CN102140555B - 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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- CN102140555B CN102140555B CN201110092257.0A CN201110092257A CN102140555B CN 102140555 B CN102140555 B CN 102140555B CN 201110092257 A CN201110092257 A CN 201110092257A CN 102140555 B CN102140555 B CN 102140555B
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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 degenerated primer, detection method and application thereof for detection.
Background technology
Comovirus (
comovirus) and fabavirus genus (
fabavirus) belong to class picornavirus order (
picornavirales), association cowpea Viraceae (
secoviridae), 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 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 wider host range, No. 1, broad bean wilt virus (
broad bean wilt virus 1, BBWV-1) be its representative species.These two Tobamovirus are that a class causes the virus of important threat to agricultural production security, and multiple virus can cause heavy losses to its host plant.According to the inward Plant Quarantine harmful organism register > > of the < < People's Republic of China (PRC) of announcement in 2007, the kind that is listed in quarantine virus 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, 3 kinds of viruses such as CPSMV).Ferrer in 2007 etc. set up 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 simultaneously for the general RT-PCR method of these two Tobamovirus.
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 rapidly and accurately judgement sample has Comovirus and fabavirus to belong to virus, carries out Identification of Species, for imports and exports safety provides assurance according to sequence.
In order to reach above-mentioned purpose, solution of the present invention is:
Comovirus and fabavirus belong to a viral general RT-PCR detection degenerated primer, by forward and reverse primer, formed, the nucleotide sequence of its forward primer see SEQ ID No.1:5 '-TGYGAYTAYDVHWSWTTYGATGG-3 '; The nucleotide sequence of reverse primer see 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: take that the total RNA of sample is template, utilize above-mentioned primer to carry out RT-PCR amplification, reaction finishes rear detected through gel electrophoresis amplified production, according to the location determination result of amplification of DNA fragments, in illness blade, all can detect the DNA fragmentation of 326 ~ 343 bp.
Described RT-PCR reaction comprises reverse transcription and two processes of pcr amplification.
The temperature of described reverse transcription reaction is 42 ℃, pcr amplification first by 42 ℃ of annealing temperatures, carry out 5 circulations, then by 52 ℃ of annealing temperatures, carry out 30 circulations, elongating temperature is 72 ℃.
A kind of Comovirus and fabavirus belong to viral general RT-PCR and detect the application with degenerated primer, the described total RNA of primer pair sample carries out after RT-PCR amplification after the further sequencing of increased DNA fragmentation, can Rapid identification Comovirus and the viral species that belongs to of fabavirus.
Comovirus and fabavirus belong to viral general RT-PCR and detect the 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 the RdRp gene order design degenerated primer on viral RNA 1 genome according to Comovirus and fabavirus, and this primer can be used for Comovirus and fabavirus belongs to viral general RT-PCR detection.And primer of the present invention and related reagent can be assembled into test kit, with easy to use.Whether detection method of the present invention rapidly and accurately judgement sample has Comovirus and fabavirus to belong to virus, carries out Identification of Species, for imports and exports safety provides assurance according to sequence.
Accompanying drawing explanation
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, 10 detected results that are healthy Broad Bean Leaves.That is, 1 be cowpea mosaic virus (CPMV) (isolate 1), 2 for cowpea mosaic virus (CPMV) (isolate 2), 3 for cowpea severe mosaic virus (CPSMV) (isolate 1), 4 for cowpea severe mosaic virus (CPSMV) (isolate 2), 5 for bean pod mottle virus (BPMV), 6 for radish mosaic virus (RaMV), 7 for red clover mottle virus (RCMV), 8 for Flos Cucurbitae mosaic virus (SqMV), 9 for broad bean wilt virus No. 1 (BBWV1), 10 be healthy Broad Bean Leaves.
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
Embodiment is used for further illustrating of the present invention below, 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 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) get the sick leaf of 0.1 g, add the TRIzol reagent (American I nvitrogen company) of 1 ml, move in 1.5 m L centrifuge tubes of sterilizing, under room temperature, keep 5 min;
2) add 0.2 mL chloroform, thermal agitation 15 s, then at room temperature keep after 2-15 min, and 4 ℃, centrifugal 15 min of 12000 g;
3) by upper water phase transition in 1.5 new m L centrifuge tubes, add 0.5 m L Virahol, put upside down and mix, under room temperature, keep 15 min;
4) 4 ℃, centrifugal 10 min of 12000 g, RNA will form precipitation in sidewall and the bottom of pipe;
5) outwell supernatant liquor, add 75% washing with alcohol precipitation, then 4 ℃, centrifugal 5 min(of 7500 g are as precipitation suspends, and use 12000 g centrifugal), discard ethanol;
6) be deposited under room temperature fully dry after, be dissolved in the water of 40 μ L nuclease free ,-20 ℃ save backup.
3, the foundation of RT-PCR amplification method
The standby total RNA in step 2 of take is template, carries out RT-PCR reaction.20 μ L reaction systems are carried out reverse transcription reaction, in the reaction tubes of 0.2 mL, add the water of 8.75 μ L nuclease free, 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 tetra-kinds of mixtures of dCTP) (10 mmol/L) (precious biotechnology (Dalian) company limited), after mixing, in 65 ℃ of maintenance 5 min, transfer to rapidly quenching 5min on ice; Then in reaction tubes, add, 4 μ L 5 * reverse transcription damping fluids (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; After 72 ℃ of 15 min deactivation ThermoScript II, obtain reverse transcription product cDNA.
PCR reaction system is 25 μ L, in the PCR of 0.2 mL reaction tubes, add the water of 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 denaturation 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 PCR instrument, complete.
PCR reaction finishes laggard row agarose gel electrophoresis and detects, and according to the DNA fragmentation of 326 ~ 343 bp that whether increase, judges in sample, whether to contain Comovirus and fabavirus belongs to virus.Whether the present embodiment detection method rapidly and accurately judgement sample has Comovirus and fabavirus to belong to virus, carries out Identification of Species, for imports and exports safety provides assurance according to sequence.Comovirus and fabavirus belong to virus RT-PCR and detect and with degenerated primer, further can make Comovirus and fabavirus 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 sick leaf and the healthy Broad Bean Leaves of 9 isolates of 7 kinds of virus such as 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), broad bean wilt virus No. 1 (BBWV1), by embodiment 1 method, extract respectively total RNA, carry out again RT-PCR amplification, agarose gel electrophoresis detected result.In result illness blade, all detect the DNA fragmentation of approximately 350 bp.Detected result as shown in Figure 1.Show that the general RT-PCR method of setting up can be used for the detection of different virus kind in these two Tobamovirus, there is 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 Nepovirus virus (being respectively ArMV, TRSV, ToRSV, TBRV, PRMV, CLRV, RpRSV) the sick leaf that belongs to cowpea mosaic virus subfamily, by embodiment 1 method, extract respectively total RNA, carry out RT-PCR amplification, carry out specific detection.Detected result as shown in Figure 2.Show that the general RT-PCR method of setting up can't produce cross reaction with the virus of same Tobamovirus, there is good specificity, meet the requirement of 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 (4)
1. a Comovirus and fabavirus belong to viral general RT-PCR detection degenerated primer, it is characterized in that being formed by forward and reverse primer, the nucleotide sequence of its forward primer see SEQ ID No.1:5 '-TGYGAYTAYDVHWSWTTYGATGG-3 '; The nucleotide sequence of reverse primer see 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. a degenerated primer as claimed in claim 1 belongs to the detection method of viral general RT-PCR for detection of Comovirus and fabavirus, it is characterized in that comprising the steps: take that the total RNA of sample is template, utilize above-mentioned primer to carry out RT-PCR amplification, reaction finishes rear detected through gel electrophoresis amplified production, according to the location determination result of amplification of DNA fragments, infect in the illness blade of these two Tobamovirus viruses and all can detect the DNA fragmentation of 326 ~ 343 bp; Described RT-PCR amplification comprises reverse transcription and two processes of pcr amplification; Wherein, the condition of reverse transcription is: 42 ℃ of reaction 50min; The condition of pcr amplification is: 95 ℃ of denaturation 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.
One kind Comovirus and fabavirus belong to viral general RT-PCR and detect the application with degenerated primer as claimed in claim 1, it is characterized in that: the described total RNA of primer pair sample carries out after RT-PCR amplification in judgement sample, whether contain fast Comovirus and fabavirus and belong to viral.
4. Comovirus and fabavirus belong to viral general RT-PCR and detect the application with degenerated primer as claimed in claim 1, 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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| CN201110092257.0A CN102140555B (en) | 2011-04-07 | 2011-04-07 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
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| CN201110092257.0A CN102140555B (en) | 2011-04-07 | 2011-04-07 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
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| CN103898237B (en) * | 2014-01-09 | 2019-08-06 | 吉林大学 | Degenerate primer RT-PCR detection method for Eimeria coccidia virus |
| CN106148567B (en) * | 2016-07-08 | 2019-07-02 | 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 its method |
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 |
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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 (2)
| Title |
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| 进境豇豆种子携带种传病毒的检测与鉴定;陈青等;《植物病理学报》;20091231;第39卷(第1期);第91-94页 * |
| 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》.2009,第39卷(第1期),第91-94页. |
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