CN102787176A - Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants - Google Patents
Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants Download PDFInfo
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Abstract
The invention provides a method for SRBSV (southern rice black-streaked dwarf virus) detection on rice plants and accurate quantification of RNA (ribose nucleic acid) copy number. The method can be used for eliminating the interference of the SRBSV, the copy number of the virus RNA in the SRBSV plants is fast obtained, and further, the basis is laid for studying the SRBSV pathogenic mechanism, the molecular biology study and the like.
Description
Technical field:
The present invention relates to southern rice black-streaked dwarf virus on the rice plant detection and viral RNA copy number accurately quantitatively, belong to the agricultural cience and farming techniques field.
Background technology:
(Southern rice black-streaked dwarf virus SRBSDV) is the suggestion novel species that Reoviridae (Reoviridae) Fijivirus belongs to (Fijivirus) the 2nd group to southern rice black-streaked dwarf virus.Mainly propagate without the mode of ovum with persistence, can infect paddy rice, corn etc. and cause southern rice black-streaked dwarf disease and maize rough dwarf virus by white backed planthopper (Sogatella furcifera).This virion is spherical, and genome is made up of 10 double-stranded RNAs.Paddy rice all can receive the dip-dye of SRBSDV each breeding time; The strumae that the field symptom shows as mainly that plant stunts, dark green leaf color, blade back and stem stalk have initial stage oyster white, later stage brown; Jointing stage diseased plant stipes portion number joint produces the living fibrous root of the gas that falls to give birth to and Gao Jiewei is tillered, and the host also comprises corn and multiple weeds except that paddy rice.Yield effect to paddy rice is very big, grave illness field even can cause not having output.Should disease spread generation in recent years in the THE LOWER YANGTZE VALLEY area; And be day by day serious trend; Area takes place and reaches 3,000,000 mu in the southern rice black-streaked dwarf virus whole nation in 2009, rises to 1,900 ten thousand mu rapidly in 2010, and only the Hunan Province just takes place nearly 1,000 ten thousand mu; 80,000 mu of total crop failure areas have brought tremendous loss to paddy rice production.Press for and set up reliable, accurate test method.
At present, the detection method that plant virus is commonly used has: the biology inoculation experiments, and serology detects, and electron microscopic observation and molecular method detect.But because SRBSDV and rice black-streaked dwarf virus (Rice black-streaked dwarf virus; RBSDV) the genome sequence homology is high; Many-sided characteristics such as symptom, virion form, size and serology are closely similar in the field, thus caused difficulty to its diagnosis and evaluation, and also there are defectives such as length consuming time, complicated operation and cost height in traditional method; The susceptibility of PCR equimolecular method is high; But can only carry out sxemiquantitative or draw the conclusion of " have or do not have ", can not be accurately quantitative viral RNA copy number, make the fundamental research etc. of pathogenesis and SRBSDV receive certain restriction.
Real time RT-PCR is a kind of method of simple and effective detection gene copy number; Realized the leap of PCR from qualitative to quantitative; Can accurately judge the copy number of viral RNA, and have advantages such as consuming time few, susceptibility is high, easy and simple to handle, safety economy.SYBR Green I is a kind of double-stranded DNA dyestuff, combines the back fluorescence intensity obviously to strengthen with double-stranded DNA, and in amplification system, adding this dyestuff can real time direct detect amplified production.This dyestuff does not have specificity, can not discern specific two strands, and different templates is not needed special customization, does not need the good probe of designs specificity, and versatility is relatively good.Can come right area branch specificity and non-specific through the melting curve of measuring amplified production.After utilizing the standard substance of known initial copy number to make typical curve, as long as obtain cycle threshold (threshold cycle, the C of unknown sample
TValue), can calculate the initial copy number of this sample from typical curve.Have the incomparable advantage of conventional round pcr: high specificity, accomplish the discriminating of template through the specific hybrid of Auele Specific Primer or probe and target gene, accuracy is very high, is not prone to non-specific products such as dimer; Sensitivity is higher; The amount that shows amplified production with the indicator that can produce fluorescent signal; Fluorescent signal embeds double-stranded DNA through optical dye, or methods acquisitions such as the sequence-specific fluorescent probe of double-tagging or energy signal transfer probe, has greatly improved the sensitivity that detects; Particularity is good, C that can the utilization index phase
TValue accurate quantification starting template amount can also detect single copy gene; Fast, easy, amplification and detect and in same pipe, to accomplish need not uncapped and carried out electrophoresis detection, has significantly reduced operation, has shortened the time, has avoided crossed contamination, environmental pollution, has solved the PCR pollution problem effectively; Level of automation is high, and spectroscopic techniques is used in combination in computer technology, and amplification and detection are synchronous completion, detect in real time through computingmachine, and the os sealing.The real-time fluorescence quantitative PCR technology is more and more widely the every field that is applied to scientific research.
Summary of the invention:
The invention provides a kind of method that detects southern rice black-streaked dwarf virus in the also accurately quantitative paddy rice diseased plant; Can get rid of the interference of rice black-streaked dwarf virus through this method, quick diagnosis goes out the copy number that whether carries southern rice black-streaked dwarf virus in the rice plant and obtain viral RNA.
The method of southern rice black-streaked dwarf virus in a kind of rapid detection provided by the present invention and the gauge water rice plants obtains through following method:
1) the southern rice black-streaked dwarf virus S9 nucleotide sequence of having reported according to NCBI; Analyze the back through software DNAstar and select relative conservative region (especially comparing) with RBSDV; Utilize corresponding zone on the EU523359.1 through Primer5 design primer, select best 2 primer SRBSDV-S9-F and SRBSDV-S9-R;
2)
Reagent (Invitrogen) extracts the total RNA of paddy rice diseased plant;
3) be template with the total RNA of paddy rice diseased plant that extracts; SRBSDV-S9-F and SRBSDV-S9-R are that primer carries out RT-PCR acquisition goal gene; SRBSDV S9 purpose fragment (141bp) is reclaimed in rubber tapping, connects pGEM-T easy and makes up cloning vector, will connect product transformed into escherichia coli (Escherichia coli) competent cell DH5 α again; Screening, evaluation obtain positive colony; Extract and the purification of Recombinant plasmid, identify recombinant plasmid once more, so far obtain to contain the segmental recombinant plasmid of purpose through RT-PCR and BlAST analysis.
4) with restriction enzyme Sal I positive recombinant plasmid dna is carried out single endonuclease digestion; Reclaim purifying and obtain the wire DNA; To contain the segmental wire DNA of purpose is template; Carry out in-vitro transcription according to the explanation of T7Transcription Kit (Fermentas) and obtain wire RNA, the RNA according to the explanation purifying in-vitro transcription of in-vitro transcription purification kit EZ-10Spin Column5 Minutes RNA Cleanup&Concentration Kit obtains the RNA standard substance;
5) reference reagent box iScript
TMThe requirement of One-Step RT-PCR Kit With SYBR Green (BIO-RAD); To obtain the RNA standard substance is template; Concentration to primer SRBSDV-S9-F and SRBSDV-S9-R is optimized with 5 * 5 matrix forms (upstream and downstream primer final concentration is respectively 100nM, 200nM, 300nM, 400nM and 500nM), confirms the optimum response system; In 55-65 ℃ of scope, annealing-extension is optimized, confirms optimum reaction condition, result data is analyzed through software I Q5 Optical System Software Version 2.0;
6) base of calculation article RNA copy number carries out 10 times of gradient dilutions (2.5 * 10 with Nuclease-free water
10-2.5 * 10
4Copies/ μ L), obtain SYBR Green I-based one-step real time RT-PCR optimum response system and condition is carried out amplified reaction, to obtain C separately with optimizing
TValue utilizes the accompanying software analysis to obtain quantitative curve and typical curve, and the logarithmic value of original template copy number is the C of X-coordinate (x axle), correspondence in the typical curve
TValue is ordinate zou (y axle);
7) after Real time RT-PCR amplification finishes,, whenever measure the light absorption value of amplified production, utilize the accompanying software analysis to obtain melting curve, judge the specificity of this method at a distance from 0.5 ℃ (80 circulations) from 55 ℃ to 95 ℃.
8) compare the susceptibility of checking SYBR Green I-based one-step real time RT-PCR with the RT-PCR method.
Primer sequence provided by the invention is following:
SRBSDV-S9-F GAGACCCACCTCCACTGATT
SRBSDV-S9-R ACGTTTACCACTGCGCCTTC
Expanding effect was best when the concentration of primer SRBSDV-S9-F and SRBSDV-S9-R all was 300nM in the SYBR Green I-based one-step real time RT-PCR reaction system, annealing-elongating temperature be 60.0 ℃ optimum.According to the typical curve of setting up can accurately quantitative field paddy rice diseased plant sample in SRBSDV RNA copy number, can judge the specificity of this method through the solubility curve of analysing amplified product.
A kind of detect and quantitatively in the plant application of southern rice black-streaked dwarf virus RNA copy number method comprise: in the quick diagnosis of doubtful diseased plants such as paddy rice and corn with quantitative.
Utilize this method can obtain SRBSDVRNA copy number in the southern rice black-streaked dwarf virus plant sample fast, for the pathogenesis of this virus disease and the fundamental research of SRBSDV etc. provide support.
Description of drawings:
(1, SRBSDV-S9-F and SRBSDV-S9-R final concentration are 300nM to the optimization of Fig. 1 primer concentration; 2, SRBSDV-S9-F final concentration 400nM and SRBSDV-S9-R final concentration 300nM; 3, SRBSDV-S9-F final concentration 500nM and SRBSDV-S9-R final concentration 300nM).
Fig. 2 thermograde is optimized (1,56.4 ℃; 2,60.0 ℃; 3,57.9 ℃; 4,55.4 ℃; 5,63.1 ℃; 6,65.3 ℃).
0 times of serial dilution RNA of Figure 31 standard substance fluorescent quantitation curve (is followed successively by 2.5 * 10 from left to right
10, 2.5 * 10
9, 2.5 * 10
8, 2.5 * 10
7, 2.5 * 10
6, 2.5 * 10
5, 2.5 * 10
4The RNA standard substance of copies/ μ L).
The typical curve (2.5 * 10 that Fig. 4 obtains with 10 times of serial gradient dilution RNA standard substance
10Copies/ μ L-2.5 * 10
4Copies/ μ L).
The melting curve of 0 times of serial dilution RNA of Figure 51 standard substance.
The practical implementation method:
Embodiment 1, and in-vitro transcription and purifying prepare the RNA standard substance
Select suitable restriction enzyme Sal I, add reagent successively according to table 1,37 ℃ of 8h carry out single endonuclease digestion to positive recombinant plasmid dna.
Table 1 restriction endonuclease analysis system
Table1?The?system?of?restriction?enzyme?analysis
The single endonuclease digestion product is through 2% agarose gel electrophoresis, reclaims according to the recovery purifying specification sheets of AxyPrep DNA Gel Extration Kit (Axygen) and obtains the wire DNA.
With the wire DNA is template, and SRBSDV-S9-F and SRBSDV-S9-R are that primer carries out PCR evaluation wire DNA, conventional PCR system (25 μ L); Amplification condition: at first,, then under following condition, carry out 35 circulations: 95 ℃ of sex change 45s at 95 ℃ of preparatory sex change 5min; 57-60 ℃ of annealing 45s, 72 ℃ are extended 30s, in the end after loop ends; 72 ℃ of insulation 10min get 2 μ L PCR products, 2% agarose gel electrophoresis and detect.
To contain the segmental wire DNA of purpose is template, carries out in-vitro transcription according to the explanation of T7 Transcription Kit (Fermentas) and obtains wire RNA.
(1) add reagent successively according to table 2 order, mixing is fully done centrifugally slightly, and 37 ℃ of 2h are transcription product;
Table 2 in-vitro transcription
Table2?In?vitro?transcription
(2) in transcription product RNA, add 2 μ L do not have the RNA enzyme DNase I (2U/ μ L, Fermentas), mixing, 37 ℃ of 15min, the DNA that is not transcribed in the digestion transcription product;
(3) add 2 μ L EDTA (50mM), mixing, the activity of 65 ℃ of effect 10min deactivation DNase I.
RNA according to the explanation purifying in-vitro transcription of in-vitro transcription purification kit EZ-10 Spin Column5 Minutes RNA Cleanup&Concentration Kit obtains the RNA standard substance.
(1) 500 μ L Solution A is added in the 55 μ L RNA solution of in-vitro transcription acquisition mixing;
(2) mixture is transferred to RNA preparation pipe (placing on the 2mL centrifuge tube), under the room temperature, 12, the centrifugal 30s of 000rpm abandons supernatant;
(3) add 700 μ L RNA Wash Sloution, under the room temperature, 12, the centrifugal 30s of 000rpm abandons liquid;
(4) under the room temperature, the centrifugal 2min of 10000rpm removes residual RNAWash Sloution;
(5) will prepare pipe and transfer in the new RNase-free centrifuge tube, and add 60 μ L RNA Elution Buffer, room temperature leaves standstill 2min;
The centrifugal 1min of (6) 12,000rpm, what obtain is exactly the RNA standard substance, be kept at-70 ℃ subsequent use.
With the concentration and the OD value of Eppendorf BioPhotometer Plus nucleic acid-protein determinator mensuration RNA standard substance, calculate copy number (Santhosh et al., 2007) according to following formula.The standard rna of transcribing acquisition is done 10 times of gradient dilutions with the 0.1%DEPC treating water, be used for the foundation of susceptibility mensuration and typical curve.
The optimization of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR reaction system and reaction conditions
In order to obtain optimum response system and parameter, according to test kit iScript
TMThe requirement of One-Step RT-PCR Kit With SYBR Green (BIO-RAD) is carried out, and concrete steps are following:
(1) gets the quantitative fluorescent PCR dedicated pipe, add the reagent in the table 3, gently mixing;
(2) seal light-transmissive film, with scraper plate that mould is tight;
The reaction system of table 3 real-time fluorescence quantitative RT-PCR
Table3?The?system?of?real?time?RT-PCR
(3) at Bio-Rad IQ
TMCarry out amplified reaction on the 5 Multicolor Real-Time PCR Detection System.At first, 50 ℃ of 10min synthesize cDNA, the activity of 95 ℃ of 5min deactivation ThermoScript II; Then carry out 40 PCR circulations: 95 ℃ of 10s, 60 ℃ of 10s (collecting signal this moment); 95 ℃ of 1min, 55 ℃ of 1min carry out 80 circulations at 55-95 ℃ then, the 0.5 ℃ of circulation in every interval, be 10s a cycling time;
(4) after reaction is accomplished, through software I Q5 Optical System Software Version 2.0 analytical data.
Annealing-elongating temperature is optimized, in 55-65 ℃ of scope, annealing-extension is optimized.
Primer concentration is optimized, under identical standard article RNA template concentrations, the upstream and downstream primer concentration is optimized with 5 * 5 matrix forms (upstream and downstream primer final concentration is respectively 100nM, 200nM, 300nM, 400nM and 500nM).
The amplification curve of primer concentration optimization experiment such as Fig. 1 can find out that the concentration of upstream primer SRBSDV-S9-F is respectively 300nM, 400nM, 500nM, the C of quantitative curve when downstream primer SRBSDV-S9-R concentration is 300nM
TValue and fluorescent value are more suitable, and amplification was best when wherein primer concentration all was 300nM.
Annealing-elongating temperature optimization experiment result shows (Fig. 2), annealing-elongating temperature when 56.4 ℃, 57.9 ℃ and 60.0 ℃, the quantitative C of curve
TBe worth basic identical and less, the time that reaches plateau early and also fluorescent value higher relatively, in view of low excessively annealing temperature is prone to produce the non-specific amplification product, thus annealing-elongating temperature be 60.0 ℃ optimum.
The 20 μ L reaction systems that obtain through optimization are: 2 * SYBR Green RT-PCR reaction mix, 10 μ L; Each 0.6 μ L (10 μ M) of SRBSDV-S9-F and SRBSDV-S9-R; Iscript reverse transcriptase for one-step RT-PCR 0.4 μ L; Template ribonucleic acid 2 μ L finally supply 20 μ L by nuclease-free water.Optimum reaction condition is 50 ℃ of reverse transcription 10min; 95 ℃ of 5min deactivation ThermoScript II; Then get into 40 circulations: 95 ℃ of 10s, 60 ℃ of 30s; 95 ℃ of 1min then, 55 ℃ of 1min, then 55-95 ℃ of every 10s raises 0.5 ℃, obtains the melting curve of amplified production.
Embodiment 3, the foundation of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR typical curve
Press the copy number that calculates among the embodiment 2, standard substance RNA is carried out 10 times of gradient dilutions (2.5 * 10 with Nuclease-free water
10-2.5 * 10
4Copies/ μ L), be that template obtains the optimum response system with embodiment 2 optimizations and condition is reacted with these group standard substance, obtain C separately
TValue utilizes the accompanying software analysis to obtain amplification curve and typical curve, and the logarithmic value of original template copy number is the C of X-coordinate (x axle), correspondence in the typical curve
TValue is ordinate zou (y axle).The result shows that the RNA standard substance of each dilution gradient measure a basic light absorption value after first amplification cycles finishes, and next considerable change does not all take place when the 4th loop finishes.Since the 5th circulation 2.5 * 10
10The light absorption value of copies/ μ LRNA standard substance begins to increase, and quantitatively curve begins to come back, the 7th circulation (C
TValue) the quantitative curve in back raises up rapidly, to the 25th circulation, reaches peak value, gets into plateau subsequently, until reaction finishes, quantitative curve is typical S type.2.5 * 10
9-2.5 * 10
4The variation tendency of the quantitative curve of copies/ μ L RNA standard substance is identical therewith, just position (the C of curve new line
TValue) reduces and wrong successively 3 to 4 circulations in back their C with concentration
TValue is followed successively by 7,10.5,14,18,21,24,27, and it is identical to be shown as one group of slope, the parallel curves (like Fig. 3) that spacing equates.
Typical curve shows the C of (Fig. 4) all RNA standard substance
TValue has no standard substance big deviation to occur basically point-blank.The amplification efficiency E=100.2% of typical curve among this figure; The coefficient of determination (R
2) greater than 0.98, be 0.996, slope is-3.317, calculation formula is y=-3.317x+30.659, can calculate the accurate copy number that obtains SRBSDV RNA in the unknown sample through this formula.
Embodiment 4, the mensuration of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR melting curve
Press the melting curve of the condition acquisition amplified production among the embodiment 2; Fig. 5 shows the melting temperature (Tm) Tm of amplified production at 78 ℃, and is unimodal, the peak is sharp and narrow, explains that set primer specificity is good; Amplified production is single, does not have the generation of primer dimer and non-specific amplification product.
Above-mentioned enforcement does not limit the present invention in any form.
Claims (1)
1. one kind is accurately detected and the quantitative method of southern rice black-streaked dwarf virus on the plant; It is characterized in that: the Auele Specific Primer that utilizes SYBR Green I-based one-step real time RT-PCR reaction; Paddy rice virus total RNA to extract is that template is carried out amplified reaction; After accomplishing, reaction passes through the software analysis data; This method can be got rid of the interference of black streaked dwarf virus of rice, accurately identifies the virus in the southern rice black-streaked dwarf virus plant, and obtains the accurate copy number of SRBSDV RNA simultaneously.
Above-mentioned " Auele Specific Primer of SYBR Green I-based one-step real time RT-PCR reaction " is meant following 2 primers:
SRBSDV-S9-F GAGACCCACCTCCACTGATT
SRBSDV-S9-R ACGTTTACCACTGCGCCTTC?。
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Cited By (3)
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| CN102952903A (en) * | 2012-12-14 | 2013-03-06 | 湖南省植物保护研究所 | Primer and reagent kit capable of being used for detecting southern rice black-streaked dwarf virus (SRBSDV) as well as quantitative detection method of SRBSDV |
| CN104830998A (en) * | 2015-05-20 | 2015-08-12 | 宋立胜 | Molecular identification method for resistance of rice black streaked dwarf virus (RBSDV) |
| CN108753928A (en) * | 2018-06-13 | 2018-11-06 | 江苏省农业科学院 | Rice black-streaked dwarf virus in plant is quickly detected using RT-RPA methods |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952903A (en) * | 2012-12-14 | 2013-03-06 | 湖南省植物保护研究所 | Primer and reagent kit capable of being used for detecting southern rice black-streaked dwarf virus (SRBSDV) as well as quantitative detection method of SRBSDV |
| CN102952903B (en) * | 2012-12-14 | 2014-04-16 | 湖南省植物保护研究所 | Primer and reagent kit capable of being used for detecting southern rice black-streaked dwarf virus (SRBSDV) as well as quantitative detection method of SRBSDV |
| CN104830998A (en) * | 2015-05-20 | 2015-08-12 | 宋立胜 | Molecular identification method for resistance of rice black streaked dwarf virus (RBSDV) |
| CN108753928A (en) * | 2018-06-13 | 2018-11-06 | 江苏省农业科学院 | Rice black-streaked dwarf virus in plant is quickly detected using RT-RPA methods |
| CN108753928B (en) * | 2018-06-13 | 2021-07-23 | 江苏省农业科学院 | Rapid detection of rice black-streaked dwarf virus in plants by RT-RPA method |
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Application publication date: 20121121 |