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 PDF

Info

Publication number
CN102140555A
CN102140555A CN2011100922570A CN201110092257A CN102140555A CN 102140555 A CN102140555 A CN 102140555A CN 2011100922570 A CN2011100922570 A CN 2011100922570A CN 201110092257 A CN201110092257 A CN 201110092257A CN 102140555 A CN102140555 A CN 102140555A
Authority
CN
China
Prior art keywords
pcr
fabavirus
comovirus
primer
belong
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2011100922570A
Other languages
Chinese (zh)
Other versions
CN102140555B (en
Inventor
林石明
廖富荣
黄蓬英
吴媛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
INSPECTION AND QUARANTINE CENTER OF XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Original Assignee
INSPECTION AND QUARANTINE CENTER OF XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by INSPECTION AND QUARANTINE CENTER OF XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU filed Critical INSPECTION AND QUARANTINE CENTER OF XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Priority to CN201110092257.0A priority Critical patent/CN102140555B/en
Publication of CN102140555A publication Critical patent/CN102140555A/en
Application granted granted Critical
Publication of CN102140555B publication Critical patent/CN102140555B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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

A kind of Comovirus and fabavirus belong to viral general RT-PCR and detect with degenerated primer, detection method and application thereof
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.
CN201110092257.0A 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 Expired - Fee Related CN102140555B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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

Publications (2)

Publication Number Publication Date
CN102140555A true CN102140555A (en) 2011-08-03
CN102140555B CN102140555B (en) 2014-09-03

Family

ID=44408349

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201110092257.0A Expired - Fee Related 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

Country Status (1)

Country Link
CN (1) CN102140555B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
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

Citations (3)

* Cited by examiner, † Cited by third party
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Title
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)

* Cited by examiner, † Cited by third party
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

Also Published As

Publication number Publication date
CN102140555B (en) 2014-09-03

Similar Documents

Publication Publication Date Title
CN101691614B (en) Method for rapidly identifying rice black-streaked dwarf virus and southern rice black-streaked dwarf virus
CN101875982B (en) Method for synchronously detecting multiple tobacco viruses
CN110567951B (en) Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof
CN108950068A (en) A kind of avian infectious bronchitis virus QX type strain identification detection kit
CN102676701B (en) Universal RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection primer and detection method for avian pneumovirus
CN104178585A (en) Potato virus detection primers and potato virus detection method
CN102605092B (en) LAMP (Loop-mediated isothermal amplification) rapid detection method of Citrus huanglongbing
CN105463136A (en) Kit for RT-PCR typing detection of avian infectious bronchitis virus
CN101875981B (en) Primer group and kit for synchronously detecting multiple tobacco viruses
CN103540680A (en) Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus
CN103642804A (en) Degenerate primer and method of detecting Y potyvirus virus of potato by using degenerate primer
CN102031313A (en) RT-PCR (reverse transcription-polymerase chain reaction) method for detecting the infection reality of host by arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus
CN102140555B (en) Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof
CN102206714B (en) Degenerate primer, detection method and application for general RT-PCR detection of comovirinae subvirus
JP6895168B2 (en) Virus diagnostic method
CN107893129A (en) The method for detecting apple green wrinkle fruit disease poison
CN103667519A (en) Polymerase chain reaction (PCR) primer pair for identifying H9 subtype avian influenza virus and application thereof
CN106521028A (en) Multiplex RT-PCR method used for synchronously detecting four kinds of viruses transmitted by insects
CN103255230B (en) One tube PCR type kit for discriminating I type duck hepatitis virus and new I type duck hepatitis virus
CN113667776A (en) Real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting plantain mosaic virus in lily
Supakitthanakorn et al. Simultaneous and sensitive detection of CVB, CChMVd and CSVd mixed infections in Chrysanthemum using multiplex nested RT-PCR.
CN106367532B (en) PCR-RFLP method for distinguishing clade2.3.4 and clade 2.3.4.4H5AIV based on sequence polymorphism
CN104498633B (en) NASBA primer, test kit and the method for detection Peach latent mosaic viroid
Pakdel et al. Molecular characterization of the complete genome of a barley yellow dwarf virus-PAV isolate from Iran.
CN102277450B (en) Primer used for multiple detection of five quarantine nepoviruses and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20140903

Termination date: 20160407

CF01 Termination of patent right due to non-payment of annual fee