CN102618667B - Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction) - Google Patents
Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention discloses a method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction). The method comprises the following steps: 1), synthesizing specific primers corresponding to a potato virus X--PVX, a potato virus Y--PVY, a potato leaf roll virus--PLRV, a potato virus A--PVA and a potato virus S--PVS respectively; 2), extracting the total RNA of a potato sample, and conducting reverse transcription on the total RNA to synthesize a cDNA; and 3), placing all the specific primers in a polyfunctional fluorescent quantitation (PCR) PCR reaction system, carrying out PCR amplification by taking the cDNA obtained in the step 2 as a template, and analyzing whether the potato sample carries at least one of the PVX, the PVY, the PLRV, the PVA and the PVS or not.
Description
Technical field
The invention belongs to plant virus detection technique field, be specifically related to a kind of method that multiple real time fluorescence PCR detects five kinds of potato viruses simultaneously.
Background technology
Potato is a kind of important worldwide food crop, and potato virus disease is a very important class disease on potato, is distributed in each potato planting district, the world, and the some of them potato virus can seriously influence output and the quality of potato.In China, main potato virus kind has potato virus X (Potato virus X, PVX), marmor upsilon (Potato virus Y, PVY), corium solani (Potato virus, PLRV), marmor solani (Potato virus, PVA) and potato virus S (Potato virus, PVS).Virus to the Influence of production maximum is PVY and PLRV, is secondly PVX and PVA, and the PVS impact is less.These viruses have a strong impact on output and the quality of potato, the general underproduction 10%～30%, and severe one reaches more than 80%.Except potato, other crops of Solanaceae as: tomato, green pepper etc. also can be infected by potato virus, and Potato viruses detection and control have caused very big attention, and exploitation is fast, sensitive, normalized detection system is the inevitable direction that virus detects development.
The at present both at home and abroad research of potato molecular detection technology relates to take protein as detection (or serological test) method on basis and take the detection method of nucleic acid as the basis more, and is more quick, sensitive than traditional detection method, accurate.Because potato tends to be infected simultaneously by several viruses, Multiple detection is even more important in the potato virus disease prevention and control.The double PCR method of utilizing Singh etc. has detected PLRV and the PVY in the potato simultaneously.Yuan Qing etc. utilize triple RT-PCR that the potato leaf, leaf stalk, stem and the potato piece that infect PVX, PVS, PVA and Combined Infection are tested, and test result shows, the method can detect the sample of the above virus of multiplicity of infection.
Real-time fluorescence quantitative PCR is that a kind of real-time quantitative that developed recently gets up detects the specific nucleic acid technology, compare with regular-PCR, have the characteristics such as quantitatively accurate, quick, highly sensitive, high specificity, it has been widely used in the every field of life science.Two large class fluorescence mode are arranged in principle: a class is the fluorescent hybridization probe, as Taqman, molecular beacon etc., based on the principle (FRET) of resonance energy transfer; Another kind of is DNA binding dye, is mainly Sybr Green I.A kind of method specificity in front is good, except needs composition sequence special primer, also needs synthetic expensive fluorescent probe, and cost is higher; Rear a kind of method economy only needs synthetic Auele Specific Primer, but is subject to the impact of primer dimer, and specificity is relatively high less than probe method.
The multiple real time fluorescence round pcr is approved widely because having efficient quick, can reduce experimental cost, accelerate the advantages such as experiment process.Due to the restriction of instrument, utilize probe method can only detect at most at present four kinds of amplified productions in a reaction system.Agindotan etc. utilize the Taqman probe to detect simultaneously PLRV, PVA, PVX and 4 kinds of viruses of PVY, but because the probe cost is higher, probe method also is not suitable for widespread use.The multiple real time fluorescence dyestuff round pcr that grows up at present can in same reaction system, be distinguished different nucleic acid fragments by the analysis of Tm value.Wang Xiaowu etc. utilize SYBR Premix ExTaq to detect simultaneously pig reproduction and breath syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV), porcine circovirus 2 type (porcine circovirus type 2, PCV-2) and porcine pseudorabies virus (porcine pseudorabies virus, PRV) 3 kinds of viruses, Chassagne etc. utilize Sybr Green can detect simultaneously colibacillary stx1 in a reaction system, these three kinds of genes of stx2 and eae.
In multiple real time fluorescence dyestuff PCR virus detects, detect simultaneously multiple virus, need designed primer amplification term harmonization, and amplified fragments Tm value is wanted to distinguish and is big or smallly made it present different peaks in a melting curve.Because dyestuff is tested specificity except also affecting with the primer dimer combination with the purpose fragment, increase difficulty to design of primers, and Sybr Green I commonly used has the defective of " dyestuff heavily distributes " that pcr amplification is had restraining effect and there is preferences in amplified production, after pcr amplification in melting curve analysis, realize that the differentiation at melting peak of many amplified productions is very difficult often.Just because of above-mentioned all restrictions, a kind of virus of every increase in one-time detection, just increased other difficulty of level on design of primers and amplification condition, this is also that the fluorescence dye multiplex PCR detection virus of reporting at present can only detect the reason of three kinds of viruses at most.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method that multiple real time fluorescence PCR detects five kinds of potato viruses simultaneously, and the method can be quick, reliable, highly sensitive, high specificity and low-cost these the five kinds of potato viruses of potato virus X PVX, marmor upsilon PVY, corium solani PLRV, marmor solani PVA and potato virus S PVS that detect simultaneously.
In order to solve the problems of the technologies described above, the invention provides a kind of method that multiple real time fluorescence PCR detects five kinds of potato viruses simultaneously, comprise the following steps:
1), synthesize respectively potato virus X--PVX, marmor upsilon--PVY, corium solani--the corresponding Auele Specific Primer of PLRV, marmor solani--PVA and potato virus S--PVS;
2), total RNA of extracting potato sample, cDNA is synthesized in reverse transcription;
3), with step 1) described all Auele Specific Primers are placed in the multiple fluorescence quantitative PCR reaction system, take step 2) described cDNA is template, carries out pcr amplification; Analyze the potato sample according to melting curve and whether carry at least a in PVX, PVY, PLRV, PVA and PVS.
Detect simultaneously the improvement of the method for five kinds of potato viruses as multiple real time fluorescence PCR of the present invention:
Described PVX amplimer is to being:
Forward: CACAGGCTGCTTGGGACTT,
Oppositely: TGCCGTTGGAATAGGGAC;
Amplified fragments Tm value is 83.9;
Described PVY amplimer is to being:
Forward: CTGAGATGCCAACTGTGATGA,
Oppositely: GTTGACATTTGGCGAGGTT;
Amplified fragments Tm value is 80.6;
Described PLRV amplimer is to being:
Forward: CGCCGAAGACGCAGAAGAGGA,
Oppositely: AGACTCGGCCCGAAGGTGAA;
Amplified fragments Tm value is 89.1;
Described PVA amplimer is to being:
Forward: CCAATGCTCAAAGGTAAG,
Oppositely: TCTACTTGCTTTGGTTTGT;
Amplified fragments Tm value is 78.2;
Described PVS amplimer is to being:
Forward: GAGATAGGTAGGCCCTCACTT,
Oppositely: CTGTGCCATTTGCTCGGT;
Amplified fragments Tm value is 86.8.
Above-mentioned fragment Tm is the concluding data of gained after amplification, is to set by designing corresponding primer in advance.
Detect simultaneously the further improvements in methods of five kinds of potato viruses as multiple real time fluorescence PCR of the present invention: step 3) the multiple fluorescence quantitative PCR reaction system consists of the following composition: 10 * PCR buffer, 5 μ L, 25mM MgCl
25 μ L, dNTP (10mM) 1.5 μ L, 20 * EvaGreen, 2.5 μ L, Taq DNA Polymerase 2.5ul, 2.5 μ L step 2) described cDNA, PVX amplimer to 0.2 μ L, PVY amplimer to 0.4 μ L, PLRV amplimer to 0.28 μ L, PVA amplimer to 0.25 μ L and PVS amplimer to 0.23 μ L, use dd H
2O is settled to 50 μ L;
The multiple fluorescence PCR response procedures is: 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s; The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
In the present invention, analyze the potato sample according to melting curve and whether carry at least a, specific as follows in PVX, PVY, PLRV, PVA and PVS: the peak shown according to melting curve, 83.9 peak the Tm value appears is, PVX virus has been described; Appearance Tm value is 80.6 peak, and PVY virus has been described; Appearance Tm value is 89.1 peak, and PLRV virus has been described; Appearance Tm value is 78.2 peak, and PVA virus has been described; Appearance Tm value is 86.8 peak, and PVS virus has been described.
Technical scheme of the present invention is specific as follows:
The first step, PCR design of primers
The present registered potato virus PVX of search, PVY, PLRV, PVA and all the sequencing results of PVS from NCBI are used primer-design software and are designed respectively primer.The design primer is by constantly adjusting, and the primer of five kinds of viral TM values can be effectively distinguished in selection, notes dimeric problem between amplified fragments length and primer self and primer in the time of the design primer.
Designed PCR primer in table 1, the present invention
Second step: a small amount of extracting of the total RNA of sample (being routine techniques)
The total RNA of potato leaf sample extracts: 500mg blade lyophilized powder (liquid nitrogen grinding) is placed in the 5ml centrifuge tube, adds immediately 5ml Trizol, 5min is placed in homogenate on ice.Add 200ul chloroform/ml Trizol (that is, the ratio according to the corresponding 200ul chloroform of every ml Trizol adds chloroform).Concuss 30s, be put in 5～10min on ice, after layering 4 ℃, 12000r/min. centrifugal 20min, get supernatant liquor to another 5ml centrifuge tube, add isopyknic Virahol (approximately 0.5ml) mixing gently, place 2h or-80 ℃ of placement 1h for-20 ℃, 4 ℃ of centrifugal 20min of 12000r/min abandon supernatant, add 1ml 75% (volumetric concentration) washing with alcohol, mixing gently, 4 ℃ of centrifugal 10min of 12000r/min blot ethanol with the rifle head, vacuum-drying 5-10min (thereby make not can overdrying), the RNA of gained is dissolved in 25 μ L DEPC. aqua sterilisas.
The total RNA of potato tuber sample extracts: (1) gets fresh tuber 500mg, adding liquid nitrogen is ground to Powdered, move in the 5mL centrifuge tube, add RNA Extraction buffer [0.25mol/L NaCl, the 0.05mol/L Tris-HCl of 750 μ L precoolings, 20mmol/L EDTA, 1% (w/v) SDS, 1% (w/v) PVP, pH7.5] and chloroform/primary isoamyl alcohol of 750 μ L (24: 1, v/v), fully vortex vibration.In 25 ℃, the centrifugal 2min of 13000r/min.
The preparation method of this RNA Extraction buffer is specially:
Contain SDS, the PVP of 10g of EDTA, 10g of Tris-HCl, 20mmol of NaCl, the 0.05mol of 0.25mol in every liter of RNA Extraction buffer base fluid, all the other are distilled water; Then be the NaOH of 0.5mM or the pH to 7.5 that HCl solution is regulated RNA Extraction buffer base fluid with concentration; Get the RNA Extraction buffer.
Shift in the new 1.5mL centrifuge tube of supernatant to, add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1, v/v), put upside down mixing.In 25 ℃, the centrifugal 2min of 13000r/min.Again shift the supernatant phase, repeat above-mentioned steps, until can be observed a clean middle layer.
Extracting supernatant is for several times gathered mutually be transferred in a new 5mL centrifuge tube.Add the 3mol/LNaAc (pH5.2) of 1/10 volume and the precooling dehydrated alcohol of 2.5 times of volumes, after putting upside down mixing, 4 ℃ of standing 30min.In 4 ℃, the 13 centrifugal 20min of 000r/min.After 75% (v/v) washing with alcohol precipitation with precooling, centrifuge tube is placed in Bechtop allows residual ethanol be evaporated completely to the greatest extent, add 0.1% (w/v, i.e. 1g/ liter) DEPC aqua sterilisa dissolution precipitation of people 40 μ L.After precipitation was dissolved fully, adding 10mol/L LiCt was 2mol/L to final concentration, ice bath 1h.4 ℃, the centrifugal 20min of 18000r/min.After 75% (v/v) washing with alcohol precipitation with precooling, centrifuge tube is placed in Bechtop allows residual ethanol be evaporated completely to the greatest extent, add 0.1% (w/v) DEPC aqua sterilisa dissolution precipitation of 25 μ L ,-80 ℃ of preservations are stand-by.
The 3rd step: cDNA is synthesized in reverse transcription
CDNA synthetic system (total reaction volume 20 μ L): total RNA2.0 μ L of above-mentioned second step gained, random primer (50 μ M) 1 μ L, 5 * RT Buffer, 4 μ L, dNTP (10mM) 1 μ L, Rnase Inhibior 0.5 μ L, RNase 1 μ L, DEPC H
2O10.5 μ L.Reaction conditions is 42 ℃ and keeps 1h.
The 4th step: multiple fluorescence quantitative PCR
Multiple fluorescence quantitative PCR reaction system (total reaction volume 50 μ L): 10 * PCR buffer, 5 μ L, 25mM MgCl
25 μ L, dNTP (10mM) 1.5 μ L, 20 * EvaGreen, 2.5 μ L, Taq DNA Polymerase 2.5ul, 2.5 the cDNA that μ L the 3rd step is synthetic and the viral primer in the first step (PVX wherein, PVY, PLRV, each 0.2,0.4,0.28,0.25 and 0.23 μ L of PVA and PVS upstream and downstream primer) supply dd H
2O to 50 μ L.Reaction is carried out on ABI7000 fluorescent quantitation instrument, and amplification program is 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s.The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
The 5th step: detected result is judged
Determine the Tm value of every kind of virus after the software analysis melting curve.
The present invention overcomes the defective of existing multi-fluorescence dyestuff round pcr, and for the multiple disposable test problems that mainly infects virus of China potato, the present invention designs 5 pairs of Auele Specific Primers, make these 5 kinds of amplified productions between the two the Tm value can obviously distinguish; The present invention designs identical annealing temperature and amplified production clip size between 60-200bp.Multi-fluorescence dyestuff PCR reaction system and the amplification system set up according to the present invention, can realize that first order fluorescence PCR reaction detects 5 kinds of potato viruses simultaneously, be potato virus X PVX, marmor upsilon PVY, corium solani PLRV, the detection effect of marmor solani PVA and potato virus S PVS.
Adopt technique scheme, outstanding technical progress of the present invention is:
(1) designed the primer of the multiple real time fluorescence PCR detection that detects simultaneously five kinds of important viruses of potato, EvaGreen multiple real time fluorescence PCR system, do not use probe, overcome the shortcomings such as the strong or repeatability of the not high or specificity of existing molecular detecting method complicated operation or detection sensitivity is bad.
(2) multiple real time fluorescence PCR detection method of the present invention can detect five kinds of important virus diseases on potato simultaneously, still belonging to precedent in the multiple real time fluorescence PCR method at home and abroad, is also the maximum different amplified productions that utilize at present multi-fluorescence dyestuff PCR method can detect in same reaction tubes.
(3) the present invention adopts a kind of novel fluorescence dyestuff EvaGreen, EvaGreen has eliminated the defective of " dyestuff heavily distributes ", intelligentized " on request discharge " DNA combination technology make its to the inhibition of PCR much smaller than Sybr Green I, also without the dye migration phenomenon between amplified production, can better compatible multiplex PCR.First Application EvaGreen real-time fluorescence quantitative PCR of the present invention detects in a reaction system by melting curve distinguishes five kinds of viruses.Set up a kind of molecular detection technology of multiple fluorescence PCR fast and accurately platform, not only the development of new detection method had important theory significance, and to promoting the cause of disease detection level, prevent that dangerous pathogenic micro-organism from importing China into and having important practice significance and application prospect.
In sum, the present invention utilizes fluorescent quantitative PCR technique, a multiple fluorescence PCR reaction detects potato virus X (Potato virus X, PVX), marmor upsilon (Potato virus Y simultaneously, PVY), corium solani (Potato virus, PLRV), marmor solani (Potato virus, PVA) and the method for these 5 kinds of potato viruses of potato virus S (Potato virus, PVS).The invention still further relates to primer design method in multiple real time fluorescence PCR reaction, and the reaction system parameter and the program parameter that detect simultaneously 5 kinds of potato viruses.The present invention is that the research of multiple fluorescence PCR is provided convenience, and is suitable for the departments such as Check and Examination of Port quarantine, agriculture production, plant protection and uses, and has simultaneously scientific research value widely and application prospect.
Description of drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is operational flowchart of the present invention.
Fig. 2 is the test-results of embodiments of the invention 1, the leaf sample that contains standard potato standard virus source, detect simultaneously 5 kinds of potato virus (PVX through a multiple fluorescence quantitative PCR, PVY, PLRV, PVA and PVS) melting curve figure, be provided with in addition a negative control, its melting curve result shows without amplified production, proves that present method specificity is good.
Fig. 3 is the wherein test-results of 4 samples of embodiments of the invention 2, and the sampling position is Hangzhou Gao Sha on the spot, and the total RNA of extracting blade chooses 20 samples and detects, and chooses the wherein melting curve figure of 4 samples after a multiple fluorescence quantitative amplification respectively.
Fig. 4 is the wherein test-results of 4 samples of embodiments of the invention 3, the sampling position is the area, Haining on the spot, extract the total RNA of potato tuber of results, choose 20 samples and detect, choose the wherein melting curve figure of 4 samples after adopting a multiple fluorescence quantitative to increase.
Fig. 5 is the test-results of reference examples 1 of the present invention.Adopt Sybr Green I dyestuff to replace EvaGreen five kinds of potatos poison source samples are carried out melting curve figure after the multi-fluorescence amplification.
Embodiment
The foundation of embodiment 1,5 kind of basic detection method of potato virus
The first step: primer is synthetic
5 kinds of viral primer sequences (seeing Table 1) that the present invention is designed entrust Chinese Shanghai to give birth to technical service company of work biotech firm synthetic primer, and resultant quantity is every primer 2 OD.
Second step: the total RNA of positive extracting in a small amount
To contain respectively potato PVX, PVY, PLRV, positive each 500mg of potato flour sample in PVA and PVS poison source is placed in the 5ml centrifuge tube, and the ratio in 1ml Trizol/100mg adds Trizo immediately, and 5min is placed in homogenate on ice.Ratio in 200ul chloroform/mlTrizol adds chloroform.Concuss 30s, be put in 5-10min on ice, after layering 4 ℃, 12000r/min. centrifugal 20min, get supernatant liquor to another 5ml centrifuge tube, add isopyknic Virahol (approximately 0.5ml) mixing gently, place 2h or-80 ℃ of placement 1h for-20 ℃, 4 ℃ of centrifugal 20min of 12000r/min abandon supernatant, add 1ml 75% washing with alcohol, mixing gently, 4 ℃ of centrifugal 10min of 12000r/min blot ethanol with the rifle head, vacuum-drying 5-10min, the RNA of last gained is dissolved in 25 μ L DEPC. aqua sterilisas.
The 3rd step: cDNA is synthesized in reverse transcription
CDNA synthetic system (total reaction volume 20 μ L): total RNA2.0 μ L, random primer (50 μ M) 1 μ L, 5 * RT Buffer4 μ L, dNTP (10mM) 1 μ L, Rnase Inhibior 0.5 μ L, RNase1 μ L, DEPC H
2O 10.5 μ L.Reaction conditions is 42 ℃ and keeps 1h.Get cDNA.
Random primer can be directly available from biotech company, and concrete sequence is: NNNNNN.
The 4th step: multiple fluorescence quantitative PCR
Multiple fluorescence quantitative PCR reaction system (total reaction volume 50 μ L): 10 * PCR buffer, 5 μ L, 25mM MgCl
25 μ L, dNTP (10mM) 1.5 μ L, 20 * EvaGreen, 2.5 μ L, Taq DNA Polymerase 2.5ul, 2.5 the cDNA that μ L the 3rd step is synthetic and the viral primer in the first step (PVX wherein, PVY, PLRV, each 0.2,0.4,0.28,0.25 and 0.23 μ L of PVA and PVS upstream and downstream primer), supply dd H
2O to 50 μ L.Establish simultaneously a negative control.Reaction is carried out on ABI7000 fluorescent quantitation instrument, and amplification program is 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s.The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
The 5th step: result detects
According to the ABI7000 quantitative real time PCR Instrument the melting curve figure that automatically generates, come analysing amplified result.
The test-results of the present embodiment is seen Fig. 2.The X-coordinate of Fig. 2 represents the Tm value, and ordinate zou represents Derivative (amplified production fluorescent signal value changes speed); Melting curve shows five peaks, the corresponding X-coordinate in each peak corresponding PVX, PVY, PLRV, the Tm value of these five kinds of potato virus amplified fragments of PVA and PVS is respectively 83.9,80.6,89.1,78.2,86.8, negative control shows that without the peak it is without amplified production.Experimental result shows that the inventive method can detect PVX accurately and effectively simultaneously, PVY, PLRV, these five kinds of potato viruses of PVA and PVS.
Annotate: the Tm value only can represent a kind of potato virus amplified production specifically.
Potato viruses detection in the potato leaf in embodiment 2, high sand ground district, Hangzhou
The first step: primer is synthetic
With embodiment 1 the first step.
Second step: blade sampling
Sampling spot is the high sand ground of Zhejiang Hangzhou district.Select to have the plant of viral reveal any symptoms on potato planting ground, with scissors clip blade, often take a Plant samples, scissors is with 75% (volumetric concentration) alcohol disinfecting, then carries out the next one and take a sample.Each sample is packed in the polyethylene food bag, then drops into sampling box and preserve and take back the laboratory.20 samples are set altogether.
The 3rd step: the total RNA of leaf sample extracting in a small amount
Operation steps is equal to embodiment 1 second step.
The 4th step: cDNA is synthesized in reverse transcription
With the 3rd step of embodiment 1.
The 5th step: multiple fluorescence quantitative PCR
With the 4th step of embodiment 1.
The 6th step: result detects
With the 5th step of embodiment 1.
In the present embodiment 2, the test-results of 4 samples is wherein seen Fig. 3.The melting curve of 4 sample complex virus infection is respectively as shown in Fig. 3 A～D.These 20 leaf sample multiple fluorescence PCRs show that every kind of corresponding Tm value of virus institute is consistent, are respectively 83.9,80.6,89.1,78.2,86.8.In 20 samples, detect 5 examples that have of independent infection PLRV, that infects separately PVX has 3 examples, 2 examples that have that infect separately PVY, 1 example that has that infects separately PVS, without infecting separately PVA, polyinfection PVX and PVY's is 4 examples, and polyinfection PVX and PVS's is 2 examples, and polyinfection PVX, PVS and PLRV's is 1 example, polyinfection PVY, PVS and PVA's is 1 example, and polyinfection PLRV, PVX, PVS, PVY and PVA's is 1 example.
The detection of potato virus in embodiment 3, potato tuber
The first step: primer is synthetic
With embodiment 1 the first step.
Second step: stem tuber sampling
Potato tuber virus detects sampling spot carries out in the Haining, and this experiment has detected 5 kinds altogether.Every kind is chosen 5 strains at random, and a stem tuber is got in every strain.After stem tuber is taken back the laboratory, first wash away earth with tap water, then clean 3 times with distilled water, room temperature is dried moisture.With pocket knife with 5 stem tubers of each kind be cut into small pieces mix after, get 500mg and be used for extracted total RNA.20 samples are set altogether.
The 3rd step: the total RNA of potato tuber sample extracting in a small amount
Get the above-mentioned potato tuber sample of 500mg grind into powder in liquid nitrogen, then it is inserted in the 5ml centrifuge tube, (24: 1, v/v), fully vortex vibrated to add the RNA Extraction buffer of 750 μ L precoolings and chloroform/primary isoamyl alcohol of 750 μ L.In 25 ℃, the 13 centrifugal 2min of 000r/min.In transfer to a new 5mL centrifuge tube, add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1, v/v), put upside down mixing.In 25 ℃, the centrifugal 2min of 13000r/min.Again shift upper phase, repeat above-mentioned steps, until can be observed a clean middle layer.In extracting upper phase transition to a new 5mL centrifuge tube for several times.Add the 3mol/LNaAc (pH5.2) of 1/10 volume and the precooling dehydrated alcohol of 2.5 times of volumes, after putting upside down mixing, 4 ℃ of standing 30min.In 4 ℃, the centrifugal 20min of 13000r/min.After 75% washing with alcohol precipitation with precooling, centrifuge tube is placed in Bechtop allows residual ethanol be evaporated completely to the greatest extent, add the 0.1%DEPC aqua sterilisa dissolution precipitation of people 40 μ L.After precipitation was dissolved fully, adding 10mol/L LiCt was 2mol/L to final concentration, ice bath 1h.4 ℃, the centrifugal 20min of 18000r/min.After 75% washing with alcohol precipitation with precooling, centrifuge tube is placed in Bechtop allows residual ethanol be evaporated completely to the greatest extent, add the DEPC aqua sterilisa dissolution precipitation of 25 μ L ,-80 ℃ of preservations are stand-by.
The 4th step: cDNA is synthesized in reverse transcription
With the 3rd step of embodiment 1.
The 5th step: multiple fluorescence quantitative PCR
With the 4th step of embodiment 1.
The 6th step: result detects
With the 5th step of embodiment 1.
The test-results of the present embodiment 4 samples is wherein seen Fig. 4.The melting curve of 4 sample complex virus infection is respectively as shown in Fig. 4 A～D.Presentation of results: these 20 stem tuber sample multiple fluorescence PCRs show that every kind of corresponding Tm value of virus institute is consistent, are respectively 83.9,80.6,89.1,78.2,86.8.In 20 samples, detect 3 examples that have of independent infection PLRV, that infects separately PVX has 6 examples, 2 examples that have that infect separately PVY, 3 examples that have that infect separately PVS, without infecting separately PVA, polyinfection PVX and PVY's is 2 examples, and polyinfection PLRV and PVS's is 1 example, and polyinfection PLRV and PVY's is 1 example, polyinfection PVY, PVS and PVA's is 1 example, and polyinfection PVX, PVS and PLRV's is 1 example.
Comparative Examples 1,
In order to prove the exactness of conclusion of the present invention, described 20 potato leafs and described 20 the potato tuber samples of embodiment 3 wherein of embodiment 2 are detected respectively according to the substance fluorescence PCR method, determine the viral species that sample infects.Concrete operation step: get in embodiment 2 and embodiment 3 that in the 4th step, every kind of synthetic cDNA of sample reverse transcription carries out separately the substance quantitative fluorescent PCR with 5 kinds of primer pairs respectively.Substance quantitative fluorescent PCR reaction system (total reaction volume 10 μ L): 10 * PCR buffer, 1 μ L, 25mM MgCl
21 μ L, dNTP (10mM) 0.3 μ L, 20 * EvaGreen, 0.5 μ L, Taq DNA Polymerase 0.1ul, the 3rd synthetic cDN of step in 1 μ L embodiment 2 or 3, the primer pair 0.5 μ L of independent a kind of virus supplies dd H
2O to 10 μ L.Reaction is carried out on ABI7000 fluorescent quantitation instrument, and amplification program is 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s.The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
Through software analysis, result is as follows:
In 20 leaf samples, detect 5 examples that have of independent infection PLRV, infect separately 3 examples that have of PVX, that infects separately PVY has 2 examples, 1 example that has that infects separately PVS, polyinfection PVX and PVY 4 examples, 2 examples of polyinfection PVX and PVS, polyinfection PVX, PVS and PLRV 1 example, 1 example of polyinfection PVY, PVS and PVA, 1 example of polyinfection PLRV, PVX, PVS, PVY and PVA.Corresponding fully one by one with the detected result of embodiment 2.Above-mentioned 20 potato tuber samples detect through the PCR of routine method, and its viral detected result is also corresponding fully one by one.
In 20 stem tuber samples, infect separately PVX 6 examples, detect independent infection PLRV 3 examples, infect separately the PVS3 example, infect separately PVY 2 examples, polyinfection PVX and PVY 2 examples, polyinfection PLRV and PVS 1 example, polyinfection PLRV and PVY 1 example, polyinfection PVY, PVS and PVA 1 example, polyinfection PVX, PVS and PLRV1 example.Corresponding fully one by one with the detected result of embodiment 3.Above-mentioned 20 stem tuber samples detect through the PCR of routine method, and its viral detected result is also corresponding fully one by one.
The coincidence rate of above-mentioned detected result and embodiment 2 and embodiment 3 detected results is 100%, thereby proof the inventive method is accurately feasible.
For the superiority of sufficient proof the inventive method, the contriver has done following contrast experiment:
Comparative Examples 2, employing Sybr Green I carry out multiple fluorescence PCR, and the 5 kind viral sample identical to embodiment 1 detect.Sybr Green I multiple fluorescence quantitative PCR reaction system (total reaction volume 50 μ L): 10 * PCR buffer, 5 μ L, 25mM MgCl
25 μ L, dNTP (10mM) 1.5 μ L, 20 * Sybr Green I, 2.5 μ L, Taq DNA Polymerase 2.5ul, 2.5 the cDNA that μ L embodiment 1 the 3rd step is synthetic and 5 kinds of viral primers (PVX wherein, PVY, PLRV, each 0.2,0.4,0.28,0.25 and 0.23 μ L of PVA and PVS upstream and downstream primer), supply dd H
2O to 50 μ L.Establish simultaneously a negative control.This reaction system is except the fluorescence dye difference, and in other reaction system, each composition all remains unchanged.Reaction is carried out on ABI7000 fluorescent quantitation instrument, and amplification program is 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s.The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
The test-results of the present embodiment is seen Fig. 5.Result is as follows: the melting curve of Sybr Green I multiple fluorescence PCR can only show three peaks, PVY Tm corresponding with PLRV position Wu Feng wherein, and Sybr Green I multiple fluorescence PCR has only detected PVA, PVX and three kinds of viruses of PVS.Because every kind of amplified fragments varies in size, and the amplification efficiency height is different under similarity condition, has competitive amplification.In these five kinds of amplified productions, PVY is relative with the PLRV amplification efficiency, and the PVY fragment is again less lower than other three kinds, a little less than competitiveness, thereby has affected the detection effect.Above-mentioned detected result confirms to select EvaGreen really can obtain better effect than Sybr Green I commonly used, and this has also illustrated the defective and the drawback that suppresses pcr amplification of Sybr Green I " dyestuff heavily distributes " simultaneously.
At last, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1. multiple real time fluorescence PCR detects the method for five kinds of potato viruses simultaneously, it is characterized in that comprising the following steps:
1), synthesize respectively potato virus X--PVX, marmor upsilon--PVY, corium solani--the corresponding Auele Specific Primer of PLRV, marmor solani--PVA and potato virus S--PVS;
Described PVX amplimer is to being:
Forward: CACAGGCTGCTTGGGACTT,
Oppositely: TGCCGTTGGAATAGGGAC;
Described PVY amplimer is to being:
Forward: CTGAGATGCCAACTGTGATGA,
Oppositely: GTTGACATTTGGCGAGGTT;
Described PLRV amplimer is to being:
Forward: CGCCGAAGACGCAGAAGAGGA,
Oppositely: AGACTCGGCCCGAAGGTGAA;
Described PVA amplimer is to being:
Forward: CCAATGCTCAAAGGTAAG,
Oppositely: TCTACTTGCTTTGGTTTGT;
Described PVS amplimer is to being:
Forward: GAGATAGGTAGGCCCTCACTT,
Oppositely: CTGTGCCATTTGCTCGGT;
2), total RNA of extracting potato sample, cDNA is synthesized in reverse transcription;
3), described all Auele Specific Primers of step 1) being placed in the multiple fluorescence quantitative PCR reaction system, take step 2) described cDNA is template, carries out pcr amplification; Analyze the potato sample according to melting curve and whether carry at least a in PVX, PVY, PLRV, PVA and PVS; Be specially: the peak shown according to melting curve, 83.9 peak the Tm value appears is, PVX virus has been described; Appearance Tm value is 80.6 peak, and PVY virus has been described; Appearance Tm value is 89.1 peak, and PLRV virus has been described; Appearance Tm value is 78.2 peak, and PVA virus has been described; Appearance Tm value is 86.8 peak, and PVS virus has been described.
2. multiple real time fluorescence PCR according to claim 1 detects the method for five kinds of potato viruses simultaneously, it is characterized in that: described step 3) multiple fluorescence quantitative PCR reaction system consists of the following composition: 10 * PCR buffer, 5 μ L, 25mM MgCl
25 μ L, dNTP (10m Μ) 1.5 μ L, 20 * EvaGreen, 2.5 μ L, Taq DNA Polymerase 2.5ul, 2.5 μ L step 2) described cDNA, PVX amplimer to 0.2 μ L, PVY amplimer to 0.4 μ L, PLRV amplimer to 0.28 μ L, PVA amplimer to 0.25 μ L and PVS amplimer to 0.23 μ L, use dd H
2O is settled to 50 μ L;
Described multiple fluorescence PCR response procedures is: 95 ℃ of 5min; 40 circulations: 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 31s; The melting curve program is 95 ℃ of 15s, 60 ℃ of 20s, 95 ℃ of 15s; Collect fluorescent signal since 60 ℃.
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| CN105039594A (en) * | 2015-07-24 | 2015-11-11 | 湖南农业大学 | Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof |
| CN105039601A (en) * | 2015-08-25 | 2015-11-11 | 广东省农业科学院植物保护研究所 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primers and kit for detecting pepper veinal mottle virus |
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| CN107012261B (en) * | 2017-06-13 | 2020-07-24 | 福建省农业科学院畜牧兽医研究所 | Duck adenovirus type A and type 2 dual EvaGreen real-time fluorescent quantitative PCR detection primers |
| CN109097492A (en) * | 2018-08-09 | 2018-12-28 | 惠州学院 | A kind of fluorescence quantifying PCR method detecting corium solani |
| CN109097493A (en) * | 2018-08-09 | 2018-12-28 | 惠州学院 | A kind of fluorescence quantification PCR primer and kit detecting corium solani |
| CN110951825A (en) * | 2019-09-25 | 2020-04-03 | 云南衡原检测技术有限公司 | Potato virus detection method |
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