden balsam necrotic spot virus of the present invention.
Described application is included in the application that detects garden balsam necrotic spot virus on butterfly orchid, spider orchid or the Sowthistle Tasselflower Herb.
Compared with prior art, this discovery has the following advantages and effect:
1. simple to operate, good stability, highly sensitive, high specificity; Show among the embodiment: three in each sample repeats resulting cycle number (Ct) value difference value 0.01 to 1.7, reliability of the present invention is described, good reproducibility; The Ct mean value difference that detection obtains on while three different host illustrates having good stability of this invention 0.25 to 0.34.Show among Fig. 4: present method can detect INSV viral level 6.99 copy μ l
-1, show that the present invention has the effect of trace detection; Among Fig. 6 at INSV, common calla chlorosis spot poison (Calla LilyChlorotic Spot Virus, CCSV), annulus zonatus (Tomato Zonate Spot Virus, TZSV) and tomato spotted wilf virus (Tomato spotted wilt tospovirus, TSWV) have only INSV that fluorescent signal is arranged in these four kinds of viruses, three kinds of viruses of all the other CCSV, TSWV and TZSV are not all found tangible amplified fluorescence curve, show the high specificity that the present invention detects INSV.
Detection time short, extract from RNA and to detect net result and only need 3h;
3. the scope of application is extensive, through overtesting, use the present invention and can detect garden balsam necrotic spot virus respectively from corresponding plant, and effect is obvious.
4. but both qualitative detection of the present invention again can the detection by quantitative garden balsam necrotic spot virus, can replace the method for traditional qualitative detection garden balsam necrotic spot virus.
In the sequence table shown in the SEQ ID NO:1 is the base sequence of the upstream primer of primer of the present invention;
In the sequence table shown in the SEQ ID NO:2 is the base sequence of the downstream primer of primer of the present invention;
In the sequence table shown in the SEQ ID NO:3 is the base sequence of TaqMan probe of the present invention, is marked with the report fluorophor at 5 of described TaqMan probe ' end, and 3 ' end is marked with the cancellation fluorophor.
Description of drawings
Fig. 1 is the specific detection agarose gel electrophoresis figure of primer of the present invention, and it is with upstream primer and base sequence downstream primer RT-PCR amplification as in sequence table SEQ ID NO:2 shown in the INSV specific nucleic acid fragment that obtain of base sequence shown in SEQ ID NO:1 in the sequence table.M is Marker; 1 and 2 is INSV two reproducible results fragments (being the purpose fragment) behind described upstream and downstream primer PCR.
Fig. 2 is that its relation conefficient is to the amplification curve of the standard substance of the real-time fluorescence quantitative RT-PCR detection of garden balsam necrotic spot virus: 0.997, and pcr amplification efficient is: 100.3%.The standard plasmid that 5 successive concentration differ 10 times from left to right is set, and each corresponding concentration is provided with 3 repetitions.
Fig. 3 is the typical curve to the standard substance of the real-time fluorescence quantitative RT-PCR detection of garden balsam necrotic spot virus, its slope :-3.315, and intercept 49.300.From left to right tiltedly to get off be respectively standard substance plasmid extent of dilution for A, B, C, D, five points of E among the figure: 10
5, 10
4, 10
3, 10
2, 10
1As can be seen: the fluorescence intensity of being gathered reach a preset value the cycle number of process high more, its copy number is low more.
Fig. 4 is the amplification curve that the real-time fluorescence quantitative PCR sensitivity signal of garden balsam necrotic spot virus is detected, curve from left to right, curve 1 is 6.99 * 10
9Copy μ l
-1, curve 2 is 6.99 * 10
8Copy μ l
-1, curve 3 is 6.99 * 10
7Copy μ l
-1, curve 4 is 6.99 * 10
6Copy μ l
-1, curve 5 is 6.99 * 10
5Copy μ l
-1, curve 6 is 6.99 * 10
4Copy μ l
-1, curve 7 is 6.99 * 10
3Copy μ l
-1, curve 8 is 699 copy μ l
-1, curve 9 is 69.9 copy μ l
-1, curve 10 is 6.99 copy μ l
-1, the negative contrast of all the other curves.
Fig. 5 is TaqMan fluorescent quantitative PCR graphic representations of spider orchid and the Sowthistle Tasselflower Herb INSV of agricultural university virus of Chenggong butterfly orchid, Yunan County, and curve 1, curve 6 and curve 9 are 3 repeat amplification protcol curves of INSV virus on agricultural university's Sowthistle Tasselflower Herb among the figure; Curve 2, curve 5 and curve 7 are 3 repeat amplification protcol curves of INSV virus on the butterfly orchid of Chenggong; Curve 3, curve 4 and curve 8 are Yunan County's 3 repeat amplification protcol curves of INSV virus on the spider orchid all.The negative contrast amplification curve of its excess-three bar curve.
Fig. 6 is the specific amplification graphic representation of TaqMan fluorescence quantitative RT-RCR, has only garden balsam necrotic spot virus (Impatiens necrotic spot virus among the figure, INSV) collect the fluorescent signal amplification, and all the other samples: common calla chlorosis spot poison (Calla Lily Chlorotic Spot Virus, CCSV), annulus zonatus (Tomato Zonate Spot Virus, TZSV) and tomato spotted wilf virus (Tomato spotted wilttospovirus TSWV) does not all have obvious fluorescent signal and takes place.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but do not constitute limiting the scope of the invention.
Embodiment 1 is used for garden balsam necrotic spot virus is carried out primer and the design and synthetic thereof that real-time fluorescence quantitative RT-PCR detects.
Described primer is made up of upstream primer and downstream primer, and the base sequence of described upstream primer is shown in SEQ ID NO:1 in the sequence table, and the base sequence of described downstream primer is shown in SEQ ID NO:2 in the sequence table.
Described primer be according to the NSs gene order on the INSV SRNA chain of Genbank report (accession number: NC_003624), the primer that uses DNAman sequence alignment software to select conservative region and the online software Primer 3.0 of design of primers and Primer 5.0 on conservative region, to design.The specificity result that this primer detects INSV positive (through INSV immunity test strip and RT-PCR tests positive) as shown in Figure 1, target stripe is clear bright, and does not have assorted band, illustrates that this is remarkable to INSV cDNA specificity to primer.
Described primer is synthetic by Bo Maide biotech firm.
Described TaqMan probe is according to the NSs gene order (accession number: NC_003624) on the INSV SRNA chain of Genbank report, use DNAman sequence alignment software to select conservative region and the online software Primer 3.0 of design of primers and Primer 5.0 designed probe on conservative region, the base sequence of described TaqMan probe is shown in SEQ ID NO:3 in the sequence table.Described TaqMan probe is between embodiment 1 described upstream primer and downstream primer, 5 ends of described TaqMan probe are marked with the report fluorophor, 3 ends are marked with the cancellation fluorophor, described report fluorophor is FAM, described cancellation fluorophor is TAMRA, and described TaqMan probe believes that by last Hypon biotech firm is synthetic.
Described test kit comprises primer and TaqMan probe; Described primer is embodiment 1 a described primer; Described TaqMan probe is embodiment 2 described TaqMan probes; The design of its primer and probe is undertaken by embodiment 1 and embodiment 2 described methods respectively;
Described test kit also comprises the positive criteria product, and it is from positive colony bacterium liquid and extracts plasmid, with the absorbancy of UV-light spectrophotometric determination D260nm and 280nm, and calculates the plasmid content that extracts.Plasmid with the gradient dilution of ultrapure water with 10 times, is diluted to 10 at last
1, 10
2, 10
3, 10
4, 10
5Times plasmid is made the positive criteria product, and each usage quantity respectively is the every reactions of 2 μ l/.
Test kit also comprises on the market can show the material that available reagent maybe can now be joined: they are INSV immunity test strips; the M-MLV ThermoScript II; 5x M-MLV Buffer; 10Mm dNTP mixture; 50 μ l random primers, six aggressiveness; 40U/ μ l RNA inhibitor; PCR quality water; Illustra RNAspin MiniRNA Isolation Kit test kit; the PCR product of hundred Tyke biotech firms reclaims purification kit; competent cell DH5@; SOC substratum and X-Gal; IPTG; the blue hickie substratum of the LB of Amp; described competent cell DH5@; SOC substratum and X-Gal; IPTG; the blue hickie substratum of the LB of Amp is according to X-Gal in the common agents preparation of TaKaRa company; IPTG; the configuration of the blue hickie culture medium preparation of the LB of Amp method forms; plasmid reclaims purification kit (purchasing in hundred Tyke Bioteke Bioisystech Co., Ltd); super mixed liquid of the Premix Ex TaqTM of TaKaRa company and PMD19 simple-T Vector, the sterilization ultrapure water.
Agents useful for same does not have explanation in following agents useful for same and the test kit, is the commercially available prod.
It is as follows that it detects step:
(1) synthetic following primer and TaqMan probe
Described primer is made up of upstream primer and downstream primer, and the base sequence of described upstream primer is shown in SEQ ID NO:1 in the sequence table, and the base sequence of described downstream primer is shown in SEQ ID NO:2 in the sequence table.
The base sequence of described TaqMan probe is shown in SEQ ID NO:3 in the sequence table, described TaqMan probe is between above-mentioned upstream and downstream primer, 5 ends of described TaqMan probe are marked with the report fluorophor, 3 ends are marked with the cancellation fluorophor, described report fluorophor is FAM, and described cancellation fluorophor is TAMRA.
Described primer is synthetic by Bo Maide biotech firm, and described TaqMan probe believes that by last Hypon biotech firm is synthetic.
(2) extraction of viral RNA and cDNA's is synthetic
1) butterfly orchid body inner virus RNA gathers and extracts: virus collection is in Dou Nan flowers market, Chenggong County, Kunming, Yunnan Province, with the sick leaf of INSV immunity test strip tests positive; It (is Illustra RNAspin Mini RNA Isolation Kit that virus is extracted according to using IllustraRNAspin Mini RNA separating kit, purchase company in GE Healthcare, GE Healthcare company is present General Electric Company, Ltd., former peace agate West Asia bio tech ltd) extract RNA from the sick leaf of butterfly orchid, operation steps is carried out according to the working instructions that this separating kit provided;
2) cDNA's is synthetic: adopt 20 μ l systems, with PCR quality water 3.5 μ l, 5xM-MLVBuffer 2.0 μ l, 10Mm dNTP mixture 2.0 μ l, 50 μ l random primers, six aggressiveness, 2.0 μ l, 40U/ μ l RNA inhibitor 1.0 μ l, M-MLV ThermoScript II 0.5 μ l and RNA 5.0 μ l are positioned over and leave standstill 9 minutes on ice after mixing, after carry out the synthetic cDNA of RT reaction, the RT response procedures: 42 ℃, 30min; 99 ℃, 5min; 5 ℃, 5min; 4 ℃, preserve, synthetic cDNA places-20 ℃ of preservations standby;
(3) foundation of the preparation of standard substance and typical curve
With the cDNA through the INSV sample of INSV immunity test strip and RT-PCR test positive is template, add the described primer of step (1) and carry out PCR, PCR reaction conditions and temperature are provided with as follows: TakaRa ExTaq (5U/ μ l) 0.25 μ l, 10 * Ex Taq Buffer, 5 μ l, dNTP Mixture (each 2.5mM) 4 μ l, template cDNA 2 μ l, upstream primer (20 μ M) 2 μ l, downstream primer (20 μ M) 2 μ l, sterile purified water up to 50 μ l
Reaction conditions:
Reclaim the test kit specification sheets according to the DNA of hundred Imtech (Bioteke) glue and reclaim pcr amplification product, Takara PMD19 simple-T Vector test kit connects, connection procedure is with the description of test book, and by the bacillus coli DH 5 alpha conversion, it is as follows to transform detailed process:
(a) 10 μ l connection product is added in the 100 μ l DH5 α competent cells, places 30 minutes in the ice.
(b) after 42 ℃ of 45 seconds of heating, in ice, placed 1 minute again.
(c) add 890 μ l SOC substratum, 37 ℃ of shaking culture 60 minutes.
(d) the SOC substratum that contains competent cell the LB-agar plate that takes a morsel to X-Gal, IPTG, Amp
Cultivate (37 ℃ on the substratum, 12H～16H. constant temperature culture), from growing the corresponding zone of hickie, tap with toothpick, the back point is put and is carried out in the PCR centrifuge tube that has prepared the PCR reaction solution whether the PCR detection is target fragment length on the LB-Agar Plating of the new X-Gal of another piece, IPTG, Amp.The bacterium liquid of the test positive absorbancy of UV-light spectrophotometric determination D260nm and 280nm, and calculate the plasmid content that extracts.Plasmid with the gradient dilution of ultrapure water with 10 times, is diluted to 10 at last
1, 10
2, 10
3, 10
4, 10
5Times plasmid is made the positive criteria product, and be template with the positive criteria product of making, each corresponding concentration is provided with 3 repetitions, (reaction system sees that specifically step 4) makes corresponding amplification curve and typical curve after real-time fluorescence quantitative PCR reaction, as Fig. 2 and Fig. 3, the amplification relation conefficient is: 0.997, and pcr amplification efficient is: 100.3%.Typical curve, its slope :-3.315, (3.0～-4.0, wherein-3.4 the best) intercept 49.300.
(4) TaqMan fluorescence quantitative RT-RCR reaction system
Adopt 25 μ l reaction systems, Premix Ex TaqTM (2x) 12.5 μ l wherein, each 0.5 μ l of upstream and downstream primer, fluorescent probe 0.25 μ l, dH
2O (aqua sterilisa) 9.25 μ l, cDNA 2 μ l (synthetic in (2) step); All reaction reagents are all in preparation on ice, and all the other operations are carried out according to the specification sheets of the Premix Ex TaqTM of precious biotechnology (Dalian) company limited (TaKaRa company); Real-time fluorescence quantitative PCR response procedures: 94 ℃ of pre-denaturation temperatures, 5min; 58 ℃ of annealing temperatures, 30s; Gather fluorescent signal 15s for 58 ℃.The results are shown in Table 2 and Fig. 5, the result shows that 3 are repeated positive and collect the cycle number that fluorescent signal produces and be respectively: 27.74,27.15 and 27.75, and its viral level is respectively: 8.11 * 10
5Copy μ l
-1, 1.25 * 10
6Copy μ l
-1, 8.08 * 10
5Copy μ l
-1And negative control does not have the generation of fluorescent signal.
Detecting above-mentioned virus with test kit is: at first extract the total RNA of plant from the sick leaf of INSV test positive, virus is extracted the Illustra RNAspin Mini RNA Isolation Kit method of using, operation steps is carried out according to the working instructions that GE Healthcare company provides, and adopts the synthetic method of above-mentioned 20 μ l system cDNA synthetic to the total RNA of the plant of being carried; Use primer and probe in this test kit, and be template with synthetic cDNA, according to the preparation of embodiment 4 described step (3) standard substance, the foundation and the reaction of step (4) TaqMan fluorescence quantitative RT-RCR of typical curve, the final absolute quantitation detection that realizes the content of virus.
Detected sample be pick up from Yunan County, Kunming, Yunnan Province can be all spider orchid (Hymenocallislittoralis Salisb), the real-time fluorescence quantitative RT-PCR detection method is identical with embodiment 4, repeats no more.This experiment is provided with 3 repetitions and 3 negative controls, the results are shown in Table 2 and Fig. 5, and curve 3, curve 4 and curve 8 are all 3 repeat amplification protcol curves of virus on the spider orchid of Yunan County among the figure.The result shows that the cycle number that 3 repetition positive collect the fluorescent signal generation is respectively: 27.91,27.10 and 28.25, and its viral level is respectively: 7.21 * 10
5Copy μ l
-1, 1.30 * 10
6Copy μ l
-1, 5.59 * 10
5Copy μ l
-1And negative control does not have the generation of fluorescent signal.
Detected sample is Sowthistle Tasselflower Herb (Emilia sonchifolia (linn.) DC) plant that INSV is infected in artificial inoculation, this plant is that this seminar cultivates in engineering center of National Laboratory of Yunnan Prov Agriculture University greenhouse, and successfully inoculates with the INSV virus that the method for frictional inoculation will be picked up from Chenggong County, Kunming, Yunnan Province.The real-time fluorescence quantitative RT-PCR detection method is identical with embodiment 4, repeats no more.This experiment is provided with 3 repetitions and 3 negative controls, the results are shown in Table 2 and Fig. 5, and curve 1, curve 6 and curve 9 are 3 repeat amplification protcol curves of virus on agricultural university's Sowthistle Tasselflower Herb among the figure; The result shows that the cycle number that 3 repetition positive collect the fluorescent signal generation is respectively: 27.86,27.05 and 28.75, and its viral level is respectively: 7.45 * 10
5Copy μ l
-1, 1.34 * 10
6Copy μ l
-1, 3.89 * 10
5Copy μ l
-1And negative control does not have the generation of fluorescent signal.
The detection of embodiment 7 specificitys of the present invention and repeatability and sensitivity:
(purchase company according to using Illustra RNAspin Mini RNA Isolation Kit in GE Healthcare, it is present General Electric Company, Ltd., former peace agate West Asia bio tech ltd) extracts RNA, the working instructions that operation steps provides according to GE Healthcare company extract total RNA (finding in Yunnan Province that at present four kinds of tomato spotted wilf viruses belong to virus) from the plant of following four kinds of tomato spotted wilf viruses genus virus infection, they are respectively INSV (picking up from Chenggong County, Kunming, Yunnan Province butterfly orchid), TSWV (deriving from Chenggong County, Kunming, Yunnan Province asparagus lettuce), TZSV (from the Jian Shuishi of Yunnan Province tomato), CCSV (picking up from spider orchid in Yunnan Prov Agriculture University's test farm booth); RNA detects its specificity with real time fluorescent quantitative, result such as Fig. 6 behind the synthetic cDNA of RT reaction.Same INSV positive is provided with 3 repetitions, estimates with real-time fluorescence quantitative PCR, as Fig. 5 and table 2.The gradient dilution of positive plasmid usefulness sterilization ultrapure water, from 10 with 10 times
1Be diluted to 10
20, detect its minimum copy number with real-time fluorescence quantitative PCR, as Fig. 4; The extraction of viral RNA and cDNA's is synthetic in the experimental implementation, and the preparation of standard substance is identical with embodiment 4 with the reaction of TaqMan fluorescence quantitative RT-RCR.
Table 2 Chenggong butterfly orchid, Yunan County are all on spider orchid and the agricultural university Sowthistle Tasselflower Herb plant
INSV viral level detected result
Annotate: repeat 1, repeat 2 and repeat 3 expression cycle number Ct values, the Chenggong butterfly orchid represents to be collected in the butterfly orchid plant in the Dou Nan flowers market, Chenggong, Kunming, Yunnan Province, and detecting through the INSV immunity test strip is the INSV positive; Yunan County all the blue expression of spider pick up from Yunan County, Kunming, Yunnan Province can be all the blue plant of spider,, detect through the INSV immunity test strip and to be the INSV positive; Agricultural university's Sowthistle Tasselflower Herb is represented the Sowthistle Tasselflower Herb plant that this seminar cultivates in engineering center of National Laboratory of Yunnan Prov Agriculture University greenhouse, and inoculation INSV virus, detects through the INSV immunity test strip to be the INSV positive.
Can find clearly that from table 1 three in each sample repeats resulting Ct value difference value 0.01 to 1.7, illustrates reliability of the present invention, good reproducibility; The Ct mean value difference that detection obtains on while three different host illustrates having good stability of this invention 0.25 to 0.34.Fig. 4 shows: the present invention can detect INSV viral level 6.99 copy μ l
-1, show that the present invention has the effect of trace detection; Have only INSV that fluorescent signal is arranged in INSV, TSWV, these four kinds of viruses of TZSV, CCSV among Fig. 6, three kinds of viruses of CCSV, TSWV and TZSV are not all found tangible amplified fluorescence curve, show the high specificity that the present invention detects INSV.
Sequence table
＜110〉Yunnan Prov Agriculture University
＜120〉a kind of real-time fluorescent quantitative RT-PCR method and primer, probe and test kit that detects garden balsam necrotic spot virus
<160>3
<170>PatentIn?version?3.3
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＜213〉garden balsam necrotic spot virus (Impatiens necrotic spot virus)
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ccagaccaac?aacaggacag?ta 22
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<211>20
<212>DNA
<213>Impatiens?necrotic?spot?virus
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<213>Impatiens?necrotic?spot?virus
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tcactggcaa?tgtctgcaac?ttc 23
Claims (10)
1. one kind is used for garden balsam necrotic spot virus is carried out the primer that real-time fluorescence quantitative RT-PCR detects, described primer is made up of upstream primer and downstream primer, the base sequence of described upstream primer is shown in SEQ ID NO:1 in the sequence table, the base sequence of described downstream primer is shown in SEQ ID NO:2 in the sequence table, and the target product fragment length is 182bp.
2. one kind is used for garden balsam necrotic spot virus is carried out the TaqMan probe that real-time fluorescence quantitative RT-PCR detects, the base sequence of described TaqMan probe is shown in SEQ ID NO:3 in the sequence table, 5 ' end at described TaqMan probe is marked with the report fluorophor, 3 ' end is marked with the cancellation fluorophor, and described TaqMan probe is between the upstream primer and downstream primer of the described primer of claim 1.
3. TaqMan probe according to claim 2 is characterized in that: described report fluorophor is FAM, and described cancellation fluorophor is TAMRA.
4. one kind is carried out the test kit that real-time fluorescence quantitative RT-PCR detects to garden balsam necrotic spot virus, comprise primer and TaqMan probe, described primer is the described primer of claim 1, and described TaqMan probe is claim 2 or 3 described TaqMan probes.
5. test kit according to claim 4, comprise the positive criteria product, it is from positive colony bacterium liquid and extracts plasmid, absorbancy with UV-light spectrophotometric determination D260nm and 280nm, and the plasmid content of calculating extraction, plasmid with the gradient dilution of ultrapure water with 10 times, is diluted to 10 at last
1, 10
2, 10
3, 10
4, 10
5Times positive criteria product that plasmid is made, each usage quantity respectively is the every reactions of 2 μ l/.
6. test kit according to claim 5; comprise the garden balsam necrotic spot virus immunity test strip; the M-MLV ThermoScript II; 5x M-MLV Buffer; 10Mm dNTP mixture; 50 μ l random primers, six aggressiveness; 40U/ μ lRNA inhibitor, PCR quality water, Illustra RNAspin Mini RNA Isolation Kit test kit; the PCR product of hundred Tyke biotech firms reclaims purification kit; competent cell DH5@, SOC substratum and X-Gal, IPTG; the blue hickie substratum of the LB of Amp; described competent cell DH5@, SOC substratum and X-Gal, IPTG; the blue hickie substratum of the LB of Amp is according to X-Gal in the common agents preparation of TaKaRa company; IPTG, the configuration of the blue hickie culture medium preparation of the LB of Amp method forms, and plasmid reclaims purification kit; super mixed liquid of the Premix Ex TaqTM of TaKaRa company and PMD19 simple-T Vector, the sterilization ultrapure water.
7. real-time fluorescent quantitative RT-PCR method that detects garden balsam necrotic spot virus, comprise the extraction of viral RNA to be measured and synthesizing of cDNA thereof, TaqMan fluorescence quantitative RT-RCR reaction system, carry out the reaction of TaqMan fluorescence quantitative RT-RCR in TaqMan fluorescence quantitative RT-RCR reaction system, by the preparation of standard substance and the foundation of typical curve, calculate detected result, it is characterized in that: the primer that uses in synthetic and described reaction is the described primer of claim 1, and the TaqMan probe that uses in synthetic and described reaction is claim 2 or 3 described TaqMan probes.
8. real-time fluorescent quantitative RT-PCR method according to claim 7, it is characterized in that: described TaqMan fluorescence quantitative RT-RCR reaction system is to adopt 25 μ l reaction systems, Premix ExTaqTM (2x) 12.5 μ l wherein, each 0.5 μ l of upstream primer and downstream primer, TaqMan probe 0.25 μ l, dH
2O9.25 μ l, cDNA 2 μ l; All reaction reagents are all in preparation on ice; The real-time fluorescence quantitative PCR response procedures is 94 ℃ of pre-denaturation temperatures, 5min; 58 ℃ of annealing temperatures, 30s; Gather fluorescent signal 15s for 58 ℃.
9. the application in detecting garden balsam necrotic spot virus of described TaqMan probe of primer according to claim 1 and claim 2 or the described primer of claim 1 and the described TaqMan probe of claim 3.
10. the application of the real-time fluorescent quantitative RT-PCR method of test kit according to claim 4 or the described detection garden balsam necrotic spot virus of claim 7 in detecting garden balsam necrotic spot virus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105671196A (en) * | 2016-04-19 | 2016-06-15 | 中华人民共和国无锡出入境检验检疫局 | Primer and probe for fluorescence quantitative PCR of phomopsis amydalina TaqMan |
| CN110036776A (en) * | 2019-04-17 | 2019-07-23 | 广西特色作物研究院 | A kind of pair of Citrus Huanglongbing pathogen be resistant or the rapid identification method of the germplasm materials of patience |
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| US20080300210A1 (en) * | 2007-05-16 | 2008-12-04 | Wisconsin Alumni Research Foundation | Method of Controlling Insects and Virus Transmission |
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| EP0426195A1 (en) * | 1989-11-03 | 1991-05-08 | S&G Seeds B.V. | Improvements in or relating to organic compounds |
| US20080300210A1 (en) * | 2007-05-16 | 2008-12-04 | Wisconsin Alumni Research Foundation | Method of Controlling Insects and Virus Transmission |
| CN101307366A (en) * | 2008-07-01 | 2008-11-19 | 云南出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting impatiens necrotic spot virus from oncidium |
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| CN105671196A (en) * | 2016-04-19 | 2016-06-15 | 中华人民共和国无锡出入境检验检疫局 | Primer and probe for fluorescence quantitative PCR of phomopsis amydalina TaqMan |
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| CN110036776A (en) * | 2019-04-17 | 2019-07-23 | 广西特色作物研究院 | A kind of pair of Citrus Huanglongbing pathogen be resistant or the rapid identification method of the germplasm materials of patience |
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