CN102888470B - RT-PCR primer of mushroom dsRNA virus and detection method - Google Patents
RT-PCR primer of mushroom dsRNA virus and detection method Download PDFInfo
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Abstract
The invention relates to a mushroom virus detection method, particularly relates to an RT-PCR primer of mushroom dsRNA virus and a detection method, and belongs to the field of biotechnology. The RT-PCR primer is shown as SEQ NO.1-2. The detection method comprises the steps of extracting mushroom dsRNA, forming cDNA through inverse transcription, using the cDNA through inverse transcription as a template, and establishing RT-PCR detection of the mushroom dsRNA virus, wherein the size of the RT-PCR output is 852bp. The method has the advantages of short detection time, high sensitivity (the primer can be detected clearly just by using 10ng of dsRNA, and the specificity and the reliability of the detection result are enhanced by using a special virus primer for inverse transcription), the detection result is reliable, the judgment is easy and the repeatability is high. The gene is the physical basis of heredity and is unlikely to be influenced by the external factor. Besides, the detection map obtained has a specificity stripe 852bp, and the dominance judgment marker is obvious.
Description
technical field
The present invention relates to a kind of Lentinula edodes Virus detection method, be specifically related to a kind of RT-PCR primer and detection method of mushroom dsRNA virus, belong to biological technical field.
background technology
Virus disease is a class disease of outbalance in Edible Fungi, different from other causal organism harm, and it has the property of hiding, may be until certain growth period just starts aobvious disease.Mushroom (
lentinus edodes (Berk.) sing) be famous edible mushrooms, its ultimate production is only second to Twospore Mushroom, is second-biggest-in-the-world mushroom class.China is the area of origin of cultivating champignon, is also maximum mushroom production and marketing distribution centre, the current whole world.But along with cultivating champignon popularization, in recent years there is the Lentinula edodes Virus in various degree evil of being critically ill, after mushroom infects virus, mycelial growth is slow, growing way weakens, sporophore deformity, Quality and yield degradation, in Hubei, once fairly large outburst Lentinula edodes Virus was sick on the ground such as Zhejiang, Fujian, bring heavy financial loss, the sound development of serious threat mushroom industry to mushroom agriculture.
Lentinula edodes Virus form is various, varies in size, and mainly comprises spherical, rod and tadpole shape, and some doubtful virions, and its genome major part belongs to dsRNA.Utilized Electron microscopy virus in the past, a large amount of difform virion of usually can having observed infected in tissues, object virus is difficult to be resolved and identify.Simultaneously because Mushroom virus does not show symptom conventionally, may be also just can encapsidate, so must depend on, heritable dsRNA detected, could prove their existence.In the more than ten years recently, Electron microscopy Mushroom virus technology is replaced by the method for typical ethidium bromide fluorescent dye dsRNA banding pattern gradually.Compare with Electron microscopy technology, dsRNA analyzes has clear superiority differentiating in virus similar in form, that heredity is different and differentiating on virus and ordinary cells composition, and the susceptibility that dsRNA analyzes is stronger, diagnosis more rapidly and reliable.
Detoxification bacterial strain is compared embodiment clear superiority at aspects such as vigor, resistance, output with band toxic bacterial strain.Because the research of Lentinula edodes Virus disease also relatively lags behind, we also do not find comparison effective means to prevent and treat at present, thereby healthy mushroom strain is provided is to guarantee that cultivating champignon obtains the key of high yield and stable yields.Therefore set up the RT-PCR detection method of mushroom dsRNA virus, for Lentnus edodes provides healthy bacterial classification, have great importance, simultaneously for the foundation of mushroom strain detoxification technology, the virus-free breed of variety of mushroom etc. are laid a good foundation.
summary of the invention
The RT-PCR primer and the detection method that the object of this invention is to provide a kind of mushroom dsRNA virus, the method is by the special primer of design Lentinula edodes Virus gene, set up the RT-PCR method of Lentinula edodes Virus, for convenience of, quickly and efficiently detect mushroom strain whether in spite of illness poison lay the first stone.
First the present invention provides a kind of RT-PCR primer of mushroom dsRNA virus, as follows:
Forward primer L1-S 5 '-ACCGCAATCAATAACT-3 ',
Reverse primer L1-A 5 '-AGACAAGATGGAGACG-3 '.
Secondly, the present invention also provides a kind of RT-PCR detection method of mushroom dsRNA virus, first extracts mushroom dsRNA, by reverse transcription, become cDNA, then the cDNA of reverse transcription of take is template, and the RT-PCR that sets up mushroom dsRNA virus detects, and RT-PCR product size is 852bp.
Described RT-PCR detection method specifically comprises the following steps:
(1) extraction of mushroom dsRNA;
(2) the mushroom dsRNA of extraction is become to cDNA by reverse transcription: in PCR pipe, add 10～100 ng/ul dsRNA 1ul, 10 umol/L reverse primer L1-A 0.3～0.5 ul, DEPC water 8.5～8.8 ul, 95～100 ℃ of insulation 5 min, then place rapidly 5 min on ice, add again 5 * RT Buffer, 4 ul, 10 mmol/L dNTPs 0.5～2 ul, 40 U/ul RNA enzyme inhibitors 0.5 ul, 100 U/ul ThermoScript II M-MLV 0.3～0.5 ul, DEPC water 3～4.7ul, place on PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min, obtain the cDNA of reverse transcription,
(3) RT-PCR detects: the cDNA of reverse transcription of take is template, and the RT-PCR that sets up mushroom dsRNA virus detects, pcr amplification system: cDNA template 1～3 ul, 10 * PCR Buffer, 3 ul, and containing 15 mmol/L MgCl
2, 2.5 mmol/L dNTPs 1～2.4 ul, 10 umol/L forward primer L1-S 0.5～0.7 ul, 10 umol/L reverse primer L1-A 0.5～0.7 ul, 5 U/ul Taq archaeal dna polymerase 0.3～0.6 ul, with DEPC water, complement to 30 ul;
PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 45second, 48.5 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min;
(4) detected result of RT-PCR product: according to the DNA collection of illustrative plates of electrophoresis detection, be that the specific DNA band of 852bp is labeled as Xianggu mushroom strain with dsRNA virus if amplify molecular weight, do not represent not carry dsRNA virus if amplify specific DNA band.
The method of utilizing RT-PCR to detect Lentinula edodes Virus provided by the invention, detects virus, dsRNA with Electronic Speculum and detects virus and detect virus with zymetology and compare, have highly sensitive, detection time is short, detected result is reliable, easily judge, the advantage such as reproducible.Specific as follows:
When 1, Electronic Speculum detects virus, virus must occur with the form of particle being just observed, and is subject to the restriction on opportunity; In addition, separated Lentinula edodes Virus particle difficulty is larger, and needs multiple purifying, thickening equipment, and electronic microscopic examination, and general production, research unit are difficult to carry out.
2, (dsRNA of Xianggu mushroom strain detects at document for Wang Li etc., edible mushrooms journal 2009.) in, Xianggu mushroom strain dsRNA virus is detected, although strains tested all detects the existence of dsRNA mostly, the size and number of the dsRNA band that amplification is arrived is different.And when application dsRNA detects virus, the impact of being extracted quality and concentration often contains other RNA in dsRNA leaching process, during detected result by gel electrophoresis, often show the unclear or disappearance of band, repeatability and sensitivity are poor.
3, zymetology detection virus is subject to the character of enzyme and extracts sample size and limit, the time-consuming manpower that takes again when this external application isozyme detects virus.
4, the RT-PCR detection method of setting up with Lentinula edodes Virus genome, detection time is shorter, highly sensitive, and (detection system only needs the dsRNA of 10ng just can clearly to detect, with the peculiar primer of virus, carry out reverse transcription, strengthened specificity and improved the reliability of detected result), reliable, the easily judgement of detected result, the advantage such as reproducible.Gene is physical basis of heredity, is not subject to the impact of extraneous factor etc., and the detection collection of illustrative plates simultaneously obtaining has specific band: 852bp, its dominant marker judges very clear.
The present invention, for Lentnus edodes provides virus-free bacterial classification, provides reliable detection method, and there is to larger using value the aspects such as the foundation of mushroom strain detoxification technology, the virus-free breed of variety of mushroom.
accompanying drawing explanation
Fig. 1 is for being used primer L1-S and L1-A to detect the DNA collection of illustrative plates of Xianggu mushroom strain amplification, and wherein M～7 are swimming lane numbering; The molecular weight of the numeral DNA fragmentation in left side, the Mark that swimming lane M is DL2000, swimming lane 1～7 is viruliferous Xianggu mushroom strain, labeled fragment is 852bp.
Fig. 2 is for being used primer L1-S and L1-A to detect the DNA collection of illustrative plates of Xianggu mushroom strain amplification, and wherein M～13 are swimming lane numbering; The molecular weight of the numeral DNA fragmentation in left side, the Mark that swimming lane M is DL2000, swimming lane 8 is viruliferous Xianggu mushroom strain, and labeled fragment is 852bp, and swimming lane 9～13 is the Xianggu mushroom strain after detoxification, the labeled fragment of 852bp do not detected.
Embodiment
RT-PCR detection method specifically comprises the following steps:
(1) extraction of mushroom dsRNA;
(2) the mushroom dsRNA of extraction is become to cDNA by reverse transcription;
Holding temperature before the RT of take reaction and the dsRNA concentration in RT reaction system, reverse primer concentration, dNTPs concentration, ThermoScript II M-MLV enzyme amount etc. are parameter, RT reaction to each parameter is optimized one by one, and the RT reaction system parameter area that obtains mushroom dsRNA is as follows: 95～100 ℃ of 10～100 ng/ul dsRNA, reverse primer 0.15～0.25 umol/ L, dNTPs 025～1 mmol/L, ThermoScript II M-MLV 2.5～7.5 U/ul, holding temperature.Through optimizing, can amplify specific band clearly.
(3) RT-PCR detects: the cDNA obtaining through RT reaction of take is template, to cDNA addition, the Mg in pcr amplification system
2+the parameters such as concentration, dNTPs concentration, forward and reverse primer concentration, Taq archaeal dna polymerase enzyme amount are optimized one by one, and each parameter area that obtains PCR optimum augumentation system is: cDNA template 1～4 ul, Mg
2+concentration 1.5～2.5 mmol/L, dNTPs concentration 0.12～0.2 mmol/L, primer concentration 0.13～0.23 umol/ L, Taq enzyme enzyme amount 0.07～0.1U/ul.Through optimizing, can amplify specific band clearly.
In order fully to disclose the detection method of a kind of Lentinula edodes Virus of the present invention, below in conjunction with embodiment, be illustrated.
Embodiment 1: a kind of RT-PCR detection method of mushroom dsRNA virus
A RT-PCR detection method for mushroom dsRNA virus, comprises the following steps:
One, Lentinula edodes Virus RT-PCR detects the design of primer
According to the Lentinula edodes Virus sequence of having delivered
lentinula edodesmycovirus HKB gene(AB429556.2), apply Primer5 or other primer-design software, obtain the detection primer of Lentinula edodes Virus RT-PCR.The feature of this primer is that Lentinula edodes Virus gene is had to specific amplified site, and the clip size obtaining is 852bp.Lentinula edodes Virus RT-PCR primer is as follows:
L1-S 5’-ACCGCAATCAATAACT-3’，
L1-A 5’-AGACAAGATGGAGACG -3’。
Two, the extraction of the dsRNA of Lentinula edodes Virus
The mushroom strain of undesired mushroom strain or generation virus disease (Wang Li etc., the dsRNA of Xianggu mushroom strain detects, edible mushrooms journal 2009.), the mushroom strain after detoxification are carried out to mycelium culture and collection, extract the dsRNA of mushroom.
(1) the mycelial cultivation of mushroom strain and collection
1, under aseptic technique, mushroom strain is transferred in PDA inclined-plane, 25 ℃ of constant temperature culture 13d-15d;
2,, under aseptic technique, with the good mushroom strain of the inoculation rake broken switching of rake, transfer into 250 ml triangular flasks, containing in 100 ml PDA liquid nutrient mediums;
3, be placed on the 25 ℃ of standing cultivation of thermostat container 20d-25d;
4, put on disposable glove, cultured shiitake mushroom hypha in triangular flask, pour two-layer gauze for filtering into, with tweezers or key, choose the agar block not consuming except, aseptic water washing, filter paper blots;
5, take 0.5～1g mycelia, with filter paper, wrap, be directly used for extracting viral dsRNA or-20 ℃ and save backup.
(2) dsRNA of mushroom extracts
1, the mushroom mycelium of having collected is put into mortar, add the rapid grind into powder of liquid nitrogen;
Remaining step reference (Guo Lingfang, Zhang Changqing, the Shandong studies of the Hongloumeng, etc. a kind of simple and quick Mushroom virus dsRNA extracting method [J]. experimental technique and management, 2010,27 (12): 51～53,57.)
Three, RT-PCR detects and sets up
The mushroom dsRNA extracting becomes cDNA by reverse transcription, and the cDNA transcribing of take is template, and the RT-PCR that sets up Lentinula edodes Virus detects.
(1) RT of mushroom dsRNA reaction
Best RT reaction conditions is: in PCR pipe, add 100 ng/ul dsRNA 1ul, 10 umol/L reverse primer L1-A 1.5 ul, DEPC water 7.5 ul, 95 ℃ of insulation 5 min, then place rapidly 5 min on ice.Add again 5 * RT Buffer, 4 ul, 10 mmol/L dNTPs 1 ul, 40 U/ul RNA enzyme inhibitors 0.5 ul, 100 U/ul ThermoScript II M-MLV 0.5 ul, DEPC water 5ul, place on PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min, obtain the cDNA of reverse transcription.
(2) pcr amplification
PCR optimum augumentation system is: cDNA template 2 ul, 10 * PCR Buffer, 3 ul(are containing 15 mmol/L MgCl
2), 2.5 mmol/L dNTPs 1.5 ul, 10 umol/L primer L1-S 5 '-ACCGCAATCAATAACT-3 ' 0.48 ul, 10 umol/L primer L1-A 5 '-AGACAAGATGGAGACG-3 ' 0.48 ul, 5 U/ul Taq archaeal dna polymerase 0.24 ul, with DEPC water, complement to 30 ul.
PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 45second, 48.5 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min.
Four, pcr amplification product detects
Specifically detection method is, gets pcr amplification product 8 ul and adds 1.5 ul 6 * tetrabromophenol sulfonphthalein sample-loading buffers, mixes, at 1.0% sepharose, containing Goldview
tMdNA dyestuff carries out electrophoresis, and 5V/cm constant voltage electrophoresis 50 min, are observed and taken pictures by ultraviolet gel imaging system.Or, get pcr amplification product 8 ul, mix with 1.5 ul 6 * tetrabromophenol sulfonphthalein sample-loading buffers, on the fine jade vinegar sugar gel of point sample in 1%, in 0.5 X tbe buffer liquid, 5V/cm constant voltage electrophoresis 0.5～1h, after electrophoresis finishes, with EB dyeing, then on gel imaging instrument, take a picture.
Pcr amplification product detected result is the single fragment that the bacterial classification that contains mushroom dsRNA virus can specific amplified goes out 852bp, as shown in Figure 1, if mushroom strain does not go out fragment with the amplification of this dsRNA virus, as shown in Figure 2.
Experiment results proved, while the Xianggu mushroom strain with dsRNA virus being carried out to RT-PCR detection with primer L1-S 5 '-ACCGCAATCAATAACT-3 ' and primer L1-A 5 '-AGACAAGATGGAGACG-3 ', the band of a 852bp can be detected, through the Xianggu mushroom strain of detoxification, can't detect this band.The RT-PCR that utilizes of the present invention detects Xianggu mushroom strain whether with the method for dsRNA virus, its detection time short, highly sensitive, detected result reliably, easily judgement.
Main medium used in the present invention, reagent and instrument are as follows: (all chemical reagent are analytical pure)
1, PDA slant medium: potato 200 g, glucose 20 g, agar 20 g, water 1000 mL, pH nature, 121 ℃ of sterilizing 30min;
2, PDA liquid nutrient medium: potato 200 g, glucose 20 g, water 1000 mL, pH nature, 121 ℃ of sterilizing 30min;
3, one-step RT-PCR test kit (RevertiAid
tMfirst Strand cDNA Synthesis Kit) purchased from Fermentas company;
4、0.5×TBE：44.5 mmol/L Tris, 50 mmol/L HBO
3、1 mmol/L EDTA；
5, sample-loading buffer: 0.1% tetrabromophenol sulfonphthalein, 40% sucrose;
6, EB:10 mg/ml ethidium bromide;
7, pcr amplification reagent is purchased from the precious biotech firm in Dalian.
<110> University Of Agriculture and Forestry In Fujian
RT-PCR primer and the detection method of a <120> mushroom dsRNA virus
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> DNA
<213> artificial sequence
<400> 1
accgcaatca ataact 16
<210> 2
<211> 16
<212> DNA
<213> artificial sequence
<400> 2
agacaagatg gagacg 16
Claims (1)
1. a RT-PCR detection method for mushroom dsRNA virus, is characterized in that: described RT-PCR detection method specifically comprises the following steps:
(1) extraction of mushroom dsRNA;
(2) the mushroom dsRNA of extraction is become to cDNA by reverse transcription: in PCR pipe, add 100 ng/ μ l dsRNA 1 μ l, 10 μ mol/L reverse primer L1-A 1.5 μ l, DEPC water 7.5 μ l, 95 ℃ of insulation 5 min, then place rapidly 5 min on ice, add again 5 * RTBuffer, 4 μ l, 10 mmol/L dNTPs 1 μ l, 40 U/ μ l RNA enzyme inhibitors 0.5 μ l, 100 U/ μ l ThermoScript II M-MLV 0.5 μ l, DEPC water 5 μ l, place on PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min, obtain the cDNA of reverse transcription, wherein reverse primer L1-A is 5 '-AGACAAGATGGAGACG-3 ',
(3) RT-PCR detects: the cDNA of reverse transcription of take is template, and the RT-PCR that sets up mushroom dsRNA virus detects, pcr amplification system: cDNA template 2 μ l, containing 15 mmol/L MgCl
210 * PCR Buffer, 3 μ l, 2.5 mmol/L dNTPs 1.5 μ l, the primer L1-S 0.48 μ l that 10 μ mol/L sequences are 5 '-ACCGCAATCAATAACT-3 ', primer L1-A 0.48 μ l, the 5 U/ μ l Taq DNA polysaccharase 0.24 μ l that 10 μ mol/L sequences are 5 '-AGACAAGATGGAGACG-3 ', with DEPC water, complement to 30 μ l;
PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 45sec, 48.5 ℃ of 45sec, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min;
(4) detected result of RT-PCR product: according to the DNA collection of illustrative plates of electrophoresis detection, if amplify molecular weight and be the specific DNA band of 852bp, be labeled as Xianggu mushroom strain with dsRNA virus, if amplify specific DNA band, do not represent not carry dsRNA virus.
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| CN103421766B (en) * | 2013-08-06 | 2015-05-27 | 大连医科大学 | Method for extracting anti-oxidant anti-tumor Latcripin-3 gene segment from mushroom C91-3 strain |
| CN114438264B (en) * | 2022-02-24 | 2023-07-21 | 福建农林大学 | Pleurotus geesteranus RNA virus detection primer set and detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000229861A (en) * | 1999-02-15 | 2000-08-22 | Nippon Kinoko Kenkyusho | Immunopotentiator, its production and usage |
| CN101812541A (en) * | 2010-04-07 | 2010-08-25 | 中国科学院武汉病毒研究所 | Lentinus edodes virus detection kit and method |
| KR20110134610A (en) * | 2010-06-09 | 2011-12-15 | 경상대학교산학협력단 | Specific primer for diagnosing lentinula edodes virus lehv, and method for diagnosing using the same |
| KR20110134616A (en) * | 2010-06-09 | 2011-12-15 | 경상대학교산학협력단 | Specific primer for diagnosing lentinula edodes virus lesv, and method for diagnosing using the same |
-
2012
- 2012-10-30 CN CN201210423457.4A patent/CN102888470B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000229861A (en) * | 1999-02-15 | 2000-08-22 | Nippon Kinoko Kenkyusho | Immunopotentiator, its production and usage |
| CN101812541A (en) * | 2010-04-07 | 2010-08-25 | 中国科学院武汉病毒研究所 | Lentinus edodes virus detection kit and method |
| KR20110134610A (en) * | 2010-06-09 | 2011-12-15 | 경상대학교산학협력단 | Specific primer for diagnosing lentinula edodes virus lehv, and method for diagnosing using the same |
| KR20110134616A (en) * | 2010-06-09 | 2011-12-15 | 경상대학교산학협력단 | Specific primer for diagnosing lentinula edodes virus lesv, and method for diagnosing using the same |
Non-Patent Citations (11)
| Title |
|---|
| 70. |
| Detection of double stranded RNA elements in cultivated Lentinula;Ohta C,et al;《Mushroom Sci Biotechnol》;20081231;第16卷;155－158 * |
| Lentinula edpdes mycovirus HKB gene for hypothetical protein, putative RNA-dependent RNA polymerase,complete cds;Magae,Y;《Genbank:AB429556.2》;20120712 * |
| Magae,Y.Lentinula edpdes mycovirus HKB gene for hypothetical protein, putative RNA-dependent RNA polymerase,complete cds.《Genbank:AB429556.2》.2012, |
| Molecular characterization of a novel mycovirus in the cultivated mushroom, Lentinula edodes;Yumi Magae;《Virology Journal》;20120331;第9卷 * |
| Ohta C,et al.Detection of double stranded RNA elements in cultivated Lentinula.《Mushroom Sci Biotechnol》.2008,第16卷155－158. |
| YumiMagae.Molecularcharacterizationofanovelmycovirusinthecultivatedmushroom Lentinula edodes.《Virology Journal》.2012 |
| 一种新香菇病毒基因组部分cDNA序列及病毒RT-PCR检测;姚立等;《微生物学通报》;20100120;第37卷(第1期);61-70 * |
| 姚立等.一种新香菇病毒基因组部分cDNA序列及病毒RT-PCR检测.《微生物学通报》.2010,第37卷(第1期),61− |
| 王丽等.香菇菌株的dsRNA检测.《食用菌学报》.2009,第16卷(第4期),10－15. |
| 香菇菌株的dsRNA检测;王丽等;《食用菌学报》;20091231;第16卷(第4期);10-15 * |
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