CN101561415B - Method for simultaneously detecting banana bunchy top virus and floral leaf heart rot virus - Google Patents
Method for simultaneously detecting banana bunchy top virus and floral leaf heart rot virus Download PDFInfo
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- CN101561415B CN101561415B CN2008100275973A CN200810027597A CN101561415B CN 101561415 B CN101561415 B CN 101561415B CN 2008100275973 A CN2008100275973 A CN 2008100275973A CN 200810027597 A CN200810027597 A CN 200810027597A CN 101561415 B CN101561415 B CN 101561415B
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Abstract
The invention discloses a method for simultaneously detecting banana bunchy top virus and floral leaf heart rot virus, which is characterized by comprising the following steps: firstly, extraction of total nucleic acid of banana diseased leaves; secondly, augmentation of RT-PCR; thirdly, electrophoretic analysis. The invention aims at overcoming the shortcomings of the prior art and providing themethod which is simple and accurate, can simultaneously detect the banana bunchy top virus and the floral leaf heart rot virus by only one time detection, effectively reduces the sample cross contamination and has high detection efficiency.
Description
Technical field
The present invention relates to a kind of plant virus detection method, particularly a kind of detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously.
Background technology
Banana is China important industrial crops in south; But virosis is one of principal element of its sustainable development of restriction always; Especially abaca bunchy top virus (banana bunchy topvirus; BBTV) banana bunchy top disease that causes and by cucumber mosaic virus (two the most general and serious diseases take place in cucumber mosaicvirus, the banana floral leaf heart rot that CMV) causes in the banana production.Because China south banana planting seedling is main with tissue cultured seedling at present; For the generation that prevents land for growing field crops banana disease viral disease with spread; Guarantee tissue culture seedlings of bananas not in spite of illness poison be the of paramount importance ring in the virus-free production of whole banana industry; Reinforcement just can be guaranteed the safety that the banana land for growing field crops produces from the diffusion of control virus effectively on the source to the detection of the early stage virus of tissue cultured seedling; Also can guarantee simultaneously external quarantine request to China's tissue culture seedlings of bananas departure, therefore detection technique and the method to banana seedlings virus just becomes the key point that virus is controlled.This paper has studied and has explored relatively more suitable basic unit actual detected and infected the technology and the methods of two kinds of topmost virus detections of banana according on the basis to the virus generaI investigation of banana planting base, Zhongshan City, Guangdong Province.
The banana disease viral disease is one of key constraints of China's banana production and tissue culture seedlings of bananas departure always at present, and the banana seedling has particularly been become the very effective measure that control banana disease viral disease spreads and ensure its smooth departure to the viral early detection of tissue culture seedlings of bananas.Abaca bunchy top virus (BBTV) and banana floral leaf heart rot virus (CMV) are two topmost viruses that infect China's banana, and their frequent compound infecting.The common method that detects them is with EUSA method (ELISA), but banana seedling tissue cultured seedling particularly, because plant is immature, body inner virus content is very low, the situation of omission and flase drop can usually can occur with the method for ELISA; And molecular biological method PCR method particularly, its sensitivity is higher more than 1000 times than ELISA method, very is fit to detect the sample of low concentration virus.There have been many bibliographical informations to detect the virus of banana seedlings with the method for PCR.But they all are to detect to single virus; Though Peng Jun etc. have also reported with multiplex PCR and have detected BBTV and CMV at " gardening journal newspaper "; But also be to extract DNA and RNA respectively, then again to the independent reverse transcription of CMV again with the sample mechanical mixture of BBTV, carry out PCR at last and detect; Not only running program is complicated, and causes the sample cross pollution easily.
Therefore, the existing detection method that is used to detect abaca bunchy top virus and floral leaf heart rot virus awaits further improvement.
Summary of the invention
The objective of the invention is in order to overcome weak point of the prior art; Provide a kind of method simple; Only need just can detect abaca bunchy top virus and floral leaf heart rot virus simultaneously, effectively reduce the sample cross pollution, detection efficiency height and accurate test method through one-time detection.
In order to achieve the above object, the present invention adopts following scheme:
A kind of detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously is characterized in that may further comprise the steps:
(1), the extraction of banana disease leaf TNA:
A, difference clip infect the banana diseased plant spire 0.1g of BBTV, CMV and MOI BBTV and CMV;
B, under liquid nitrogen (196 ℃), be ground into powder, add the CTAB extraction buffer of 900 μ L rapidly, change in the centrifuge tube of 2mL through 65 ℃ of preheatings, 65 ℃ of water-bath 30min, during shake up frequently;
C, add 900 μ Ll chloroform/isoamylol (V: V/24: 1), leniently shake up, under the room temperature with the centrifugal 8min of small desk hydro-extractor 12000rpm;
D, supernatant is forwarded in the new 2mL centrifuge tube, add the 3mol/LNaAc (pH5.2) and the ice-cold absolute ethyl alcohol of 2.5 * volume of 1/10 volume, place 2hr for-20 ℃, then at 4 ℃ of centrifugal 10min of following 12000rpm;
E, abandon supernatant, 70% the ethanol that does not have RNase with 400 μ L suspends the RNA vibration, makes salt ion in the RNA deposition by fully dissolving; Centrifugal once more 10min carefully outwells supernatant with precipitated rna, quick subsequently centrifugal 5s; Ethanol on the tube wall is collected pipe at the end,, place and blow about 5min on the super-clean bench with the exhaustion of lancet head; With the water-soluble deposition of separating of 50 μ L DEPC ,-20 ℃ of storages are subsequent use at last;
(2), RT-PCR amplification
The reaction primer
BBTV primer: BBTV-P1:5 '-ATGTGGTATGCTGGATGTTC-3 '
BBTV-P2：5’-GTTCATATTTCCCGCTTTGA-3’
Amplified production is 748bp;
CMV primer: CMV-P1:5 '-CACCCAACCTTTGTGGGTAG-3 '
CMV-P2：5’-CAACACTGCCAACTCAGCTC-3’
Amplified production is 557bp;
Upstream and downstream primer (the P1 that in the thin-walled PCR of 0.2mL pipe, adds deionized water 3.5 μ L, 2 * buffer12.5 μ L, amplification enzyme mixed liquor 1 μ L, amplification BBTV and CMV genetic fragment respectively; P2) each 1 μ L (10 μ mol/L), banana disease leaf TNA 1 μ L (0.5 μ g); After mixing; Carry out single stage method RT-PCR amplification, and the negative contrast of TNA from the virus-free banana blade that infects of health, to extract; RT-PCR amplification procedure wherein: 50 ℃ of 30min, 94 ℃ of 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, totally 35 circulations;
(3), electrophoretic analysis
Get 5 μ L reaction product and on 1% Ago-Gel, carry out electrophoretic analysis with 1 * TAE damping fluid preparation.
Aforesaid a kind of detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously, wherein the CTAB extraction buffer described in the step (1) is the Tris-HCl (pH8.0) of 0.1M; 0.02M EDTA (pH8.0); 1.4M NaCl; The composition of 2% CTAB (W/V) and 0.2% beta-mercaptoethanol.
The original producton location of the part material that the present invention uses:
1, the susceptible material of banana: pick up from banana planting base, Zhongshan City, Guangdong Province;
2, RT-PCR kit PrimeScript
TMOne-Step Ver.2.0: available from precious bioengineering (Dalian) company limited,
3, reverse transcriptase, Taq enzyme, CTAB (Cetyltriethyl ammonium bromide), Trizol RNA extraction reagent and other biological reagent are all available from the general rich bio tech ltd in Guangzhou.
In sum, beneficial effect of the present invention:
(1), detection method of the present invention adopts 2.5 * the absolute ethyl alcohol isopropyl alcohol that replaces in the CTAB method, using usually precipitate TNA, both can obtain total DNA, also obtained complete total RNA.With this TNA is template, and is used in combination RT-PCR single stage method kit, promptly can detect CMV separately, also can detect BBTV separately, more can detect this two kinds of viruses simultaneously.
(2), RT-PCR of the present invention is reflected at reverse transcription RT and PCR in the same pipe to carry out; So not only can reduce the cross pollution between the sample; Also, make detection sensitivity more improve, and can improve detection efficiency because the reaction product of whole RT all is used as the template of PCR.
(3) the present invention can disposable extraction sample TNA with carry out disposable RT-PCR and detect; Can detect single or compound these two kinds of viruses; Thereby improved detection efficiency greatly; And the unnecessary pollution of having avoided artificial hybrid dna and RNA nucleic acid samples to be caused, be practicality detecting of banana virus and method quickly and accurately.
Description of drawings
Fig. 1 is testing result figure of the present invention.
Wherein, M:100bp DNA marker; 1: negative control; 2:CMV PCR detects; 3:BBTV PCR detects; 4:CMV, BBTV PCR simultaneously detect; 5: the total DNA of banana disease Ye; 6: banana disease leaf TNA.
Embodiment
Below in conjunction with embodiment the present invention is done and to further describe:
Embodiment 1
A kind of detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously of the present invention may further comprise the steps:
(1), the extraction of banana disease leaf TNA:
Clip infects the banana diseased plant spire 0.1g of BBTV, CMV and MOI BBTV and CMV respectively; Under liquid nitrogen (196 ℃), be ground into powder, add the CTAB extraction buffer of 900 μ L rapidly, change in the centrifuge tube of 2mL through 65 ℃ of preheatings, 65 ℃ of water-bath 30min, during shake up frequently; Add 900 μ Ll chloroform/isoamylol (V: V/24: 1), leniently shake up, under the room temperature with the centrifugal 8min of small desk hydro-extractor 12000rpm; Supernatant is forwarded in the new 2mL centrifuge tube, add the 3mol/L NaAc (pH5.2) and the ice-cold absolute ethyl alcohol of 2.5 * volume of 1/10 volume, place 2hr for-20 ℃, then at 4 ℃ of centrifugal 10min of following 12000rpm; Abandon supernatant, 70% the ethanol that does not have RNase with 400 μ L suspends the RNA vibration, makes salt ion in the RNA deposition by fully dissolving; Centrifugal once more 10min carefully outwells supernatant with precipitated rna, quick subsequently centrifugal 5s; Ethanol on the tube wall is collected pipe at the end,, place and blow about 5min on the super-clean bench with the exhaustion of lancet head; With the water-soluble deposition of separating of 50 μ L DEPC ,-20 ℃ of storages are subsequent use at last;
(2), RT-PCR amplification
Upstream and downstream primer (the P1 that in the thin-walled PCR of 0.2mL pipe, adds deionized water 3.5 μ L, 2 * buffer12.5 μ L, amplification enzyme mixed liquor 1 μ L, amplification BBTV and CMV genetic fragment respectively; P2) each 1 μ L (10 μ mol/L), banana disease leaf TNA 1 μ L (0.5 μ g); After mixing; Carry out single stage method RT-PCR amplification, and the negative contrast of TNA from the virus-free banana blade that infects of health, to extract; RT-PCR amplification procedure wherein: 50 ℃ of 30min, 94 ℃ of 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, totally 35 circulations; Wherein the upstream and downstream primer of BBTV and CMV genetic fragment is respectively
BBTV primer: BBTV-P1:5 '-ATGTGGTATGCTGGATGTTC-3 '
BBTV-P2：5’-GTTCATATTTCCCGCTTTGA-3’
CMV primer: CMV-P1:5 '-CACCCAACCTTTGTGGGTAG-3 '
CMV-P2：5’-CAACACTGCCAACTCAGCTC-3’；
(3), electrophoretic analysis
Get 5 μ L reaction product and on 1% Ago-Gel, carry out electrophoretic analysis with 1 * TAE damping fluid preparation.
Analysis result is as shown in Figure 1, and when extracting the total DNA of banana disease Ye with conventional CTAB method, the result shows a complete total DNA of 23kb plant, like the 5th electrophoresis band; And use the inventive method to extract identical banana sample blade; The result shows except the complete total DNA of 23kb plant, also includes complete 28S and 18S rRNA band, explains and not only contains complete DNA in this sample; And also have complete total RNA, like the 6th electrophoresis band.With this TNA is template, when having only the CMV primer, can amplify the special band of 557bp of CMV, like the 2nd electrophoresis band; And when only adding the BBTV primer, also can amplify the special band of its 748bp, like the 3rd electrophoresis band; When adding CMV and BBTV primer simultaneously, under identical reaction conditions, can amplify their special band simultaneously, like the 4th electrophoresis band; And negative control has no band, like the 1st electrophoresis band.
This shows that detection method of the present invention can just can detect abaca bunchy top virus and floral leaf heart rot virus through one-time detection fast and accurately simultaneously.
Claims (2)
1. detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously is characterized in that may further comprise the steps:
(1), the extraction of banana disease leaf TNA:
A, difference clip infect the banana diseased plant spire 0.1g of BBTV, CMV and MOI BBTV and CMV;
B, under liquid nitrogen-196 ℃, be ground into powder, add the CTAB extraction buffer of 900 μ L rapidly, change in the centrifuge tube of 2mL through 65 ℃ of preheatings, 65 ℃ of water-bath 30min, during shake up frequently;
C, add 900 μ Ll chloroform/isoamylol V: V/24: 1, leniently shake up, under the room temperature with the centrifugal 8min of small desk hydro-extractor 12000rpm;
D, supernatant is forwarded in the new 2mL centrifuge tube, add the 3mol/LNaAc of 1/10 volume, the absolute ethyl alcohol that pH5.2 and 2.5 * volume are ice-cold is placed 2hr for-20 ℃, then at 4 ℃ of centrifugal 10min of following 12000rpm;
E, abandon supernatant, 70% the ethanol that does not have RNase with 400 μ L suspends the RNA vibration, makes salt ion in the RNA deposition by fully dissolving; Centrifugal once more 10min carefully outwells supernatant with precipitated rna, quick subsequently centrifugal 5s; Ethanol on the tube wall is collected pipe at the end,, place and blow about 5min on the super-clean bench with the exhaustion of lancet head; With the water-soluble deposition of separating of 50 μ L DEPC ,-20 ℃ of storages are subsequent use at last;
(2), RT-PCR amplification
The reaction primer
BBTV primer: BBTV-P1:5 '-ATGTGGTATGCTGGATGTTC-3 '
BBTV-P2：5’-GTTCATATTTCCCGCTTTGA-3’
CMV primer: CMV-P1:5 '-CACCCAACCTTTGTGGGTAG-3 '
CMV-P2：5’-CAACACTGCCAACTCAGCTC-3’
The upstream and downstream primer P1 that in the thin-walled PCR of 0.2mL pipe, adds deionized water 3.5 μ L, 2 * buffer12.5 μ L, amplification enzyme mixed liquor 1 μ L, amplification BBTV and CMV genetic fragment respectively; Each 1 μ L of P2; 10 μ mol/L, banana disease leaf TNA 1 μ L, 0.5 μ g is after mixing; Carry out single stage method RT-PCR amplification, and the negative contrast of TNA from the virus-free banana blade that infects of health, to extract; RT-PCR amplification procedure wherein: 50 ℃ of 30min, 94 ℃ of 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, totally 35 circulations;
(3), electrophoretic analysis
Get 5 μ L reaction product and on 1% Ago-Gel, carry out electrophoretic analysis with 1 * TAE damping fluid preparation.
2. a kind of detection method that detects abaca bunchy top virus and floral leaf heart rot virus simultaneously according to claim 1, wherein the CTAB extraction buffer described in the step (1) is the Tris-HCl of 0.1M, pH8.0; 0.02M EDTA, pH8.0; 1.4M NaCl; The composition of 2% CTAB (W/V) and 0.2% beta-mercaptoethanol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103290138B (en) * | 2012-02-23 | 2015-08-19 | 北京农学院 | The PCR in real time method for quick of abaca bunchy top virus |
| CN102703604B (en) * | 2012-05-14 | 2013-08-21 | 中国热带农业科学院环境与植物保护研究所 | Kit for rapidly detecting banana bunchy top virus by isothermal gene amplification and use method of kit |
| AU2013310861B2 (en) * | 2012-09-03 | 2019-02-14 | Qiagen Gmbh | Method for isolating RNA including small RNA with high yield |
| CN112779342A (en) * | 2019-11-11 | 2021-05-11 | 杨雷亮 | Loop-mediated isothermal amplification detection method for banana bacterial wilt bacteria |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5756708A (en) * | 1994-02-24 | 1998-05-26 | Queensland University Of Technology | DNA sequences of banana bunchy top virus |
| US5846705A (en) * | 1995-04-06 | 1998-12-08 | Development Center For Biotechnology | Nucleotide sequence of two circular SSDNA associated with banana bunchy top virus and method for detection of banana bunchy top virus |
| CN1506469A (en) * | 2002-12-10 | 2004-06-23 | 福建省农业科学院生物技术中心 | PCR kit for testing banana apical tuft virus and its test method |
| CN1600865A (en) * | 2003-09-23 | 2005-03-30 | 北京金长河科技发展有限公司 | Gene chip for detecting plant virus |
-
2008
- 2008-04-18 CN CN2008100275973A patent/CN101561415B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5756708A (en) * | 1994-02-24 | 1998-05-26 | Queensland University Of Technology | DNA sequences of banana bunchy top virus |
| US5846705A (en) * | 1995-04-06 | 1998-12-08 | Development Center For Biotechnology | Nucleotide sequence of two circular SSDNA associated with banana bunchy top virus and method for detection of banana bunchy top virus |
| CN1506469A (en) * | 2002-12-10 | 2004-06-23 | 福建省农业科学院生物技术中心 | PCR kit for testing banana apical tuft virus and its test method |
| CN1600865A (en) * | 2003-09-23 | 2005-03-30 | 北京金长河科技发展有限公司 | Gene chip for detecting plant virus |
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