CN106148567A - A kind of cowpea mosaic virus subfamily virus wide spectrum detection kit and method thereof - Google Patents
A kind of cowpea mosaic virus subfamily virus wide spectrum detection kit and method thereof Download PDFInfo
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Abstract
The invention discloses a kind of cowpea mosaic virus subfamily virus wide spectrum detection kit and detection method thereof.Containing the primer set being made up of three primers in cowpea mosaic virus subfamily virus wide spectrum detection kit provided by the present invention.Experiment proves, the features such as primer set for detecting cowpea mosaic virus subfamily virus provided by the present invention and test kit thereof have that broad spectrum activity is good, highly sensitive, high specificity, detection cycle are short, it is adaptable to the quickly detection of cowpea mosaic virus subfamily virus and monitoring in agricultural production and port of passing in and out.
Description
Technical field
The invention belongs to technical field of plant quarantine, relate to a kind of cowpea mosaic virus subfamily virus wide spectrum detection kit
And detection method, particularly to primer set, test kit and detection method thereof for cowpea mosaic virus subfamily virus.
Background technology
Cowpea mosaic virus subfamily (Comovirinae) belongs to picornavirus mesh (Picornavirales), association Semen vignae sinensis
Viraceae (Secoviridae), belongs to (Fabavirus) and nematicide containing Comovirus (Comovirus), fabavirus
Pass polyhedral virus and belong to (Nepovirus), contain 15 kinds, 5 kinds and 36 kinds of viruses respectively.In Comovirus virus,
Having 11 kinds of viral host ranges narrow, only infect leguminous plant, other kinds then infect Solanaceae, Cruciferae, calabash
The plants such as section.Comovirus virus mainly causes floral leaf, mottled symptom, generally will not cause ring speckle symptom.Favism
Poison belongs to virus and has the widest host range, including dicotyledon, and the monocotyledon of some sections.Fabavirus belongs to
The symptom that virus causes includes ring speckle, mottled, floral leaf, deformity, wilting and acronecrosis.Nepovirus virus
Host is different because of virus, has width to have narrow, is distributed widely in Temperate Region in China.Ring speckle is Nepovirus virus infection
Classical symptom, but generally with mottled and speckle symptom.Comovirus virus is only propagated by beetle, the most chrysomelid
The kind of section.Fabavirus belongs to virus and then passes through aphis propagation.12 kinds of viruses in Nepovirus pass through minute hand
Nematicide is propagated, and 3 kinds are passed through pollen transmission, propagated by acarid for a kind, and the vector of other viral species is the most unknown.This Asia
In coe virus, the viral species of nearly half can also pass through seed dispersal.
Multiple virus panel in cowpea mosaic virus subfamily has highly important Economic Importance, pacifies agricultural production
Complete and ecological safety causes huge threat.In this subfamily virus, there are 8 kinds of viruses to be classified as by China and forbid that the quarantine entered the territory has
Evil biology, including: arabis mosaic virus (Arabis mosaic virus, ArMV), bean pod mottle virus (Bean pod
Mottle virus, BPMV), broad bean dyeing virus (Broad bean stain virus, BBSV), cowpea severe mosaic virus
(Cowpea severe mosaic virus, CPSMV), peach rosette mosaic virus (Peach rosette mosaic virus,
PRMV), nepovirus (Tobacco ringspot virus, TRSV), annulus zonatus (Tomato black
Ring virus, TBRV), Fructus Lycopersici esculenti black speck virus (Tomato ringspot virus, ToRSV).Additionally, also more than half
Viral species not yet report generation in China.
At present, in cowpea mosaic virus subfamily virus routine testing, quick detection method commonly use serology and
Molecular biology method, particularly elisa (ELISA) method and inverse transcription polymerase chain reaction (RT-
PCR) method.Wherein, ELISA detection needs special antibody, is the most all to be provided by the Reagent Company of specialty.At present, domestic
The cowpea mosaic virus subfamily Viral diagnosis antibody type that outer detectable company provides is the most extremely limited, is confined to the most several
Plant virus.RT-PCR method is to use widely another the quickly side of detection in cowpea mosaic virus subfamily Viral diagnosis
Method.At present, the most simply set up the viral species of the most several close attentions, some viral species its gene even also unknown
Group sequence.Therefore, in cowpea mosaic virus subfamily virus, most viral species does not has corresponding serology, the fastest yet
The molecular detecting method of speed.
Summary of the invention
First purpose of the present invention is to provide a kind of detection or auxiliary detects the complete of cowpea mosaic virus subfamily virus
Primer.
Detection or the primer set of auxiliary detection cowpea mosaic virus subfamily virus that the present invention provides are following 1) or 2):
1) it is made up of primer 1, primer 2 and primer 3；
2) it is made up of primer 1, primer 2, primer 4 and primer 5；
Described primer 1 is the single strand dna shown in sequence 1；
Described primer 2 is the single strand dna shown in sequence 2；
Described primer 3 is the single strand dna shown in sequence 3；
Described primer 4 is the single strand dna shown in sequence 4；
Described primer 5 is the single strand dna shown in sequence 5.
Second object of the present invention is to provide a kind of detection or the PCR examination of auxiliary detection cowpea mosaic virus subfamily virus
Agent.
Detection or the PCR reagent of auxiliary detection cowpea mosaic virus subfamily virus that the present invention provides include above-mentioned complete draw
Thing.
In above-mentioned PCR reagent, described primer 1, described primer 2, described primer 3, described primer 4 and described primer 5 are in institute
State the final concentration in PCR reagent and be 10 μm ol/L.
Detection provided by the present invention or the PCR reagent of auxiliary detection cowpea mosaic virus subfamily virus, specifically can include
Described primer set, and M-MLV reverse transcription, 5 × M-MLV buffer, dNTPs, RNase inhibitor, 10 × Taq DNA are poly-
Synthase buffer is (containing Mg2+) and Taq archaeal dna polymerase.
Third object of the present invention is to provide a kind of detection or the reagent of auxiliary detection cowpea mosaic virus subfamily virus
Box.
Detection or the test kit of auxiliary detection cowpea mosaic virus subfamily virus that the present invention provides include above-mentioned complete draw
Thing or above-mentioned PCR reagent.
Detection provided by the present invention or the test kit of auxiliary detection cowpea mosaic virus subfamily virus, specifically can contain
State primer set or described PCR reagent, and the sick leaf freeze-dried powder of the cowpea mosaic virus subfamily virus as positive control
(the blade freeze-dried powder as containing cowpea mosaic virus)；And/or the water as negative control.
Fourth object of the present invention is to provide the new use of above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box
On the way.
The invention provides above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box to be measured in detection or auxiliary detection
Whether virus is the application in cowpea mosaic virus subfamily virus.
Present invention also offers above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box in preparation detection or auxiliary inspection
Survey the application in the product whether virus to be measured be cowpea mosaic virus subfamily virus.
Present invention also offers above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box to treat in detection or auxiliary detection
Whether test sample product contain the application in cowpea mosaic virus subfamily virus.
Present invention also offers above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box in preparation detection or auxiliary inspection
Survey the application in the product whether containing cowpea mosaic virus subfamily virus in testing sample.
Five purposes of the present invention are to provide in a kind of detection or auxiliary detection testing sample whether contain Cowpea
The method of poison subfamily virus.
Detection or auxiliary that the present invention provides detect the side whether containing cowpea mosaic virus subfamily virus in testing sample
Method comprises the steps:
(1) from described testing sample, extract total serum IgE, use above-mentioned primer 1 to carry out reverse transcription reaction, obtain to be measured
cDNA；
(2) using described cDNA to be measured as template, above-mentioned primer 1 and above-mentioned primer 2 is used to carry out the reaction of first round PCR,
Obtain first round PCR primer；
(3) using first round PCR primer as template, use above-mentioned primer 1 and above-mentioned primer 3 to carry out second and take turns PCR reaction,
Obtain second and take turns PCR primer；
Or using first round PCR primer as template, use above-mentioned primer 4 and above-mentioned primer 5 to carry out second and take turns PCR reaction,
Obtain second and take turns PCR primer；
(4) determine in described testing sample whether contain as follows according to the described second size taking turns PCR primer
Cowpea mosaic virus subfamily virus:
If described second takes turns in PCR primer containing the DNA fragmentation that size is 400～440bp, the most described testing sample contains
Have or candidate contain cowpea mosaic virus subfamily virus, otherwise the most described testing sample does not contains or candidate do not contain Semen vignae sinensis flower
Mosaic virus subfamily virus.
In said method, the temperature of described reverse transcription reaction is 42 DEG C.
In said method, described first round PCR reaction carries out 35 circulations altogether, and the annealing temperature of front 5 circulations is 42 DEG C,
The annealing temperature of rear 30 circulations is 50 DEG C.
In said method, described second takes turns PCR reaction carries out 40 circulations altogether, and the annealing temperature of front 5 circulations is 42 DEG C,
Rear 35 circulation annealing temperature be 52 DEG C.
In the present invention, above all of described testing sample can be all plant sample, as infected cowpea mosaic virus
The plant leaf blade of subfamily virus.
In the present invention, above all of described cowpea mosaic virus subfamily virus can be all following in any one or many
Kind: Aegean Jerusalem artichoke ring spot virus, peace younger brother's Si Rhizoma Solani tuber osi mottle virus, Anatolia's Fructus Vitis viniferae ring spot virus, the pale reddish brown lucerne in Australia
Mu cryptovirus, Bulgaria's Fructus Vitis viniferae cryptovirus, Kidney bean rugose mosaic poison, bean pod mottle virus, broad bean dyeing be viral,
Broad bean wilt virus 1, Broad bean wilt virus 2, echtes ackerbohnemosaik virus, quail marmor leguminosarum, Glycine mosaic virus,
Yunnan rhizome of chuanxiong A virus, tomato black ring virus, annulus zonatus, Herba Pogostemonis mild mosaic poison, Ribes nigrum L. degeneration virus, Trifolium pratense
Grass mottle virus, cowpea mosaic virus, cowpea severe mosaic virus, Trifolium incarnatum cryptovirus, Jerusalem artichoke yellow ringspot virus, lettuce Huang
Mottle virus, cocoa necrosis virus, tuber Herba basellae rubrae C virus, Blue berry leaf pinta poison, Li Qianyin ring spot virus, Radix Gentianae mosaic disease
Poison, radish mosaic virus, Rhizoma Solani tuber osi U virus, Rhizoma Solani tuber osi black ring virus, America cassava latent virus, Hibiscus syriacus L. hide ring spot virus,
The green mottle virus of Maninot esculenta crantz., Flos Cucurbitae mosaic virus, arabis mosaic virus, grapevine chrome mosaic nepovirus, Fructus Vitis viniferae curl virus, Fructus Vitis viniferae
Upper lower solution, mulberry ring spot virus, sago cycas necrosis stunt virus, peach rosette mosaic virus, beet ring spot virus, Tunisia Fructus Vitis viniferae
The green mottle virus of ring spot virus, Semen Pisi sativi, the light mosaic virus of Semen Pisi sativi, Radix Brassicae rapae ring spot virus, raspberry ring spot virus, tobacco ring spot virus
Poison, Herba lamii barbati mild mosaic Jerusalem artichoke cryptovirus malicious, Italian, mirabalan cryptovirus, Fructus Pruni pseudocerasi leaf roll virus, Fructus oleae europaeae are dived
Hidden ring spot virus.
It is experimentally confirmed, the primer set for wide spectrum detection cowpea mosaic virus subfamily virus provided by the present invention
And detection kit has the features such as highly sensitive, high specificity, broad spectrum activity are good, the detection cycle is short, it is adaptable to agricultural production
And the quick of cowpea mosaic virus subfamily virus detects and monitoring on port of passing in and out.
Accompanying drawing explanation
Fig. 1 is that the broad spectrum activity of cowpea mosaic virus subfamily virus wide spectrum detection kit and specific detection are (in test kit
Primer be: SecoV-Fat, SecoV-Rat and SecoV-R4at).Wherein, M:DNA molecular weight standard 100bp DNA
Ladder；1. peace younger brother's Si Rhizoma Solani tuber osi mottle virus (DSMZ PV-0057)；2. arabis mosaic virus (Agdia LPC 23203
(C1954))；3. echtes ackerbohnemosaik virus (DSMZ PV-0098)；4. broad bean wilt virus (ACD V012-P1)；5. Semen Viciae fabae withers
Listless viral No. 1 (DSMZ PV-0067)；6. Broad bean wilt virus 2 (ACD V212-P1)；7. Broad bean wilt virus 2 (DSMZ
PV-0862)；8. broad bean wilt virus 1&2 (Agdia LPC 46202 (C1543))；9. broad bean wilt virus 1&2
(Agdia LPC 46202(C1643))；10. Blue berry leaf pinta poison (Agdia LPC 88001 (C1796))；11. Kidney bean pod speckles
Refute virus (ACD V082-P1)；12. bean pod mottle virus (Agdia LPC 46400 (C1858))；13. cowpea mosaic virus
(Agdia LPC 24200(C1319))；14. cowpea severe mosaic virus (ATCC PV-273)；15. grapevine fanleaf virus (Adgen
1147-11)；16. Rhizoma Solani tuber osi black ring spots poison (Adgen 1330-11)；17. strawberry latent ringspot viruses (Adgen 1160-
11)；18. Flos Cucurbitae mosaic virus (Agdia LPC 26400 (C2013))；19. tomato black ring viruses (Adgen 1068-11)；
20. annulus zonatuses (Agdia LPC 22000 (C1545))；21. annulus zonatuses (the numbered LPC of Agdia 22000
(C1939))；22. nepovirus (Agdia LPC 64001 (C1714))；23. nepovirus (Agdia LPC
64001(C2009))；24. radish mosaic viruses (DSMZ PV-0355)；25. peach rosette mosaic viruses (Agdia LPC 87700
(C1973))；26. raspberry ring spot viruses (DSMZ PV-0429)；27. broad bean dyeings virus (LOEWE 07013PC)；28. is red
Clover mottle virus (LOEWE 07040PC)；29. cherry rasp leaf viruses (Adgen 1303-11)；30. Maize Chlorotic stunt
Virus (Adgen 1327-11)；31. satsuma orange dwarf viruses (Citrus Research Institute Of Chinese Academy Of Agricultural Sciences)；32. is blank right
According to.
Fig. 2 be cowpea mosaic virus subfamily virus wide spectrum detection kit (primer in test kit is: SecoV-Fat,
SecoV-Rat, SecoV-F-T3 and SecoV-R4-M13-47) broad spectrum activity and specific detection.Wherein, M:DNA molecular weight mark
Quasi-100bp DNA Ladder；1. peace younger brother's Si Rhizoma Solani tuber osi mottle virus (DSMZ PV-0057)；2. arabis mosaic virus
(Agdia LPC 23203(C1954))；3. echtes ackerbohnemosaik virus (DSMZ PV-0098)；4. broad bean wilt virus (ACD
V012-P1)；5. broad bean wilt virus 1 (DSMZ PV-0067)；6. Broad bean wilt virus 2 (ACD V212-P1)；7. silkworm
Bean wilting No. 2 (DSMZ PV-0862) of virus；8. broad bean wilt virus 1&2 (Agdia LPC 46202 (C1543))；9. silkworm
Bean wilting No. 1&2 (Agdia LPC 46202 (C1643)) of virus；10. Blue berry leaf pinta poison (Agdia LPC 88001
(C1796))；11. bean pod mottle virus (ACD V082-P1)；12. bean pod mottle virus (Agdia LPC 46400
(C1858))；13. cowpea mosaic virus (Agdia LPC 24200 (C1319))；14. cowpea severe mosaic virus (ATCC PV-
273)；15. grapevine fanleaf viruses (Adgen1147-11)；16. Rhizoma Solani tuber osi black ring spots poison (Adgen 1330-11)；17. Fructus Fragariae Ananssaes
Hide ring spot virus (Adgen1160-11)；18. Flos Cucurbitae mosaic virus (Agdia LPC 26400 (C2013))；19. Fructus Lycopersici esculenties are black
Circovirus virus (Adgen1068-11)；20. annulus zonatuses (Agdia LPC 22000 (C1545))；21. annulus zonatuses
(the numbered LPC of Agdia 22000 (C1939))；22. nepovirus (Agdia LPC 64001 (C1714))；23. Nicotiana tabacum L.s
Ring spot virus (Agdia LPC 64001 (C2009))；24. radish mosaic viruses (DSMZ PV-0355)；25. Fructus Persicae clump bunch mosaic disease
Poison (Agdia LPC 87700 (C1973))；26. raspberry ring spot viruses (DSMZ PV-0429)；27. broad bean dyeing viruses
(LOEWE 07013PC)；28. red Herba Trifolii Pratentis mottle virus (LOEWE 07040PC)；29. cherry rasp leaf virus (Adgen
1303-11)；30. maize chlorotic dwarf virus (Adgen 1327-11)；31. satsuma orange dwarf virus (Scientia Agricultura Sinicas
Institute's mandarin orange institute)；32. blanks.
Fig. 3 is the specificity verification of cowpea mosaic virus subfamily virus wide spectrum detection kit.Wherein, M:DNA molecular weight
Standard 100bp DNA Ladder；1. tobacco mosaic virus (TMV)；2. lily asymptomatic virus；3. Bulbus Lilii healthy leaves；4. common cigarette is good for
Health blade；5. Semen Viciae fabae healthy leaves；6. Fructus Lycopersici esculenti healthy leaves；7. Kidney bean healthy leaves；8. Semen vignae sinensis healthy leaves；9. Fructus Cucumidis sativi is healthy
Blade；10. health pumpkin blade；11. sweet bean healthy leaveses；12. blanks.
Fig. 4 is the sensitivity technique of cowpea mosaic virus subfamily virus wide spectrum detection kit.Wherein, M:DNA molecular weight
Standard 100bp DNA Ladder；1～10 are respectively Flos Cucurbitae mosaic virus 10-1～10-10The cDNA of dilution again.
Fig. 5 is the screening of the primer of detection cowpea mosaic virus subfamily virus.Fig. 5 A is that primer is to SecoV-F6/SecoV-
The testing result of R5；Fig. 5 B is the primer testing result to SecoV-F6-at/SecoV-R5-at；Fig. 5 C is that primer is to SecoV-
The testing result of Fat/SecoV-Rat.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Quantitative test in following embodiment, is respectively provided with three times and repeats experiment, results averaged.
Peace younger brother's Si Rhizoma Solani tuber osi mottle virus: Germany living resources preservation center (DSMZ), deposit number is PV-0057.
Bean pod mottle virus: AC Diagnostics, Inc. Products, numbered V082-PV.
Bean pod mottle virus: Agdia, Inc. Products, numbered LPC 46400 (C1858).
Broad bean dyeing virus: Loewe Biochemica GmbH Products, numbered 07013PC
Broad bean wilt virus: AC Diagnostics, Inc. Products, numbered V012-P1.
Broad bean wilt virus 1, No. 2: Agdia, Inc. Products, numbered LPC 46202 (C1543).
Broad bean wilt virus 1, No. 2: Agdia, Inc. Products, numbered LPC 46202 (C1643).
Broad bean wilt virus 1: Germany living resources preservation center (DSMZ), deposit number is PV-0067.
Broad bean wilt virus 2: AC Diagnostics, Inc. Products, numbered V212-P1.
Broad bean wilt virus 2: Germany living resources preservation center (DSMZ), deposit number is PV-0862.
Echtes ackerbohnemosaik virus: Germany living resources preservation center (DSMZ), deposit number is PV-0098.
Tomato black ring virus: An Dezhen biotechnology (Beijing) company limited product, numbered 1068-11.
Annulus zonatus: Agdia, Inc. Products, numbered LPC 22000 (C1545).
Annulus zonatus: Agdia, Inc. Products, numbered LPC 22000 (C1939).
Red Herba Trifolii Pratentis mottle virus: Loewe Biochemica GmbH Products, numbered 07040PC.
Cowpea mosaic virus: Agdia, Inc. Products, numbered LPC 24200 (C1319).
Cowpea severe mosaic virus: American Type Culture preservation center (ATCC), deposit number is PV-273.
Blue berry leaf pinta poison: Agdia, Inc. Products, numbered LPC 88001 (C1796).
Radish mosaic virus: Germany living resources preservation center (DSMZ), deposit number is PV-0355.
Rhizoma Solani tuber osi black ring spot poison: An Dezhen biotechnology (Beijing) company limited product, numbered 1330-11.
Flos Cucurbitae mosaic virus: Agdia, Inc. Products；Numbered LPC 26400 (C2013).
Arabis mosaic virus: Agdia, Inc. Products, numbered LPC 23203 (C1954).
Grapevine fanleaf virus: An Dezhen biotechnology (Beijing) company limited product, numbered 1147-11.
Peach rosette mosaic virus: Agdia, Inc. Products, numbered LPC 87700 (C1973).
Raspberry ring spot virus: Germany living resources preservation center (DSMZ), deposit number is PV-0429.
Nepovirus: Agdia, Inc. Products, numbered LPC 64001 (C1714).
Nepovirus: Agdia, Inc. Products, numbered LPC 64001 (C2009).
Cherry rasp leaf virus: An Dezhen biotechnology (Beijing) company limited product, numbered 1303-11.
Maize chlorotic dwarf virus: An Dezhen biotechnology (Beijing) company limited product, numbered 1327-11.
Satsuma orange dwarf virus: Citrus Research Institute Of Chinese Academy Of Agricultural Sciences.
Fructus Fragariae Ananssae is dived ring spot virus: An Dezhen biotechnology (Beijing) company limited product, numbered 1160-11.
5 × reverse transcription buffer: Promega Corporation Products, its catalog number (Cat.No.) is M1705.
M-MLV reverse transcription: Promega Corporation Products, its catalog number (Cat.No.) is M1705.
RNase inhibitor: Promega Corporation Products, its catalog number (Cat.No.) is N2111.
PCR buffer: Beijing Quanshijin Biotechnology Co., Ltd's product, its catalog number (Cat.No.) is AP111-03.
Taq archaeal dna polymerase: Beijing Quanshijin Biotechnology Co., Ltd's product, its catalog number (Cat.No.) is AP111-03.
Embodiment 1, cowpea mosaic virus subfamily virus wide spectrum detection kit and detection method thereof
One, the screening of the first round primer of detection cowpea mosaic virus subfamily virus
1, the extraction of RNA
Extract respectively containing arabis mosaic virus (Agdia LPC 23203 (C1954)), Broad bean wilt virus 2
(ACD V212-P1), bean pod mottle virus (Agdia LPC 46400 (C1858)), cowpea mosaic virus (Agdia LPC
24200 (C1319)), cowpea severe mosaic virus (ATCC PV-273), raspberry ring spot virus (DSMZ PV-0429), radish flower
Mosaic virus (DSMZ PV-0355) and the total serum IgE of the sick leaf sample of Flos Cucurbitae mosaic virus (Agdia LPC 26400 (C2013)).
2, reverse transcription reaction
In 0.6mL centrifuge tube, add testing sample total serum IgE 4 μ L, primer SecoV-Rat (10 μm ol/L) 2 μ L, DEPC
The ddH2O 8 μ L processed, after 95 DEG C of water-bath 10min, is immediately placed in 5min on ice.Wink from, continuously add 5 × M-MLV reversion
Record buffer 4 μ L, dNTPs (10mmol/L) 1 μ L, M-MLV reverse transcription (200U/ μ L) 0.5 μ L, RNase inhibitor (40U/ μ
L) 0.5 μ L, after 42 DEG C of water-bath 1h, 70 DEG C of water-bath 15min, the cDNA of synthesis, save backup in-20 DEG C of refrigerators.
3, PCR reaction
10 × PCR buffer is (containing Mg2+) 2.5 μ L, dNTPs (10mmol/L) 0.5 μ L, forward primer SecoV-Fat (10 μ
Mol/L) 2 μ L, downstream primer SecoV-Rat (10 μm ol/L) 2 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.25 μ L, cDNA template 3
μ L, ddH2O 14.75 μ L, totally 25 μ L.Response procedures: 95 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 42 DEG C of annealing 30s, 72 DEG C
Extend 45s, 5 circulations；94 DEG C of degeneration 30s, 50 DEG C of annealing 30s, 72 DEG C extend 45s, 30 circulations；72 DEG C of overall elongation 7min.
First round PCR reaction is replaced respectively with primer SecoV-F6/SecoV-R5 and SecoV-F6-at/SecoV-R5-at
In primer SecoV-Fat/SecoV-Rat expand.Primer sequence is as follows:
SecoV-F6:TGYGAYTAYDVNNVNTTYGAYGG；
SecoV-R5:KHYTTRTCNNYNCCRTCNGT；
SecoV-F6-at:AATAAATCATAATGYGAYTAYDVNNVNTTYGAYGG；
SecoV-R5-at:AATAAATCATAAKHYTTRTCNNYNCCRTCNGT.
5, gel electrophoresis
After reaction terminates, the pcr amplification product taking 5 μ L carries out agarose gel electrophoresis detection, testing result such as Fig. 5 institute
Show: it can be seen that compare with primer SecoV-F6/SecoV-R5 with SecoV-F6-at/SecoV-R5-at, primer
SecoV-Fat/SecoV-Rat can amplify the purpose fragment of most sample, the most finally chooses primer SecoV-Fat/
The primer that SecoV-Rat expands as first round PCR.
Two, cowpea mosaic virus subfamily virus wide spectrum detection kit
1, the primer of detection cowpea mosaic virus subfamily virus
The sequence designing following five amplimers according to the conservative region on cowpea mosaic virus subfamily viral RNA 1 is:
SecoV-Fat:AATAAATCATAATGTGAYTAYDVNNVNTTYGATGG (sequence 1)；
SecoV-Rat:AATAAATCATAAYTTRTCHNTNCCRTCNGT (sequence 2)；
SecoV-R4at:AATAAATCATAAADNAVRTTRTCRTCNCCRTA (sequence 3)；
SecoV-F-T3:ATTAACCCTCACTAAAGGGAATGTGAYTAYDVNNVNTTYGATGG (sequence 4)；
SecoV-R4-M13-47:CGCCAGGGTTTTCCCAGTCACGACADNAVRTTRTCRTCNC CRTA (sequence 5)；
Wherein, Y=C or T, D=G or A or T, V=A or G or C, N=A or G or T or C, R=A or G, H=A or T or C.
Reverse transcription primer is SecoV-Rat, first round PCR primer SecoV-Fat/SecoV-Rat, and second takes turns PCR primer
SecoV-Fat/SecoV-R4at or SecoV-F-T3/SecoV-R4-M13-47.
2, cowpea mosaic virus subfamily virus wide spectrum detection kit
The configuration (50 detection limits) of cowpea mosaic virus subfamily virus wide spectrum detection kit:
1) reverse transcription buffer: 5 ×, 1 pipe (300 μ L)；
2) dNTPs: concentration is 10mmol/L, 1 pipe (100 μ L)；
3) M-MLV reverse transcription: 200U/ μ L, 1 pipe (30 μ L)；
4) RNase inhibitor: 40U/ μ L, 1 pipe (30 μ L)；
5) the PCR buffer (Mg containing 25mmol/L2+): 10-, 1 pipe (300 μ L)；
6) Taq archaeal dna polymerase: 5U/ μ L, 1 pipe (30 μ L)；
7) without RNase ddH2O:1 manages (1mL)；
8) primer SecoV-Fat: concentration is 10 μm ol/L, 1 pipe (450 μ L)；
9) primer SecoV-Rat: concentration is 10 μm ol/L, 1 pipe (350 μ L)；
10) primer SecoV-R4at: concentration is 10 μm ol/L, 1 pipe (250 μ L)；
11) primer SecoV-F-T3: concentration is 10 μm ol/L, 1 pipe (250 μ L)；
12) primer SecoV-R4-M13-47: concentration is 10 μm ol/L, 1 pipe (250 μ L)；
13) positive control sample: containing the freeze-dried powder of arabis mosaic virus disease leaf, 1 pipe (0.05g).
Three, the detection method of cowpea mosaic virus subfamily virus wide spectrum detection kit
The detection method of the cowpea mosaic virus subfamily virus wide spectrum detection kit in step 2 is based on degenerate primer
Semi-nested RT-PCR method.Concrete detection method is as follows:
1, reverse transcription reaction
In 0.6mL centrifuge tube, add testing sample total serum IgE 4 μ L, primer SecoV-Rat (10 μm ol/L) 2 μ L, DEPC
The ddH processed2O 8 μ L, after 95 DEG C of water-bath 10min, is immediately placed in 5min on ice.Wink from, continuously add 5 × M-MLV reversion
Record buffer 4 μ L, dNTPs (10mmol/L) 1 μ L, M-MLV reverse transcription (200U/ μ L) 0.5 μ L, RNase inhibitor (40U/ μ
L) 0.5 μ L, after 42 DEG C of water-bath 1h, 70 DEG C of water-bath 15min, the cDNA of synthesis, save backup in-20 DEG C of refrigerators.
2, first round PCR reaction
The cDNA obtained in step 1, as template, uses primer SecoV-Fat/SecoV-Rat to carry out PCR amplification, obtains
First round pcr amplification product.First round PCR amplification system: 10 × PCR buffer is (containing Mg2+) 2.5 μ L, dNTPs (10mmol/L)
0.5 μ L, forward primer SecoV-Fat (10 μm ol/L) 2 μ L, downstream primer SecoV-Rat (10 μm ol/L) 2 μ L, Taq DNA are poly-
Synthase (5U/ μ L) 0.25 μ L, cDNA template 3 μ L, ddH2O 14.75 μ L, totally 25 μ L.Response procedures: 95 DEG C of denaturations 3min；94
DEG C degeneration 30s, 42 DEG C of annealing 30s, 72 DEG C extend 45s, 5 circulations；94 DEG C of degeneration 30s, 50 DEG C of annealing 30s, 72 DEG C of extensions
45s, 30 circulations；72 DEG C of overall elongation 7min.After reaction terminates, as the second reaction template taking turns PCR.
3, second PCR reaction is taken turns
With first round pcr amplification product as template, primer SecoV-Fat/SecoV-R4at is used to carry out PCR amplification,
Pcr amplification product is taken turns to second.Second takes turns PCR amplification system: 10 × PCR buffer is (containing Mg2+) 2.5 μ L, dNTPs (10mmol/
L) 0.5 μ L, forward primer SecoV-Fat (10 μm ol/L) 2 μ L, downstream primer SecoV-R4at (10 μm ol/L) 2 μ L, Taq
Archaeal dna polymerase (5U/ μ L) 0.25 μ L, first round PCR primer 1 μ L, ddH2O 16.75 μ L, totally 25 μ L.Response procedures: 95 DEG C of changes
Property 3min；94 DEG C of degeneration 30s, 42 DEG C of annealing 30s, 68 DEG C extend 60s, 5 circulations；94 DEG C of degeneration 30s, 52 DEG C of annealing 30s, 68
DEG C extend 60s, 35 circulations；72 DEG C of overall elongation 7min, reaction terminates.
Second takes turns pcr amplification reaction can also replace primer with primer SecoV-F-T3 and SecoV-R4-M13-47
SecoV-Fat and SecoV-R4at expands.
4, detected through gel electrophoresis
Take the second of 5 μ L to take turns pcr amplification product and carry out agarose gel electrophoresis detection, if second takes turns in PCR primer and contain
Size is the DNA fragmentation of 400～440bp, then contain in testing sample or candidate contains cowpea mosaic virus subfamily virus, instead
It, do not contain in testing sample or candidate does not contains cowpea mosaic virus subfamily virus.
Embodiment 2, the broad spectrum activity of cowpea mosaic virus subfamily virus wide spectrum detection kit and specific detection
One, the broad spectrum activity of cowpea mosaic virus subfamily virus wide spectrum detection kit and specific detection
1, peace younger brother's Si Rhizoma Solani tuber osi mottle virus (DSMZ PV-0057) containing cowpea mosaic virus subfamily, south are extracted respectively
Mustard mosaic virus (Agdia LPC 23203 (C1954)), echtes ackerbohnemosaik virus (DSMZ PV-0098), Semen Viciae fabae wilt disease
Poison (ACD V012-P1), broad bean wilt virus 1 (DSMZ PV-0067), Broad bean wilt virus 2 (ACD V212-P1), silkworm
Bean wilting virus No. 2 (DSMZ PV-0862), broad bean wilt virus 1&2 (Agdia LPC 46202 (C1543)), Semen Viciae fabaes wither
Listless virus 1&2 (Agdia LPC 46202 (C1643)), Blue berry leaf pinta poison (Agdia LPC 88001 (C1796)), Kidney bean
Pod mottle virus (ACD V082-P1), bean pod mottle virus (Agdia LPC 46400 (C1858)), cowpea mosaic virus
(Agdia LPC 24200 (C1319)), cowpea severe mosaic virus (ATCC PV-273), grapevine fanleaf virus (Adgen 1147-
11), Rhizoma Solani tuber osi black ring spot poison (Adgen 1330-11), Flos Cucurbitae mosaic virus (Agdia LPC 26400 (C2013)), Fructus Lycopersici esculenti
Black ring virus (Adgen1068-11), annulus zonatus (Agdia LPC 22000 (C1545)), annulus zonatus
(the numbered LPC of Agdia 22000 (C1939)), nepovirus (Agdia LPC 64001 (C1714)), tobacco ring spot virus
Poison (Agdia LPC 64001 (C2009)), radish mosaic virus (DSMZ PV-0355), peach rosette mosaic virus (Agdia LPC
87700 (C1973)), raspberry ring spot virus (DSMZ PV-0429), broad bean dyeing virus (LOEWE07013PC), Trifolium pratense
Grass mottle virus (LOEWE 07040PC)；And belong to the strawberry latent ringspot virus (Adgen of association Semen vignae sinensis Viraceae together
1160-11), cherry rasp leaf virus (Adgen 1303-11), maize chlorotic dwarf virus (Adgen 1327-11), satsuma orange
Dwarf virus (Citrus Research Institute Of Chinese Academy Of Agricultural Sciences), totally 31 virosis leaf sample total serum IgE.
2, the test kit in embodiment 1 and the detection method RNA sample respectively to each virosis leaf in step 1 is utilized
Detect, the broad spectrum activity of checking test kit.Each sample duplicate detection three times.Experiment arranges blank, i.e. with water simultaneously
Substitute PCR reaction template.
The second testing result taking turns PCR is carried out, such as Fig. 1 institute after first round PCR, with primer SecoV-Fat/SecoV-R4at
Show.Result shows, 27 cowpea mosaic virus subfamily virus sample all detect the band of about 400bp, and it is sick to belong to association Semen vignae sinensis together
Other virus sample of poison section does not expands the band of about 400bp, demonstrates good broad spectrum activity and specificity.Therefore, primer
SecoV-Fat, SecoV-Rat and SecoV-R4at composition primer set, it is adaptable to cowpea mosaic virus subfamily virus fast
Speed detection.
The second testing result taking turns PCR is carried out after first round PCR, with primer SecoV-F-T3/SecoV-R4-M13-47,
As shown in Figure 2.Result shows, 27 cowpea mosaic virus subfamily virus sample all detect the band of about 400bp, and belong to association together
Other virus sample of Semen vignae sinensis Viraceae does not expands the band of about 400bp, demonstrates good broad spectrum activity and specificity.Cause
This, the primer set of primer SecoV-Fat, SecoV-Rat, SecoV-F-T3 and SecoV-R4-M13-47 composition, it is adaptable to cowpea
The quick detection of bean mosaic virus subfamily virus.
Two, the specific detection of cowpea mosaic virus subfamily virus wide spectrum detection kit
1, extract containing tobacco mosaic virus (TMV), lily asymptomatic virus respectively, and Bulbus Lilii, common cigarette, Semen Viciae fabae, Fructus Lycopersici esculenti, dish
The total serum IgE of the healthy leaves of bean, Semen vignae sinensis, Fructus Cucumidis sativi, Fructus Cucurbitae moschatae and sweet bean.
2, utilize the test kit in embodiment 1 and detection method to the virosis leaf in step 1 and the RNA sample of healthy leaves
Product detect, and verify the specificity of test kit further.Each sample duplicate detection three times.Experiment arranges blank right simultaneously
According to, i.e. substitute PCR reaction template with water.
Testing result is as shown in Figure 3.Result shows, all samples does not all amplify band, and the Semen vignae sinensis flower of the present invention is described
Mosaic virus subfamily virus wide spectrum detection kit will not produce cross reaction with other viral species and host plant, demonstrates good
Good specificity.
Embodiment 3, the sensitivity technique of cowpea mosaic virus subfamily virus wide spectrum detection kit
1, the total serum IgE containing the sick leaf sample of Flos Cucurbitae mosaic virus (Agdia LPC 26400 (C2013)) is extracted.
2, synthesize cDNA template according to the method for 1 in the step 3 of embodiment 1, and the cDNA template of synthesis is carried out 10
Times serial dilution becomes 10-1～10-10The cDNA template of totally 10 gradient concentrations, according still further to the test kit in embodiment 1 and detection side
The cDNA template of 10 gradient concentrations is detected by method respectively, the sensitivity of checking test kit.Each sample duplicate detection three
Secondary.
Testing result is as shown in Figure 4.Result shows, the detection kit of the present invention can detect 10-8The cDNA of concentration
Template, illustrates that the primer of the present invention has higher sensitivity, it is sufficient to meet the needs of routine testing.
Claims (10)
1. detection or the primer set of auxiliary detection cowpea mosaic virus subfamily virus, for following 1) or 2):
1) it is made up of primer 1, primer 2 and primer 3；
2) it is made up of primer 1, primer 2, primer 4 and primer 5；
Described primer 1 is the single strand dna shown in sequence 1；
Described primer 2 is the single strand dna shown in sequence 2；
Described primer 3 is the single strand dna shown in sequence 3；
Described primer 4 is the single strand dna shown in sequence 4；
Described primer 5 is the single strand dna shown in sequence 5.
2. detection or a PCR reagent for auxiliary detection cowpea mosaic virus subfamily virus, complete including described in claim 1
Primer.
PCR reagent the most according to claim 2, it is characterised in that: described primer 1, described primer 2, described primer 3, institute
State primer 4 and the described primer 5 final concentration in described PCR reagent is 10 μm ol/L.
4. detection or a test kit for auxiliary detection cowpea mosaic virus subfamily virus, complete including described in claim 1
PCR reagent described in primer or Claims 2 or 3.
5. the primer set described in claim 1 or the PCR reagent described in Claims 2 or 3 or the reagent described in claim 4
In detection or auxiliary, box detects whether virus to be measured is the application in cowpea mosaic virus subfamily virus；
Or the primer set described in claim 1 or the PCR reagent described in Claims 2 or 3 or the reagent described in claim 4
Box application in preparation detection or auxiliary detect the product whether virus to be measured be cowpea mosaic virus subfamily virus.
6. the primer set described in claim 1 or the PCR reagent described in Claims 2 or 3 or the reagent described in claim 4
Whether box contains the application in cowpea mosaic virus subfamily virus in detection or auxiliary detection testing sample；
Or the primer set described in claim 1 or the PCR reagent described in Claims 2 or 3 or the reagent described in claim 4
Whether box contains the application in the product of cowpea mosaic virus subfamily virus in preparation detection or auxiliary detection testing sample.
7. whether detection or auxiliary detection testing sample contain the method that cowpea mosaic virus subfamily is viral, including as follows
Step:
(1) from described testing sample, extract total serum IgE, use the primer 1 in claim 1 to carry out reverse transcription reaction, treated
Survey cDNA；
(2) using described cDNA to be measured as template, the primer 1 in claim 1 and primer 2 is used to carry out the reaction of first round PCR,
Obtain first round PCR primer；
(3) using first round PCR primer as template, using the primer 1 in claim 1 and primer 3 to carry out second, to take turns PCR anti-
Should, it is thus achieved that second takes turns PCR primer；
Or using first round PCR primer as template, use the primer 4 in claim 1 and primer 5 to carry out second and take turns PCR reaction,
Obtain second and take turns PCR primer；
(4) determine in described testing sample whether contain Semen vignae sinensis as follows according to the described second size taking turns PCR primer
Mosaic virus subfamily virus:
If described second take turns in PCR primer containing size be 400～440bp DNA fragmentation, in the most described testing sample containing or
Candidate contains cowpea mosaic virus subfamily virus, otherwise does not contains in the most described testing sample or candidate does not contains Cowpea
Poison subfamily virus.
Method the most according to claim 7, it is characterised in that: the temperature of described reverse transcription reaction is 42 DEG C.
9. according to the method described in claim 7 or 8, it is characterised in that: described first round PCR reaction carries out 35 circulations altogether,
The annealing temperature of front 5 circulations is 42 DEG C, and the annealing temperature of rear 30 circulations is 50 DEG C.
10. according to described method arbitrary in claim 7-9, it is characterised in that: described second takes turns PCR reaction carries out 40 altogether
Circulation, front 5 circulation annealing temperatures be 42 DEG C, rear 35 circulation annealing temperature be 52 DEG C.
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| 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 |
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN102206714A (en) * | 2011-04-08 | 2011-10-05 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primer, detection method and application for general RT-PCR detection of comovirinae subvirus |
| WO2014155078A1 (en) * | 2013-03-25 | 2014-10-02 | Arcis Biotechnology Holdings Limited | Method of dna/rna extraction using silylated quaternary ammonium compounds (siqac) |
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| 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 |
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN102206714A (en) * | 2011-04-08 | 2011-10-05 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primer, detection method and application for general RT-PCR detection of comovirinae subvirus |
| WO2014155078A1 (en) * | 2013-03-25 | 2014-10-02 | Arcis Biotechnology Holdings Limited | Method of dna/rna extraction using silylated quaternary ammonium compounds (siqac) |
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