CN105177188A - Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses - Google Patents
Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses Download PDFInfo
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Abstract
The invention discloses quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and a method capable of simultaneously detecting multiple chili viruses. The method adopts four pairs of primers disclosed as SEQ ID NO:1-SEQ ID NO:8; and one RT-PCR process can simultaneously detect four chili viruses: Broad bean wilt virus 2 (BBWV 2), Pepper vein yellows virus (PeVYV), Chilli veinal mottle virus (ChiVMV) and Pepper mildmottle virus (PMMoV). Compared with the existing single RT-PCR and duplex RT-PCR detection methods, the method disclosed by the invention can simultaneously detect the four chili viruses by one RT-PCR process, and is capable of detecting more virus varieties, greatly enhancing the detection efficiency and obviously lowering the detection cost.
Description
Technical field
The invention belongs to crop virus detection techniques field, be specifically related to one and detect multiple capsicum virus quadruple RT-PCR detection primer and detection method simultaneously.
Background technology
Capsicum (CapsicumannuumL.) is Solanaceae capsicum plants, be 1 year or limited per nnial herb, fruit usually conically or Long Circle, in green during prematurity, bright red, green or purple is become after maturation, the most common with redness, range of distribution originally in Mexico to Ge Lunbiya; All there is cultivation all over the world now.Capsicum has centuries cultivation history in China, is important vegetables and seasonings, is one of Main Vegetable Species Suitable For Culture of China's establishing in large scale at present.Capsicum is except being a kind of important vegetable crop, its nutritive value is very high, Vitamin C content accounts for first place in vegetables, it is 7-15 times of tomato, capsaicine in addition in capsicum and Dihydrocapsaicin are main pungent flavor material sources, haematochrome in capsicum is one of current the most widely used natural food Agent, and capsicum also has the effect of sweating, expelling parasite simultaneously.
Since 20 century 70s, because the interracial interchange in various places is frequent, and interchange scope constantly expands, and the reason such as cropping system, and China's pepper virus disease occurrence scope becomes wide, and hazard rating increases the weight of, and has had a strong impact on the yield and quality of capsicum.The people such as Yang Yonglin are in Beijing, Tianjin, the Liao Dynasty, Soviet Union, lucky, new six areas, gathering peppery (sweet) green pepper Virus Sample 3618 adopts three routines (biological, serum, Electronic Speculum) identification of means identifies tobacco mosaic virus (TMV) (Tobaccomosaicvirus, TMV), cucumber mosaic virus (Cucumbermosaicvirus, CMV), potato virus X (PotatovirusX, PVX), marmor upsilon (PotatovirusY, PVY), marmor erodens (Tobaccoetchvirus, TEV), alfalfa mosaic virus (AlfalfaMosaicVirus, the multiple virus such as AMV).According to current, have at least the virus of kind more than 40 can infect peppery (sweet) green pepper, and majority state infect the most general with tobacco mosaic virus (TMV) (TMV) and cucumber mosaic virus (CMV).The RNA viruses that China peppery (sweet) green pepper has identified has 17 kinds, comprise CMV, TMV, marmor erodens (Tobaccoetchvirus, TEV), marmor upsilon (PotatovirusY, PVY), potato virus X (PotatovirusX, PVX), capsaicinoid ointment (Peppermildmottlevirus, PMMoV), alfalfa mosaic virus (Alfalfamosaicvirus, AMV), Broad bean wilt virus 2 (Broadbeanwiltvirus2, BBWV2), Tobacco rattle virus (Tobaccorattlevirus, TRV), capsicum ring spot virus (Chilliringspotvirus, ChiRSV), tomato spotted wilf virus (Tomatospottedwiltvirus, TSWV), peanut macula lutea virus (Groundnutyellowspotvirus, GYSV), Capsicum chlorosis virus (Capsicumchlorosisvirus, CaCV), capsicum arteries and veins mottle virus (Chilliveinalmottlevirus, ChiVMV), the light-duty green mosaic virus (Tobaccomildgreenmosaicvirus of tobacco, TMGMV), the tomato chlorisis virus (Tomatochlorosisvirus found on pimento first this year, and the capsicum vein yellows poison (Pepperveinyellowsvirus that detects of Hunan ToCV), PeVYV), those virus diseases generally cause the harm of 10% ~ 30%, can the underproduction 50% ~ 80% time serious.
Plant virus detection method conventional at present has biological method (plant indicator method), serological method (euzymelinked immunosorbent assay (ELISA)), electron microscope method, inverse transcription polymerase chain reaction (Reversetranscriptionpolymerasechainreaction, RT-PCR).Wherein, biology method measuring length consuming time, workload is large, sensitivity is poor, also usually shows similar symptoms after different virus infects, and symptom display also can be subject to the impact of Combined Infection.May be there is false negative and false-positive phenomenon in Serology test, and for detecting the less test of sample, the cost of test kit is higher within the specific limits.The detection each time of biological method and serological method can only for single virus, and detection efficiency is low.Electron microscopy is for Viraceae or belong to following classification and can not judge, and apparatus expensive, sample size is unsuitable excessive.And RT-PCR is because its specificity is the strongest, sensitivity is the highest, become the prefered method of current Pathogen test, and be widely used in plant virus detection.For multiple viral Combined Infection, can detect multiple virus simultaneously, can detect great amount of samples at short notice, improve detection efficiency, reduce testing cost, and testing cost is low by multiplex RT-PCR method, be that other detection method can not be compared.But capsicum virus substance RT-PCR detect delay is more, and multiple RT-PCR research is less.KumarS etc. establish a kind of dual RT-PCR can detect TMV and ToMV two-strain in capsicum or tomato simultaneously.Develop multiple multiple detection method both at home and abroad to the common virus such as TMV, CMV, PVY, ToMV, however domestic report do not find corresponding multiple detection method with the capsicum virus of latest find.
The conserved sequence of the viral full-length genome that the present invention issues according to Genbank or partial sequence, design universal primer, be intended to set up one simply, efficiently, sensitive quadruple RT-PCR detection technique, the capsicum simultaneously detecting domestic discovery compares the virus that common virus and latest find may exist outburst trend: Broad bean wilt virus 2 (Broadbeanwiltvirus2, BBWV2), capsaicinoid ointment (Peppermildmottlevirus, PMMoV), capsicum arteries and veins mottle virus (Chilliveinalmottlevirus, ChiVMV), capsicum vein yellows poison (Pepperveinyellowsvirus), for the Viral diagnosis studying domestic pepper virus disease popularity and detoxification capsicum kind provides technical support.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, a kind of quadruple RT-PCR simultaneously detecting multiple capsicum virus is efficiently provided to detect primer and method, capsicum 4 kinds of viruses for domestic discovery: Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV), these the 4 kinds of viral full-length genome issued according to Genbank or the conserved sequence of partial sequence, design primer, and provide quadruple RT-PCR detection method of the present invention to detect this 4 kinds of viruses simultaneously.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: the genome conserved sequence for China's 4 kinds of capsicum viruses (Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV)) designs 4 pairs of primers, and set up quadruple RT-PCR reaction system and program, to detect this 4 kinds of capsicum viruses simultaneously.
Detection method is specific as follows:
1. design of primers:
The above-mentioned 4 kinds of capsicums virus full-length genome issued according to Genbank or the conserved sequence of partial sequence, and according to the requirement that quadruple RT-PCR product varies in size, devise four pairs of primers (table 1), wherein primer pair BBWV2-F and BBWV2-R is the primer detecting Broad bean wilt virus 2 (BBWV2), and clip size is 693bp; Primer pair PevYV-F and PevYV-R is the primer detecting capsicum vein yellows poison (PeVYV), and clip size is 383bp; Primer pair ChiVMV-F and ChiVMV-R is the primer detecting capsicum vein mottle virus (ChiVMV), and clip size is 513bp; Primer pair PMMoV-F and PMMoV-R is the primer detecting capsaicinoid ointment (PMMoV), and clip size is 236bp.
Table 1 quadruple RT-PCR detects the primer of capsicum virus and the virus of detection thereof
2.RT-PCR process:
1) acquisition of pepper plant blade RNA: the capsicum young leaflet tablet getting field flower leaf paresthesia, preserves on ice, takes Trizol method to extract total serum IgE;
2) with step 1) RNA that obtains is template, with 6 base random primer RandomPrimers for reverse primer, by reverse transcription, obtains cDNA;
3) with step 2) cDNA that obtains is template, with pair primer pair of four in table 1, it carries out pcr amplification.
Above-mentioned Quadruple-PCR reaction system, mends to volume required by 50 μ l: 10xPCRBUFFER5 μ l, dNTPs5 μ l, PCRTaq2 μ l, template (cDNA) 2.5 μ l, Broad bean wilt virus 2 forward primer and each 0.5 μ l of reverse primer, capsicum vein yellows poison forward primer and each 1.25 μ l of reverse primer, capsicum vein mottle virus forward primer and each 1 μ l of reverse primer, capsaicinoid ointment forward primer and each 1.25 μ l of reverse primer, ultrapure water.
PCR program: 94 DEG C of 10min denaturations; 40 loop parameters are, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 1min; Last 72 DEG C of 10min.
4) PCR primer gel electrophoresis, gel imaging system is taken pictures, and according to object clip size and positive control, judges Virus Infection.
The present invention has following beneficial effect: detect 4 kinds of common capsicum viruses with quadruple RT-PCR, compare with dual RT-PCR detection method with current substance RT-PCR, can detect 4 kinds of viruses with a RT-PCR, the viral species of detection increases, detection efficiency improves, and the cost of detection significantly reduces.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that substance and quadruple RT-PCR detect Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV).
Wherein, M:DNAmarker; 1 is the Quadruple-PCR product of Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV); 2,3,4,5 PCR primer being respectively Broad bean wilt virus 2 (BBWV2), capsicum vein mottle virus (ChiVMV), capsaicinoid ointment (PMMoV) and capsicum vein yellows poison (PeVYV).
Fig. 2 is the electrophorogram that under different annealing temperature, quadruple RT-PCR detects Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV).
Wherein, M:DNAmarker; The temperature of 1-6 representative is followed successively by 60 DEG C, 58 DEG C, 56 DEG C, 54 DEG C, 52 DEG C and 50 DEG C.
Fig. 3 is the electrophorogram that different extension rate template quadruple RT-PCR detects Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV).
Wherein, M:DNAmarker; Template starting point concentration is that the RNA concentration of 125ng/ μ l, 1-6 representative is followed successively by 10 of starting point concentration
0, 10
-1, 10
-2, 10
-3, 10
-4with 10
-5doubly.
Fig. 4 is the electrophorogram that quadruple RT-PCR detects Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV) in the pepper plant sample of Hunan.
Wherein, M:DNAladder; Sun represents positive control; CK represents negative control; Swimming lane 1-10 is Liuyang City of Hunan Province sample, and swimming lane 11-15 is Furong District, Changsha, Hunan capsicum sample.
Embodiment
Below in conjunction with specific embodiment, the present invention is explained further, but specific embodiment does not do any restriction to the present invention.
Embodiment 1
1, design of primers
Due to needs detection 4 kinds of capsicum viruses, in order to set up, the viral species with detection is many, detecting step simple, accurate, economic dispatch feature multiple RT-PCR detection method, conserved sequence according to Genbank Broad bean wilt virus 2 (BBWV2) virus designs design of primers primer pair (forward primer BBWV2-F and reverse primer BBWV2-R) with Primer6.0, and the object clip size of amplification is 693bp; According to the conserved sequence of capsicum vein yellows poison (PeVYV), design primer pair (forward primer PevYV-F and reverse primer PevYV-R), the object clip size of amplification is 383bp; Primer pair (forward primer ChiVMV-F and reverse primer ChiVMV-R is designed respectively equally according to the conserved sequence of capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV), and forward primer PMMoV-F and reverse primer PMMoV-R), the object clip size of amplification is respectively 513bp and 236bp; Primer sequence is shown in as table 1.The clip size of each pair of primer amplification is respectively 693,383,513 and 236bp, clip size differs greatly, there will not be overlap, can clearly separate, during design primer, eliminate the complementation between mispairing between each pair of primer and other several virus and each primer.Object fragment, through cloning and sequencing, is the specific fragment of each virus.
Above-mentioned 4 pairs of primers are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
2, the extraction of capsicum total serum IgE
Variable rate technology is taked to go out the Pepper Leaves sample of Ming Mai, floral leaf, dwarfing, shrinkage, curling, yellow, the symptom such as leaf, Cong Zhi of fainting, 2.5g got by each sample, after adding liquid nitrogen grinding, RNA test kit is adopted to extract total serum IgE, the quality of electrophoresis and UV spectrophotometer measuring RNA and concentration.
3, respectively substance RT-PCR and quadruple RT-PCR amplification is carried out to the total serum IgE extracted with above-mentioned primer.
Transcriptive process,reversed is as follows:
RNA sex change: get RNA2.5 μ l65 DEG C temperature bath 8 minutes, cooled on ice;
Configuration inverse transcription reaction liquid (7.5 μ l): 5 × reaction buffer 2 μ l, 100mMDTT1 μ l, 10mMdNTPs1 μ l, RNA enzyme inhibitors 0.125 μ l, ThermoScript II 0.5 μ l, 10 μMs of random primer 0.5 μ l, ultrapure water is mended to 7.5 μ l.
Join in 2.5 μ lRNA of sex change by 7.5 μ l reaction solutions, mixing, 42 DEG C extend 1 hour, 95 DEG C of sex change 2 minutes.
PCR process:
Substance PCR reaction system (50 μ l): 10xPCRBUFFER5 μ l, dNTPs5 μ l, PCRTaq2 μ l, template (cDNA) 2.5 μ l, add forward primer in table 1 and each 2 μ l of reverse primer, ultrapure water are mended to volume required respectively.
Quadruple-PCR reaction system (50 μ l): 10xPCRBUFFER5 μ l, dNTPs5 μ l, PCRTaq2 μ l, template (cDNA) 2.5 μ l, Broad bean wilt virus 2 forward primer and each 0.5 μ l of reverse primer, capsicum vein yellows poison forward primer and each 1.25 μ l of reverse primer, capsicum vein mottle virus forward primer and each 1 μ l of reverse primer, capsaicinoid ointment forward primer and each 1.25 μ l of reverse primer, ultrapure water are mended to volume required.
PCR program: 94 DEG C of 2min denaturations; 40 loop parameters are, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 1min; Last 72 DEG C of 10min.
4, gel electrophoresis
Get 10 μ lPCR products with 2.0% agarose gel electrophoresis, gel imaging system take a picture.The results are shown in Figure 1.Result shows that quadruple RT-PCR can detect Broad bean wilt virus 2 (BBWV2), capsicum vein yellows poison (PeVYV), capsicum vein mottle virus (ChiVMV) and capsaicinoid ointment (PMMoV) simultaneously.
Embodiment 2 different annealing temperature and different templates extension rate are on the impact of quadruple RT-PCR Detection results
Because the Tm value of often pair of primer is not quite similar, therefore, need to screen suitable annealing temperature, make quadruple RT-PCR obtain four object fragments, improve detection sensitivity.Annealing temperature is set to 60 DEG C, 58 DEG C, 56 DEG C, 54 DEG C, 52 DEG C and 50 DEG C totally 6 thermogrades, and PCR primer electrophoresis result is shown in Fig. 2.Result shows that annealing temperature is when 58-54 DEG C, and quadruple RT-PCR can obtain 4 object fragments clearly.
For the sensitivity that inspection quadruple RT-PCR detects, Initial R NA concentration 125ng/ μ l, dilute 10 respectively, 100,1000 and 10000 times and 100000, namely RNA concentration is respectively 10 of starting point concentration
-1, 10
-2, 10
-3with 10
-4with 10
-5doubly.PCR primer electrophoresis result is shown in Fig. 3, when visible Initial R NA dilutes 10-100 times, can also amplify 4 object fragments, the highly sensitive of this RT-PCR detection technique is described.
Embodiment 3 quadruple RT-PCR detects field pepper plant sample
In Furong District, Changsha City, Hunan Province and Liuyang City's capsicum main producing region field investigation with take the pepper plant with yellow and flower leaf paresthesia, plant band soil zone goes back to laboratory, choose young leaflet tablet, RNA is extracted after liquid nitrogen grinding, detect its Virus Infection with the quadruple RT-PCR established, detected result is shown in Fig. 4.Result shows that quadruple RT-PCR well can detect the virus of main producing region, Hunan capsicum sample, the situation of the pepper plant sample ubiquity 3-4 kind virus Combined Infection of Changsha and Liang Ge main producing region, Liuyang.
Claims (5)
1. the quadruple RT-PCR simultaneously detecting multiple capsicum virus detects primer, this capsicum virus is: Broad bean wilt virus 2, capsicum vein yellows poison, capsicum vein mottle virus and capsaicinoid ointment, it is characterized in that, described primer comprises following 4 pairs of primers:
The primer pair detecting Broad bean wilt virus 2 is:
BBWV2-F：CTCCTGTGGTGGTTATTCA，
BBWV2-R：TTCCTATGCGTGTTATCGT；
The primer pair detecting capsicum vein yellows poison is:
PevYV-F：GAAGAAGCCGAGATAGAGTT，
PevYV-R：ATAGAGCAGCCTGAATTGAT；
The primer pair detecting capsicum vein mottle virus is:
ChiVMV-F：AACCTTGAACACCTTCTTGA，
ChiVMV-R：GTCCAGTCCGAACATCCT；
The primer pair detecting capsaicinoid ointment is:
PMMoV-F：GTCCAACAGCAGTTCTCT，
PMMoV-R：CTAATAGCCACCGTAGCAT。
2. detect a quadruple RT-PCR detection method for multiple capsicum virus simultaneously, it is characterized in that, the method extracts capsicum total serum IgE; With the RNA obtained for template, by reverse transcription, obtain cDNA; With the cDNA obtained for template, with 4 pairs of primer pairs according to claim 1, it carries out pcr amplification, last electroresis appraisal.
3. detect the quadruple RT-PCR detection method of multiple capsicum virus as claimed in claim 2 simultaneously, it is characterized in that: described PCR reaction system is: 10xPCRBUFFER5 μ l, dNTPs5 μ l, PCRTaq2 μ l, cDNA template 2.5 μ l, Broad bean wilt virus 2 forward primer and each 0.5 μ l of reverse primer, capsicum vein yellows poison forward primer and each 1.25 μ l of reverse primer, capsicum vein mottle virus forward primer and each 1 μ l of reverse primer, capsaicinoid ointment forward primer and each 1.25 μ l of reverse primer, ultrapure water are mended to 50 μ l.
4. detect the quadruple RT-PCR detection method of multiple capsicum virus as claimed in claim 2 simultaneously, it is characterized in that: described PCR response procedures is: 92 DEG C of 10min; 94 DEG C of 30s, 54-58 DEG C of 30s, 72 DEG C of 1min, 40 circulations; 72 DEG C of 10min.
5. containing the quadruple RT-PCR detection kit detecting multiple capsicum virus while primer pair described in claim 1.
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Cited By (9)
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| CN105695626A (en) * | 2016-01-25 | 2016-06-22 | 湖南农业大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer and method for detecting pepper vein yellows virus (PeVYV) |
| CN105755177A (en) * | 2016-04-27 | 2016-07-13 | 湖南农业大学 | Quadruple RT-PCR method simultaneously detecting multiple kinds of pepper viruses and application of method |
| CN106258076A (en) * | 2016-08-16 | 2017-01-04 | 新疆农业大学 | A kind of remove the method for Broad bean wilt virus 2 in pepper seed |
| CN106755597A (en) * | 2017-02-23 | 2017-05-31 | 中国农业科学院植物保护研究所 | A kind of 4 kinds of multiple PCR methods of capsicum virus of synchronous detection |
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| CN105695626A (en) * | 2016-01-25 | 2016-06-22 | 湖南农业大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer and method for detecting pepper vein yellows virus (PeVYV) |
| CN105755177A (en) * | 2016-04-27 | 2016-07-13 | 湖南农业大学 | Quadruple RT-PCR method simultaneously detecting multiple kinds of pepper viruses and application of method |
| CN106258076B (en) * | 2016-08-16 | 2019-12-06 | 新疆农业大学 | Method for removing broad bean wilting virus No. 2 from pepper seeds |
| CN106258076A (en) * | 2016-08-16 | 2017-01-04 | 新疆农业大学 | A kind of remove the method for Broad bean wilt virus 2 in pepper seed |
| CN106755597A (en) * | 2017-02-23 | 2017-05-31 | 中国农业科学院植物保护研究所 | A kind of 4 kinds of multiple PCR methods of capsicum virus of synchronous detection |
| CN107937609A (en) * | 2017-12-13 | 2018-04-20 | 沈阳大学 | One kind detection capsicum arteries and veins mottle virus primer special and its detection method |
| CN107937609B (en) * | 2017-12-13 | 2020-06-02 | 沈阳大学 | Special primer for detecting pepper vein mottle virus and detection method thereof |
| CN109061161B (en) * | 2018-11-14 | 2019-02-15 | 湖南省植物保护研究所 | For detecting the detection kit and its application of capsicum arteries and veins flavivirus |
| CN109061161A (en) * | 2018-11-14 | 2018-12-21 | 湖南省植物保护研究所 | For detecting the detection kit and its application of capsicum arteries and veins flavivirus |
| CN111808992A (en) * | 2020-07-15 | 2020-10-23 | 福建农林大学 | Hot pepper vein mottle virus polygene joint detection and identification method |
| CN112029907A (en) * | 2020-09-04 | 2020-12-04 | 云南省农业科学院生物技术与种质资源研究所 | Method for synchronously detecting five important virus pathogens in ranunculus asiaticus |
| CN112029907B (en) * | 2020-09-04 | 2023-04-11 | 云南省农业科学院生物技术与种质资源研究所 | Method for synchronously detecting five important virus pathogens in ranunculus asiaticus |
| CN112195288A (en) * | 2020-11-19 | 2021-01-08 | 广西壮族自治区农业科学院 | Multiple RT-PCR primer group for simultaneously detecting four viruses of hot pepper and method thereof |
| CN112195288B (en) * | 2020-11-19 | 2024-04-26 | 广西壮族自治区农业科学院 | Multiplex RT-PCR primer group for simultaneously detecting four viruses of capsicum and method thereof |
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