CN105176986A - RT-LAMP reagent kit for detecting feathery mottle viruses of sweet potatoes and primer special for RT-LAMP reagent kit - Google Patents
RT-LAMP reagent kit for detecting feathery mottle viruses of sweet potatoes and primer special for RT-LAMP reagent kit Download PDFInfo
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Abstract
The invention discloses an RT-LAMP reagent kit for detecting feathery mottle viruses of sweet potatoes and a primer special for the RT-LAMP reagent kit. The primer special for the RT-LAMP reagent kit for detecting the feathery mottle viruses of the sweet potatoes is composed of primer SPFMV-F3, primer SPFMV-B3, primer SPFMV-FIP, primer SPFMV-BIP, primer SPFMV-LF and primer SPFMV-LB, and the nucleotide sequences are the sequence 1, the sequence 2, the sequence 3, the sequence 4, the sequence 5 and the sequence 6 in a sequence table in sequence. It is proved through experiments that by means of the RT-LAMP reagent kit for detecting the feathery mottle viruses of the sweet potatoes and the primer special for the RT-LAMP reagent kit, the feathery mottle viruses of the sweet potatoes can be detected in a specificity mode, cross reactions with other viruses do not exist, and the minimum detection limit of the RNA of tje feathery mottle viruses of the sweet potatoes is 25 fg.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of the RT-LAMP test kit and the primer special thereof that detect Sweet Potato Feathery Mottle Virus.
Background technology
Sweet Potato Feathery Mottle Virus (Sweetpotatofeatherymottlevirus, SPFMV) be RNA viruses, one of main virus infecting sweet potato, be distributed in each sweet potato producing region, the world, belong to marmor upsilon section (Potyviridae), Potyvirus (Potyvirus).Sweet Potato Feathery Mottle Virus (Sweetpotatofeatherymottlevirus, SPFMV) can with sweet potato chlorotic stunt virus (Sweetpotatochloroticstuntvirus, SPCSV) assist life to infect sweet potato altogether and cause sweet potato viruses disease (Sweetpotatovirusdisease, SPVD), be a kind of destructive disease of sweet potato.After sweet potato infects sweet potato viruses disease, the underproduction can reach 50%-98%, even has no harvest.At present, the method detecting Sweet Potato Feathery Mottle Virus (Sweetpotatofeatherymottlevirus, SPFMV) mainly contains: the methods such as ELISA, RT-PCR, and the drawback of these methods is length consuming time, relies on expensive test set.
Ring mediated isothermal amplification (the loopmediatedisothermalamplification developed in recent years, LAMP) be a kind of new nucleic acid amplification technologies, this technology depends on the archaeal dna polymerase (BstDNApolymerase) that the primer that can identify 6 specific regions on target sequence and 1 have strand displacement characteristic, efficient amplification target gene under isothermal conditions, sensitivity and specificity high, the method has been widely used in virus, viroid, bacterium, fungi and genetically modified detection, and not yet applies the report of this technology for detection Sweet Potato Feathery Mottle Virus so far.
Summary of the invention
The present invention want technical solution problem to be how to detect Sweet Potato Feathery Mottle Virus (Sweetpotatofeatherymottlevirus, SPFMV).
The present invention provide firstly a kind of primer set, be made up of primer SPFMV-F3, primer SPFMV-B3, primer SPFMV-FIP, primer SPFMV-BIP, primer SPFMV-LF and primer SPFMV-LB, they are single strand dna, and nucleotide sequence is followed successively by sequence 1, sequence 2, sequence 3, sequence 4, sequence 5 and sequence 6 in sequence table.Wherein, the sequence 1 of sequence table is made up of 20 Nucleotide, the sequence 2 of sequence table is made up of 21 Nucleotide, the sequence 3 of sequence table is made up of 43 Nucleotide, the sequence 4 of sequence table is made up of 42 Nucleotide, the sequence 5 of sequence table is made up of 20 Nucleotide, and the sequence 6 of sequence table is made up of 20 Nucleotide.Described primer set can carry out specific reverse transcription loop-mediated isothermal amplification to the total serum IgE of Sweet Potato Feathery Mottle Virus.
In described primer set, the mol ratio of described primer SPFMV-F3, described primer SPFMV-B3, described primer SPFMV-FIP, described primer SPFMV-BIP, described primer SPFMV-LF and described primer SPFMV-LB can be 1:1:8:8:4:4.
In described primer set, the amount of each primer is as follows: primer SPFMV-LB described in primer SPFMV-LF, 0.8 μm of ol described in primer SPFMV-BIP, 0.8 μm of ol described in primer SPFMV-FIP, 1.6 μm of ol described in primer SPFMV-B3,1.6 μm of ol described in primer SPFMV-F3,0.2 μm of ol described in 0.2 μm of ol.
Described mol ratio is the ratio of total mole number, and described total mole number is various single stranded DNA mole number sum in primer.
The preparation method of described primer set also belongs to protection scope of the present invention.The preparation method of described primer set can be (I), (II) or (III) as follows:
(I) in described primer set, each bar primer is individually packed;
(II) in described primer set, each bar primer is according to described mixed in molar ratio together;
(III) in described primer set, each bar primer mixes according to described amount.
The purposes of described primer set be following a) or b) or c) or d): a) for the preparation of detecting or the test kit of auxiliary detection Sweet Potato Feathery Mottle Virus; B) detect or whether contain Sweet Potato Feathery Mottle Virus in auxiliary detection testing sample; C) for the preparation of qualification or the test kit of assistant identification Sweet Potato Feathery Mottle Virus; D) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
The present invention also protects the application of described primer set, for following a) or b) or c) or d): a) for the preparation of detecting or the test kit of auxiliary detection Sweet Potato Feathery Mottle Virus; B) detect or whether contain Sweet Potato Feathery Mottle Virus in auxiliary detection testing sample; C) for the preparation of qualification or the test kit of assistant identification Sweet Potato Feathery Mottle Virus; D) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
The present invention also protects the test kit containing above arbitrary described primer set.The purposes of described test kit be following e) or f): e) detect or in auxiliary detection testing sample whether containing or doubtful containing Sweet Potato Feathery Mottle Virus; F) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
Described test kit also can comprise 10 × reaction buffer, BstDNA polysaccharase, ThermoScript II and/or trimethyl-glycine.Described 10 × reaction buffer can be NewEnglandBiolabs Products.Described ThermoScript II specifically can be M-MLV ThermoScript II.
Described test kit also can comprise fluorescent color-developing agent.Described fluorescent color-developing agent specifically can be SYBRGreenI nucleic acid dye or fluorexon fluorescence dye.
The present invention also protects the application of described test kit, for following e) or f): e) detect or in auxiliary detection testing sample whether containing or doubtful containing Sweet Potato Feathery Mottle Virus; F) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
Present invention also offers a kind of method detecting testing sample and whether contain Sweet Potato Feathery Mottle Virus, comprise the steps:
Extract the total serum IgE of testing sample, reverse transcription loop-mediated isothermal amplification is carried out with above-mentioned arbitrary described primer set, then pass judgment on as follows: if described primer set can realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then in testing sample containing or doubtful containing Sweet Potato Feathery Mottle Virus; If described primer set can not realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
Described " detecting the method whether testing sample infects Sweet Potato Feathery Mottle Virus " specifically can be method one, comprise the steps: the total serum IgE extracting testing sample, ring mediated isothermal amplification is carried out with the reverse transcription loop-mediated isothermal of described detection Sweet Potato Feathery Mottle Virus amplification primer set, if amplification curve be rendered as typically " S type ", in testing sample containing or doubtful containing Sweet Potato Feathery Mottle Virus, if amplification curve be rendered as horizontal linear, described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
Described " detecting the method whether testing sample infects Sweet Potato Feathery Mottle Virus " specifically can be method two, comprise the steps: the total serum IgE extracting testing sample, ring mediated isothermal amplification (containing fluorexon fluorescence dye in reaction system) is carried out with the reverse transcription loop-mediated isothermal of described detection Sweet Potato Feathery Mottle Virus amplification primer set, obtain testing sample reaction solution, the colour-change of range estimation testing sample reaction solution, if testing sample reaction solution is green, then in testing sample containing or doubtful containing Sweet Potato Feathery Mottle Virus, if testing sample reaction solution is orange, then in described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
The present invention also protects and a kind ofly identifies that whether virus to be measured be the method for the Sweet Potato Feathery Mottle Virus of candidate, comprises the steps:
Extract the total serum IgE of virus to be measured, reverse transcription loop-mediated isothermal amplification is carried out with above-mentioned arbitrary described primer set, then pass judgment on as follows: if described primer set can realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate; If described primer set can not realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then described virus to be measured is the non-Sweet Potato Feathery Mottle Virus of candidate.
Described " identifying that whether virus to be measured be the method for Sweet Potato Feathery Mottle Virus " specifically can be method three, comprise the steps: the total serum IgE extracting testing sample, ring mediated isothermal amplification is carried out with the reverse transcription loop-mediated isothermal of described detection Sweet Potato Feathery Mottle Virus amplification primer set, if amplification curve be rendered as typically " S type ", virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate, if amplification curve be rendered as horizontal linear, described virus to be measured is the non-Sweet Potato Feathery Mottle Virus of candidate.
Described " identifying that whether virus to be measured be the method for Sweet Potato Feathery Mottle Virus " specifically can be method four, comprise the steps: the total serum IgE extracting testing sample, ring mediated isothermal amplification (containing fluorexon fluorescence dye in reaction system) is carried out with the reverse transcription loop-mediated isothermal of described detection Sweet Potato Feathery Mottle Virus amplification primer set, obtain testing sample reaction solution, the colour-change of range estimation testing sample reaction solution, if testing sample reaction solution is green, then virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate, if testing sample reaction solution is orange, then described virus to be measured is the non-Sweet Potato Feathery Mottle Virus of candidate.
Above-mentioned arbitrary described fluorexon fluorescence dye can be the dyestuff adding 100ml physiological saline and obtain in 100mg fluorexon (Thermofisher Products, catalog number is S-34854).
Above-mentioned arbitrary described ring mediated isothermal amplification can carry out under 60-65 DEG C of condition.More specifically can carry out under 63 DEG C of conditions.
Above-mentioned arbitrary described testing sample can be plant sample or microbiological specimens.Described testing sample specifically can be the healthy Sweet Potato Leaf not infecting Sweet Potato Feathery Mottle Virus or the Sweet Potato Leaf infecting Sweet Potato Feathery Mottle Virus.
Above-mentioned arbitrary described virus to be measured can be Sweet Potato Feathery Mottle Virus, sweet potato C virus, sweet potato G virus, sweet potato Potato Leaf Roll Virus (PLRV), No. 2, sweet potato viruses or sweet potato without syndrome virus 1.
Experiment proves, provided by the invention a kind of detect Sweet Potato Feathery Mottle Virus RT-LAMP test kit and primer special can detect Sweet Potato Feathery Mottle Virus specifically, and with other viral no cross reactions, as sweet potato Potato Leaf Roll Virus (PLRV), sweet potato C virus or sweet potato G virus.Meanwhile, the invention provides a kind of detect Sweet Potato Feathery Mottle Virus RT-LAMP test kit and the minimum detection of primer special to the RNA of Sweet Potato Feathery Mottle Virus be limited to 25fg.
Accompanying drawing explanation
Fig. 1 is the specificity of the RT-LAMP test kit detecting Sweet Potato Feathery Mottle Virus.
Fig. 2 is the sensitivity of the RT-LAMP test kit detecting Sweet Potato Feathery Mottle Virus.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Sweet Potato Feathery Mottle Virus (Sweetpotatofeatherymottlevirus in following embodiment; SPFMV), sweet potato G virus (SweetpotatovirusG; and sweet potato Potato Leaf Roll Virus (PLRV) (Sweetpotatoleafcurlvirus SPVG); SPLCV) be documented in as in Publication about Document: Qiao Qi; Zhang Zhen's minister; Zhang Desheng etc. the serology of Viruses On Sweet Potato In China kind and Molecular Detection [J]. Plant Pathology; 2012; 42 (1): 10-16., the public can obtain above-mentioned materials from Beijing Plant Protection Station (i.e. applicant).
Sweet potato C virus (SweetpotatovirusC; and sweet potato viruses No. 2 (Sweetpotatovirus2 SPVC); SPV2) be documented in as in Publication about Document: Bao Gaili; Zuo Ruijuan; Rao Weili etc. the detection of Yunnan sweet potato viruses and the diversity analysis [J] of main virus. microbiology is circulated a notice of; 2013,40 (2): 236-248., the public can obtain above-mentioned materials from Beijing Plant Protection Station (i.e. applicant).
Sweet potato is without syndrome virus 1 (Sweetpotatosymptomlessvirus1; SPSMV-1) be documented in as in Publication about Document: Mbanzibwa; D.R., Tairo, F.; Gwandu; C., Kullaya, A.; 2011.Firstreportofsweetpotatosymptomlessvirus1andsweetpo tatovirusAinsweetpotatoesinTanzania.PlantDis.95:224., the public can obtain above-mentioned materials from Beijing Plant Protection Station (i.e. applicant).
Infect the Sweet Potato Leaf of Sweet Potato Feathery Mottle Virus in following embodiment and do not infect the healthy Sweet Potato Leaf of Sweet Potato Feathery Mottle Virus, the public can obtain from Beijing Plant Protection Station (i.e. applicant).
Fluorexon fluorescence dye is in 100mg fluorexon (Thermofisher Products, catalog number is S-34854), add the dyestuff that 100ml physiological saline obtains.
Fluorescent reagent is in 1 μ lSYBRGreenI nucleic acid dye (Thermofisher Products, catalog number is S-7563), add the reagent that 49 μ l deionized waters obtain.
10 × reaction buffer is NewEnglandBiolabs Products, and catalog number is B9004S; BstDNA polysaccharase is NewEnglandBiolabs Products, and catalog number is M0537S; M-MLV ThermoScript II is Takara Products, and catalog number is 2641Q; Trimethyl-glycine is Sigma Products, and catalog number is 61962-50G; MgSO
4for Sigma Products, catalog number is 746452-500G; DNTP is Takara Products, and catalog number is 4030Q.
The preparation of the RT-LAMP primer set of embodiment 1, detection Sweet Potato Feathery Mottle Virus
The RT-LAMP primer set of the detection Sweet Potato Feathery Mottle Virus of the present embodiment is made up of primer SPFMV-F3, primer SPFMV-B3, primer SPFMV-FIP, primer SPFMV-BIP, primer SPFMV-LF and primer SPFMV-LB, each bar primer is single strand dna, and their nucleotide sequence is successively as shown in the sequence 1 in sequence table, sequence 2, sequence 3, sequence 4, sequence 5 and sequence 6.
Prepare primer SPFMV-F3, primer SPFMV-B3, primer SPFMV-FIP, primer SPFMV-BIP, primer SPFMV-LF and primer SPFMV-LB.
The RT-LAMP test kit that embodiment 2, utilization detect Sweet Potato Feathery Mottle Virus detects testing sample
One, the preparation of the RT-LAMP test kit of Sweet Potato Feathery Mottle Virus is detected
Preparation detects the RT-LAMP test kit of Sweet Potato Feathery Mottle Virus, comprises test kit C or test kit D:
Reaction reagent C, blank and negative control are fitted together the product obtained by test kit C;
Reaction reagent D, blank and negative control are fitted together the product obtained by test kit D.
Wherein, described reaction reagent C, comprises 2.5 μ l10 × reaction buffers, 8UBstDNA polysaccharase, 200UM-MLV ThermoScript II, 25 μm of ol trimethyl-glycines, fluorescent reagent 1 μ l, 0.15 μm of olMgSO
4, 0.04 μm of oldNTP, primer SPFMV-F3 and each 5pmol of primer SPFMV-B3, primer SPFMV-FIP and each 40pmol of primer SPFMV-BIP, primer SPFMV-LF and each 20pmol of primer SPFMV-LB, mend to 24 μ l with deionized water.
Described reaction reagent D, comprises 2.5 μ l10 × reaction buffers, 8UBstDNA polysaccharase, 200UM-MLV ThermoScript II, 25 μm of ol trimethyl-glycines, fluorexon fluorescence dye 5pmol, 0.15 μm of olMgSO
4, 0.04 μm of oldNTP, primer SPFMV-F3 and each 5pmol of primer SPFMV-B3, primer SPFMV-FIP and each 40pmol of primer SPFMV-BIP, primer SPFMV-LF and each 20pmol of primer SPFMV-LB, mend to 24 μ l with deionized water.
Described negative control is the total serum IgE of the healthy Sweet Potato Leaf not infecting Sweet Potato Feathery Mottle Virus, adds 1 μ l during use.
Described blank is sterilizing ultrapure water, adds 1 μ l during use.
Two, the test kit D utilizing step one to prepare detects testing sample
1, extract the total serum IgE of the Sweet Potato Leaf total serum IgE infecting Sweet Potato Feathery Mottle Virus and the healthy Sweet Potato Leaf not infecting Sweet Potato Feathery Mottle Virus respectively with TaKaRaMiniBESTPlantRNAExtractionKit (leading to Trade Co., Ltd. purchased from Beijing six directions), the concentration of RNA is 1ng/ μ l.
2、RT-LAMP
Concrete detection method is as follows:
(1) total serum IgE adding the Sweet Potato Leaf of the infection Sweet Potato Feathery Mottle Virus of 24 μ l reaction reagent D and the extraction of 1 μ l step 1 in test tube obtains reaction solution, is then reacted 60 minutes in 63 DEG C by reaction solution.After reaction terminates, the colour-change of observing response liquid.
According to the method described above, the total serum IgE of Sweet Potato Leaf infecting Sweet Potato Feathery Mottle Virus is replaced with respectively the total serum IgE not infecting the healthy Sweet Potato Leaf of Sweet Potato Feathery Mottle Virus and sterilizing ultrapure water that step 1 extracts, other step is all constant.After reaction terminates, the colour-change of observing response liquid.
(2) result is observed and is judged: if the visualization of color of reaction solution for green, contain in testing sample or doubtfully contain Sweet Potato Feathery Mottle Virus; If the visualization of color of reaction solution be orange, in described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
Experimental result shows: after the test tube of total serum IgE adding the Sweet Potato Leaf infecting Sweet Potato Feathery Mottle Virus carries out RT-LAMP, reaction solution visualization of color be green; And after the test tube of the total serum IgE or sterilizing ultrapure water that add the healthy Sweet Potato Leaf not infecting Sweet Potato Feathery Mottle Virus carries out RT-LAMP, reaction solution visualization of color is orange.Result shows, the RT-LAMP test kit of detection Sweet Potato Feathery Mottle Virus provided by the invention can detect Sweet Potato Feathery Mottle Virus accurately.
The specificity of the RT-LAMP test kit of the detection Sweet Potato Feathery Mottle Virus of embodiment 3, embodiment 2
One, carry out specificity experiments with the RT-LAMP test kit C of the detection Sweet Potato Feathery Mottle Virus of embodiment 2, in triplicate, each step repeated is as follows in experiment:
1, extract the total serum IgE of the total serum IgE of SPFMV, the total serum IgE of SPVC, the total serum IgE of SPVG, the total serum IgE of SPLCV, the total serum IgE of SPSMV-1 and SPV2 respectively with TaKaRaMiniBESTPlantRNAExtractionKit (Beijing six directions leads to Trade Co., Ltd.'s product), the concentration of RNA is 1ng/ μ l.
2、RT-LAMP
(1) total serum IgE adding the SPFMV of 24 μ l reaction reagent C and the extraction of 1 μ l step 1 in test tube obtains reaction solution, is then reacted 60 minutes in 63 DEG C by reaction solution.Take cycle number as X-coordinate, the amount of PCR primer is ordinate zou, obtains the amplification curve that fluorescence detecting system is drawn.2 test tubes are set as repetition.
According to the method described above, the total serum IgE of SPFMV is replaced with respectively the total serum IgE of SPVC, the total serum IgE of SPVG, the total serum IgE of SPLCV, the total serum IgE of SPSMV-1, the total serum IgE of SPV2 and sterilizing ultrapure water, other step is all constant, obtains corresponding amplification curve.
(2) result observe and judge: if amplification curve present typically " S type ", in testing sample containing or doubtful contain Sweet Potato Feathery Mottle Virus; If amplification curve be rendered as horizontal linear, described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
RT-LAMP the results are shown in Figure A in 1, and the test tube only adding the total serum IgE of SPFMV carries out RT-LAMP and obtains typically " S type " amplification curve.
Two, carry out specificity experiments with the RT-LAMP test kit D of the detection Sweet Potato Feathery Mottle Virus of embodiment 2, in triplicate, each step repeated is as follows in experiment:
1, extract SPFMV total serum IgE, SPVC total serum IgE, SPVG total serum IgE, SPLCV total serum IgE, SPSMV-1 total serum IgE and SPV2 total serum IgE respectively with TaKaRaMiniBESTPlantRNAExtractionKit (Beijing six directions leads to Trade Co., Ltd.'s product), the concentration of RNA is 1ng/ μ l.
2、RT-LAMP
(1) the SPFMV total serum IgE adding 24 μ l reaction reagent D and the extraction of 1 μ l step 1 in EP pipe obtains reaction solution, is then reacted 60 minutes in 63 DEG C by reaction solution.After reaction terminates, the colour-change of observing response liquid.2 EP pipes are set as repetition.
According to the method described above, SPFMV total serum IgE is replaced with respectively SPVC total serum IgE, SPVG total serum IgE, SPLCV total serum IgE, SPSMV-1 total serum IgE, SPV2 total serum IgE and sterilizing ultrapure water, other step is all constant.After reaction terminates, observe the colour-change of respective reaction liquid.
(2) result is observed and is judged: if the visualization of color of reaction solution for green, contain in testing sample or doubtfully contain Sweet Potato Feathery Mottle Virus; If the visualization of color of reaction solution be orange, in described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
Experimental result is shown in B in Fig. 1 (1,2 is sterilizing ultrapure water, and 3,4 is SPLCV, and 5,6 is SPVC, and 7,8 is SPVG, and 9,10 is SPSMV-1, and 11,12 is SPFMV, and 13,14 is SPV2).After the EP pipe only adding SPFMV total serum IgE carries out RT-LAMP, reaction solution is green.
The above results shows, the detection Sweet Potato Feathery Mottle Virus that the RT-LAMP test kit of detection Sweet Potato Feathery Mottle Virus provided by the invention can be special.
The sensitivity of the RT-LAMP test kit of the detection Sweet Potato Feathery Mottle Virus of embodiment 4, embodiment 2
One, carry out sensitivity experiment with the RT-LAMP test kit C of the detection Sweet Potato Feathery Mottle Virus of embodiment 2, in triplicate, each step repeated is as follows in experiment:
1, SPFMV total serum IgE is extracted, called after RNA1 with TaKaRaMiniBESTPlantRNAExtractionKit (Beijing six directions leads to Trade Co., Ltd.'s product).In RNA1, the concentration of RNA is 1ng/ μ l.
2, draw 1mlRNA1 to add in the test tube filled in the aseptic ultrapure water of 9ml and fully mix, obtain RNA2; Make RNA3, RNA4, RNA5, RNA6, RNA7 and RNA8 by that analogy.Use BeckmanUV-800 ultraviolet spectrophotometer to measure the RNA concentration of dilution each gradient rear, be respectively 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 100fg/ μ l, 10fg/ μ l, 1fg/ μ l and 100ag/ μ l.
2、RT-LAMP
(1) RNA1 adding 24 μ l reaction reagent C and the preparation of 1 μ l step 1 in test tube obtains reaction solution, is then reacted 60 minutes in 63 DEG C by reaction solution.Take cycle number as X-coordinate, the amount of PCR primer is ordinate zou, obtains the amplification curve that fluorescence detecting system is drawn.2 test tubes are set as repetition.
According to the method described above, RNA1 is replaced with respectively RNA2, RNA3, RNA4, RNA5, RNA6, RNA7, RNA8 and sterilizing ultrapure water prepared by step 1, other step is all constant, obtains corresponding amplification curve.
(2) result observe and judge: if amplification curve present typically " S type ", in testing sample containing or doubtful contain Sweet Potato Feathery Mottle Virus; If amplification curve be rendered as horizontal linear, described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
RT-LAMP result shows, in reaction reagent, add RNA1, RNA2, RNA3, RNA4, RNA5, RNA6 or RNA7 carry out RT-LAMP and all obtain typically " S type " amplification curve, and in reaction reagent, add RNA8 or sterilizing ultrapure water carry out the straight line that amplification curve that RT-LAMP obtains is level.Show, the minimum detection of RT-LAMP test kit to Sweet Potato Feathery Mottle Virus RNA of detection Sweet Potato Feathery Mottle Virus provided by the invention is limited to 25fg.
Two, carry out sensitivity experiment with the RT-LAMP test kit D of the detection Sweet Potato Feathery Mottle Virus of embodiment 2, in triplicate, each step repeated is as follows in experiment:
1, SPFMV total serum IgE is extracted respectively, called after RNA1 with TaKaRaMiniBESTPlantRNAExtractionKit (Beijing six directions leads to Trade Co., Ltd.'s product).In RNA1, the concentration of RNA is 1ng/ μ l.
2, draw 1mlRNA1 to add in the test tube filled in the aseptic ultrapure water of 9ml and fully mix, obtain RNA2; Make RNA3, RNA4, RNA5, RNA6, RNA7 and RNA8 by that analogy.Use BeckmanUV-800 ultraviolet spectrophotometer to measure the RNA concentration of dilution each gradient rear, be respectively 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 100fg/ μ l, 10fg/ μ l, 1fg/ μ l and 100ag/ μ l.
2、RT-LAMP
(1) RNA1 adding 24 μ l reaction reagent D and the preparation of 1 μ l step 1 in EP pipe obtains reaction solution, is then reacted 60 minutes in 63 DEG C by reaction solution.After reaction terminates, the colour-change of observing response liquid.2 EP pipes are set as repetition.
According to the method described above, RNA1 is replaced with respectively RNA2, RNA3, RNA4, RNA5, RNA6, RNA7, RNA8 and sterilizing ultrapure water prepared by step 1, other step is all constant, and other step is all constant.After reaction terminates, observe the colour-change of respective reaction liquid.
(2) result is observed and is judged: if the visualization of color of reaction solution for green, contain in testing sample or doubtfully contain Sweet Potato Feathery Mottle Virus; If the visualization of color of reaction solution be orange, in described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
Experimental result is shown in Fig. 2 (1,2 is sterilizing ultrapure water, and 3,4 is RNA1, and 5,6 is RNA2, and 7,8 is RNA3, and 9,10 is RNA4, and 11,12 is RNA5, and 13,14 is RNA6, and 15,16 is RNA7, and 17,18 is RNA8).After the EP pipe only adding RNA8 or sterilizing ultrapure water carries out RT-LAMP, reaction solution is green.Result shows, the minimum detection of RT-LAMP test kit to Sweet Potato Feathery Mottle Virus RNA of detection Sweet Potato Feathery Mottle Virus provided by the invention is limited to 25fg.
Claims (10)
1. a primer set, be made up of primer SPFMV-F3, primer SPFMV-B3, primer SPFMV-FIP, primer SPFMV-BIP, primer SPFMV-LF and primer SPFMV-LB, each bar primer is single strand dna, and nucleotide sequence is followed successively by sequence 1, sequence 2, sequence 3, sequence 4, sequence 5 and sequence 6 in sequence table.
2. primer set as claimed in claim 1, it is characterized in that: in described primer set, the mol ratio of described primer SPFMV-F3, described primer SPFMV-B3, described primer SPFMV-FIP, described primer SPFMV-BIP, described primer SPFMV-LF and described primer SPFMV-LB is 1:1:8:8:4:4.
3. primer set as claimed in claim 2, it is characterized in that: in described primer set, the amount of each primer is as follows: primer SPFMV-LB described in primer SPFMV-LF, 0.8 μm of ol described in primer SPFMV-BIP, 0.8 μm of ol described in primer SPFMV-FIP, 1.6 μm of ol described in primer SPFMV-B3,1.6 μm of ol described in primer SPFMV-F3,0.2 μm of ol described in 0.2 μm of ol.
4. the preparation method of primer set described in claim 1 is following (I), (II) or (III):
(I) in described primer set, each bar primer is individually packed;
(II) in described primer set, each bar primer mixes according to ratio described in claim 2;
(III) in described primer set, each bar primer mixes according to amount described in claim 3.
5. the application of arbitrary described primer set in claims 1 to 3, for following a) or b) or c) or d):
A) for the preparation of detecting or the test kit of auxiliary detection Sweet Potato Feathery Mottle Virus;
B) detect or in auxiliary detection testing sample whether containing or doubtful containing Sweet Potato Feathery Mottle Virus;
C) for the preparation of qualification or the test kit of assistant identification Sweet Potato Feathery Mottle Virus;
D) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
6. the test kit containing arbitrary described primer set in claims 1 to 3.
7. the application of test kit described in claim 6, for following e) or f):
E) detect or in auxiliary detection testing sample whether containing or doubtful containing Sweet Potato Feathery Mottle Virus;
F) to identify or whether assistant identification virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate.
8. detect the method whether testing sample contains Sweet Potato Feathery Mottle Virus, comprise the steps:
Extract the total serum IgE of testing sample, reverse transcription loop-mediated isothermal amplification is carried out with described primer set arbitrary in claim 1-3, then pass judgment on as follows: if described primer set can realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then in testing sample containing or doubtful containing Sweet Potato Feathery Mottle Virus; If described primer set can not realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then described testing sample not containing or doubtful not containing Sweet Potato Feathery Mottle Virus.
9. the application as described in claim 5 or 7, or method according to claim 8, is characterized in that: and described testing sample is plant sample or microbiological specimens.
10. identify that whether virus to be measured be a method for the Sweet Potato Feathery Mottle Virus of candidate, comprise the steps:
Extract the total serum IgE of virus to be measured, reverse transcription loop-mediated isothermal amplification is carried out with described primer set arbitrary in claim 1-3, then pass judgment on as follows: if described primer set can realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then virus to be measured is the Sweet Potato Feathery Mottle Virus of candidate; If described primer set can not realize increasing to the reverse transcription loop-mediated isothermal of described total serum IgE, then described virus to be measured is the non-Sweet Potato Feathery Mottle Virus of candidate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105886499A (en) * | 2016-01-29 | 2016-08-24 | 中华人民共和国东港出入境检验检疫局 | LAMP kit for detecting phytophthora sojae kaufmann et gerdemann and special primer of LAMP kit |
| CN111057799A (en) * | 2019-11-22 | 2020-04-24 | 浙江大学 | Method for rapidly detecting pinnate mottle virus of sweet potato by using micro-fluidic chip and used primer |
| CN113322353A (en) * | 2021-06-15 | 2021-08-31 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
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| CN104450977A (en) * | 2015-01-06 | 2015-03-25 | 西南大学 | Primer for sweet potato virus real-time fluorescence quantification PCR detection and detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886499A (en) * | 2016-01-29 | 2016-08-24 | 中华人民共和国东港出入境检验检疫局 | LAMP kit for detecting phytophthora sojae kaufmann et gerdemann and special primer of LAMP kit |
| CN111057799A (en) * | 2019-11-22 | 2020-04-24 | 浙江大学 | Method for rapidly detecting pinnate mottle virus of sweet potato by using micro-fluidic chip and used primer |
| CN113322353A (en) * | 2021-06-15 | 2021-08-31 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
| CN113322353B (en) * | 2021-06-15 | 2022-08-26 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
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