CN102140555A - Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof - Google Patents
Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof Download PDFInfo
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Abstract
The invention discloses degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, a detection method and application thereof. The degenerate primers consist of a positive primer and a negative primer; the nucleotide sequence of the positive primer is shown as SEQIDNo.1:5'-TGYGAYTAYDVHWSWTTYGATGG-3'; and the nucleotide sequence of the negative primer is shown as SEQIDNo.2: 5'-AKYARRTTRTCATCHCCATA-3', wherein Y=C/T, D=G/A/T, V=G/A/C, H=A/T/C, W=A/T, S=G/C, K=G/T and R=A/G. The detection method can quickly and accurately judge whether a sample contains the cowpea mosaic virus and fabavirus, variety identification is carried out according to a sequence, and guarantee for import and export safety is provided.
Description
Technical field
The present invention relates to a kind of Comovirus and fabavirus and belong to viral general RT-PCR detection degenerated primer, detection method and application thereof.
Background technology
Comovirus (
Comovirus) and the fabavirus genus (
Fabavirus) belong to class picornavirus order (
Picornavirales), association cowpea Viraceae (
Secoviridae), the cowpea mosaic virus subfamily (
Comovirinae) in 2 Tobamovirus.Comovirus contains 15 kinds of viruses, and the host range of single virus is narrow, and majority only limits to the minority leguminous plants, cowpea mosaic virus (
Cowpea mosaic virus, CPMV) be its representative species.Fabavirus belongs to and to contain 4 kinds of viruses, has the host range of broad, No. 1, broad bean wilt virus (
Broad bean wilt virus 1, BBWV-1) be its representative species.To be classes cause the virus of important threat to agriculture production safety to these two Tobamovirus, and multiple virus can cause heavy losses to its host plant.According to " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register " that announced in 2007, the kind that is listed in the venereal disease poison of quarantining in these two Tobamovirus has: broad bean dyeing virus (
Broad bean stain virus, BBSV), bean pod mottle virus (
Bean pod mottle virus, BPMV), cowpea severe mosaic virus (
Cowpea severe mosaic virus, CPSMV) wait 3 kinds of viruses.Ferrer in 2007 etc. set up the fabavirus genus (
Fabavirus) viral general RT-PCR detects authentication method, but also do not set up Comovirus (
Comovirus) general RT-PCR detection method or can be used for the general RT-PCR method of these two Tobamovirus simultaneously.
Summary of the invention
Having the object of the present invention is to provide a kind of Comovirus and fabavirus to belong to viral general RT-PCR detects with degenerated primer, detection method and application thereof.Whether detection method of the present invention judgement sample rapidly and accurately has Comovirus and fabavirus to belong to virus, carries out kind according to sequence and identifies, for imports and exports safety provides assurance.
In order to reach above-mentioned purpose, solution of the present invention is:
A kind of Comovirus and fabavirus belong to viral general RT-PCR detection and use degenerated primer, are made up of forward and reverse primer, and the nucleotide sequence of its forward primer is seen SEQ ID No.1:5 '-TGYGAYTAYDVHWSWTTYGATGG-3 '; The nucleotide sequence of reverse primer is seen SEQ ID No.2:5 '-AKYARRTTRTCATCHCCATA-3 '; Y=C/T wherein, D=G/A/T, V=G/A/C, H=A/T/C, W=A/T, S=G/C, K=G/T, R=A/G.
A kind of Comovirus and fabavirus belong to the detection method that viral general RT-PCR detects, comprise the steps: that with the total RNA of sample be template, utilize above-mentioned primer to carry out the RT-PCR amplification, reaction finishes back detected through gel electrophoresis amplified production, according to the location determination result of amplification of DNA fragments, all can detect the dna fragmentation of 326 ~ 343 bp in the illness blade.
Described RT-PCR reaction comprises reverse transcription and two processes of pcr amplification.
The temperature of described reverse transcription reaction is 42 ℃, and the elder generation of pcr amplification carries out 5 circulations with 42 ℃ of annealing temperatures, carries out 30 circulations with 52 ℃ of annealing temperatures then, and elongating temperature is 72 ℃.
A kind of Comovirus and fabavirus belong to viral general RT-PCR and detect the application of using degenerated primer, described primer carries out RT-PCR amplification back to behind the further sequencing of the dna fragmentation that is increased to the total RNA of sample, the viral species that energy Rapid identification Comovirus and fabavirus belong to.
A kind of Comovirus and fabavirus belong to viral general RT-PCR and detect application with degenerated primer, and described Comovirus and fabavirus belong to viral general RT-PCR and detect with degenerated primer and can be applied to Comovirus and fabavirus belongs to viral general RT-PCR detection kit.
Beneficial effect of the present invention is: the present invention belongs to RdRp gene order design degenerated primer on viral RNA 1 genome according to Comovirus and fabavirus, and this primer can be used for the general RT-PCR that Comovirus and fabavirus belong to virus and detects.And primer of the present invention and related reagent can be assembled into test kit, uses with convenient.Whether detection method of the present invention judgement sample rapidly and accurately has Comovirus and fabavirus to belong to virus, carries out kind according to sequence and identifies, for imports and exports safety provides assurance.
Description of drawings
Fig. 1 is that Comovirus and fabavirus belong to the versatility test-results of viral general RT-PCR method to various viruses.1-8 is the detected result of Comovirus virus, and 9 for broad bean wilt virus belongs to viral detected result, and 10 is the detected result of healthy broad bean blade.That is, 1 is that cowpea mosaic virus (CPMV) (isolate 1), 2 is healthy broad bean blade for broad bean wilt virus No. 1 (BBWV1), 10 for Flos Cucurbitae mosaic virus (SqMV), 9 for red clover mottle virus (RCMV), 8 for radish mosaic virus (RaMV), 7 for bean pod mottle virus (BPMV), 6 for cowpea severe mosaic virus (CPSMV) (isolate 2), 5 for cowpea severe mosaic virus (CPSMV) (isolate 1), 4 for cowpea mosaic virus (CPMV) (isolate 2), 3.
Fig. 2 is that Comovirus and fabavirus belong to viral general RT-PCR method specific detection result.1-7 is Nepovirus virus (being respectively ArMV, TRSV, ToRSV, TBRV, PRMV, CLRV, RpRSV).
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
Embodiment 1
1, design of primers and synthetic
According to the RNA1 genome sequence design primer of RaMV, SqMV, RCMV, CPMV, CPSMV, BPMV, BBWV1 virus in Comovirus and the fabavirus genus, primer sequence is:
SEQ?ID?No.1：5′-?TGYGAYTAYDVHWSWTTYGATGG?-3′；
SEQ?ID?No.2：5′-?AKYARRTTRTCATCHCCATA?-3′。
2, the extraction of total RNA
1) gets the sick leaf of 0.1 g, add the TRIzol reagent (American I nvitrogen company) of 1 ml, move in the 1.5 m L centrifuge tubes of sterilization, keep 5 min under the room temperature;
2) add 0.2 mL chloroform, thermal agitation 15 s, at room temperature keep 2-15 min then after, 4 ℃, centrifugal 15 min of 12000 g;
3) with the upper water phase transition in 1.5 new m L centrifuge tubes, add 0.5 m L Virahol, put upside down mixing, keep 15 min under the room temperature;
4) 4 ℃, centrifugal 10 min of 12000 g, RNA will form precipitation in the sidewall and the bottom of pipe;
5) outwell supernatant liquor, add 75% washing with alcohol precipitation, 4 ℃ then, centrifugal 5 min(of 7500 g suspend as precipitation, then use 12000 g centrifugal), discard ethanol;
6) be deposited under the room temperature after the thorough drying, be dissolved in the water of 40 μ L nuclease free ,-20 ℃ of preservations are standby.
3, the foundation of RT-PCR amplification method
With total RNA standby in the step 2 is template, carries out the RT-PCR reaction.20 μ L reaction systems are carried out reverse transcription reaction, promptly add the water of 8.75 μ L nuclease free in the reaction tubes of 0.2 mL, the total RNA of 4 μ L, the oligo of 1 μ L (d T)
15Primer (10 μ mol/L) (precious biotechnology (Dalian) company limited), 1 μ L dNTP mixture (being specially dATP, dGTP, dTTP and four kinds of mixtures of dCTP) (10 mmol/L) (precious biotechnology (Dalian) company limited), in 65 ℃ of maintenance 5 min, transfer to quenching 5min on ice rapidly behind the mixing; In reaction tubes, add then, 4 μ L, 5 * reverse transcription damping fluid (U.S. Pu Luomaige company), 1 μ L RNA enzyme inhibitors (40 U/ μ L) (precious biotechnology (Dalian) company limited), 0.25 μ L AMV ThermoScript II (200 U/ μ L) (U.S. Pu Luomaige company), 42 ℃ of reaction 50 min; Obtain reverse transcription product cDNA after 72 ℃ of 15 min deactivation ThermoScript II.
The PCR reaction system is 25 μ L, the water that promptly in the PCR of 0.2 mL reaction tubes, adds 13.25 μ L nuclease free, 3 μ L cDNA, 2 μ L forward primer SEQ ID No.1(10 μ mol/L), 2 μ L reverse primer SEQ ID No.2(10 μ mol/L), the precious biotechnology (Dalian) of 10 * PCR damping fluid, 2.5 μ L(company limited), 2 μ L dNTP(2.5 mmol/L) (precious biotechnology (Dalian) company limited), 0.25 μ L Taq archaeal dna polymerase (5 U/ μ L) (precious biotechnology (Dalian) company limited).Reaction conditions is: 95 ℃ of pre-sex change 3 min; 94 ℃ of sex change 50 s, 42 ℃ of annealing 50 s, 72 ℃ of extension 50 s, 5 circulations; Follow 94 ℃ of sex change 50 s, 52 ℃ of annealing 50 s, 72 ℃ of extension 50 s, 30 circulations; Last 72 ℃ are extended 7 min.On the PCR instrument, finish.
PCR reaction finishes laggard row agarose gel electrophoresis and detects, and judges whether contain Comovirus in the sample and fabavirus belongs to virus according to the dna fragmentation of 326 ~ 343 bp that whether increase.Whether present embodiment detection method judgement sample rapidly and accurately has Comovirus and fabavirus to belong to virus, carries out kind according to sequence and identifies, for imports and exports safety provides assurance.Comovirus and fabavirus belong to virus RT-PCR and detect and further can make Comovirus and fabavirus with degenerated primer and belong to viral general RT-PCR detection kit.
Embodiment 2
The versatility that Comovirus and fabavirus belong to general RT-PCR method detects
Get the sick leaf and the healthy broad bean blade of cowpea mosaic virus (CPMV), cowpea severe mosaic virus (CPSMV), bean pod mottle virus (BPMV), radish mosaic virus (RaMV), red clover mottle virus (RCMV), Flos Cucurbitae mosaic virus (SqMV), No. 19 isolates of 7 kinds of virus such as (BBWV1) of broad bean wilt virus, extract total RNA respectively by embodiment 1 method, carry out the RT-PCR amplification again, the agarose gel electrophoresis detected result.All detect the dna fragmentation of about 350 bp as a result in the illness blade.Detected result as shown in Figure 1.Show that the general RT-PCR method of foundation can be used for the detection of different virus kind in these two Tobamovirus, have good versatility, can be used for the general detection that Comovirus and fabavirus belong to virus.
Embodiment 3
Comovirus and fabavirus belong to general RT-PCR method specific detection get the sick leaf of Nepovirus virus (being respectively ArMV, TRSV, ToRSV, TBRV, PRMV, CLRV, RpRSV) that belongs to the cowpea mosaic virus subfamily, extract total RNA respectively, carry out the RT-PCR amplification by embodiment 1 method, carry out specific detection.Detected result as shown in Figure 2.The general RT-PCR method that shows foundation can't produce cross reaction with the virus of same Tobamovirus, has good specificity, the requirement of coincidence detection.
Sequence table
＜110〉Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau
＜120〉Comovirus and fabavirus belong to viral general RT-PCR detection degenerated primer
<130>
<160> 2
<170> PatentIn?version?？
<210> 1
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 1
TGYGAYTAYD?VHWSWTTYGA?TGG 23
<210> 2
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 2
AKYARRTTRT?CATCHCCATA 20
Claims (6)
1. Comovirus and fabavirus belong to viral general RT-PCR and detect and use degenerated primer, it is characterized in that being made up of forward and reverse primer, the nucleotide sequence of its forward primer is seen SEQ ID No.1:5 '-TGYGAYTAYDVHWSWTTYGATGG-3 '; The nucleotide sequence of reverse primer is seen SEQ ID No.2:5 '-AKYARRTTRTCATCHCCATA-3 '; Y=C/T wherein, D=G/A/T, V=G/A/C, H=A/T/C, W=A/T, S=G/C, K=G/T, R=A/G.
2. Comovirus as claimed in claim 1 and fabavirus belong to the detection method that viral general RT-PCR detects, it is characterized in that comprising the steps: that with the total RNA of sample be template, utilize above-mentioned primer to carry out the RT-PCR amplification, reaction finishes back detected through gel electrophoresis amplified production, according to the location determination result of amplification of DNA fragments, infect the dna fragmentation that all can detect 326 ~ 343 bp in the illness blade of these two Tobamovirus viruses.
3. a kind of Comovirus as claimed in claim 2 and fabavirus belong to viral general RT-PCR and detect the detection method of using degenerated primer, it is characterized in that: described RT-PCR reaction comprises reverse transcription and two processes of pcr amplification.
4. a kind of Comovirus as claimed in claim 3 and fabavirus belong to the detection method that viral general RT-PCR detects, it is characterized in that: the temperature of described reverse transcription reaction is 42 ℃, the elder generation of pcr amplification carries out 5 circulations with 42 ℃ of annealing temperatures, carry out 30 circulations with 52 ℃ of annealing temperatures then, elongating temperature is 72 ℃.
One kind Comovirus and fabavirus belong to viral general RT-PCR and detect application with degenerated primer according to claim 1, it is characterized in that: described primer carries out RT-PCR amplification back to behind the further sequencing of the dna fragmentation that is increased to the total RNA of sample, can Rapid identification Comovirus and the viral kind of fabavirus genus.
One kind according to claim 1 Comovirus and fabavirus belong to viral general RT-PCR and detect application with degenerated primer, it is characterized in that: described Comovirus and fabavirus belong to viral general RT-PCR and detect with degenerated primer and can be applied to Comovirus and fabavirus belongs to viral general RT-PCR detection kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898237A (en) * | 2014-01-09 | 2014-07-02 | 吉林大学 | Degenerate primer RT-PCR detection method for eimeria viruses |
| CN106148567A (en) * | 2016-07-08 | 2016-11-23 | 厦门出入境检验检疫局检验检疫技术中心 | A kind of cowpea mosaic virus subfamily virus wide spectrum detection kit and method thereof |
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| CN101215611A (en) * | 2008-01-11 | 2008-07-09 | 中华人民共和国厦门出入境检验检疫局 | Primer for detecting southern cowpea mosaic virus |
| CN101818211A (en) * | 2009-12-16 | 2010-09-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Molecule detection method for cowpea severe mosaic virus |
| CN101824485A (en) * | 2009-12-30 | 2010-09-08 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215611A (en) * | 2008-01-11 | 2008-07-09 | 中华人民共和国厦门出入境检验检疫局 | Primer for detecting southern cowpea mosaic virus |
| CN101818211A (en) * | 2009-12-16 | 2010-09-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Molecule detection method for cowpea severe mosaic virus |
| CN101824485A (en) * | 2009-12-30 | 2010-09-08 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit and method for detecting reversely transcribed PCR molecule of cowpea severe mosaic virus |
Non-Patent Citations (1)
| Title |
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| 陈青等: "进境豇豆种子携带种传病毒的检测与鉴定", 《植物病理学报》, vol. 39, no. 1, 31 December 2009 (2009-12-31), pages 91 - 94 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898237A (en) * | 2014-01-09 | 2014-07-02 | 吉林大学 | Degenerate primer RT-PCR detection method for eimeria viruses |
| CN103898237B (en) * | 2014-01-09 | 2019-08-06 | 吉林大学 | Degenerate primer RT-PCR detection method for Eimeria coccidia virus |
| CN106148567A (en) * | 2016-07-08 | 2016-11-23 | 厦门出入境检验检疫局检验检疫技术中心 | A kind of cowpea mosaic virus subfamily virus wide spectrum detection kit and method thereof |
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