CN102888470A - RT-PCR primer of mushroom dsRNA virus and detection method - Google Patents

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

A kind of RT-PCR primer and detection method of mushroom dsRNA virus

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 the Edible Fungi, and is different from other causal organism harm, and it has the property of hiding, may be until certain growth period just begins 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, also is maximum mushroom production and marketing distribution centre, the present whole world.But along with the cultivating champignon popularization, in various degree the Lentinula edodes Virus evil of being critically ill appears in recent years, mycelial growth was slow after mushroom infected virus, growing way weakens, sporophore deformity, the Quality and yield degradation is 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 for the mushroom farming.

The Lentinula edodes Virus form is various, varies in size, and mainly comprises sphere, 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, purpose virus is difficult to be resolved and identify.Simultaneously because Mushroom virus does not show symptom usually, may be also just can encapsidate, detect heritable dsRNA so must depend on, could prove their existence.In the recently more than ten years, Electron microscopy Mushroom virus technology is replaced by the method for typical ethidium bromide fluorescent dye dsRNA banding pattern gradually.Compare with the Electron microscopy technology, dsRNA analyzes on the virus similar on the resolution form, that heredity is different and in resolution virus and ordinary cells composition has clear superiority, and the susceptibility that dsRNA analyzes is stronger, diagnosis is rapider and reliable.

The detoxification bacterial strain is compared the embodiment clear superiority at aspects such as vigor, resistance, output with the band toxic bacterial strain.Because the research of Lentinula edodes Virus disease also relatively lags behind, we also do not find the 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, have great importance for Lentnus edodes provides healthy bacterial classification, lay a good foundation for the foundation of mushroom strain detoxification technology, the virus-free breed of variety of mushroom etc. simultaneously.

Summary of the invention

The RT-PCR primer and the detection method that the purpose 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 easily and fast, effectively detect mushroom strain and whether beat in spite of illness lower basis cruelly.

The present invention at first provides a kind of RT-PCR primer of mushroom dsRNA virus, and is 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, at first extracts mushroom dsRNA, become cDNA by reverse transcription, then take the cDNA of reverse transcription as template, the RT-PCR that sets up mushroom dsRNA virus detects, and RT-PCR product size is 852bp.

Described RT-PCR detection method specifically may further comprise the steps:

(1) extraction of mushroom dsRNA;

(2) the mushroom dsRNA that extracts is become cDNA by reverse transcription: in the 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 inhibitorss, 0.5 ul, 100 U/ul ThermoScript II M-MLV, 0.3～0.5 ul, DEPC water 3～4.7ul, place on the PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min namely obtain the cDNA of reverse transcription;

(3) RT-PCR detects: take the cDNA of reverse transcription as template, the RT-PCR that sets up mushroom dsRNA virus detects, and the pcr amplification system: cDNA template 1～3 ul, 10 * PCR Buffer, 3 ul contain 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 polymerases, 0.3～0.6 ul, complement to 30 ul with DEPC water;

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, then do not represent not carry dsRNA virus if amplify the 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, and has highly sensitive, the advantages such as detection time short, detected result reliable, easily judgement, good reproducibility.Specific as follows:

When 1, Electronic Speculum detected virus, virus must occur with the form of particle just being observed, and is subjected to the restriction on opportunity; In addition, separation Lentinula edodes Virus particle difficulty is larger, and needs multiple purifying, thickening equipment, and the 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 using dsRNA and detecting virus, extracted quality and concentration as influencing factor, often contain other RNA in the dsRNA leaching process, often show the unclear or disappearance of band during detected result by gel electrophoresis, repeatability and sensitivity are relatively poor.

3, zymetology detects the viral character of enzyme and the extraction sample size of being subjected to and limits the time-consuming manpower that takes again when this external application isozyme detects virus.

4, the RT-PCR detection method of setting up with the Lentinula edodes Virus genome, detection time is short, highly sensitive (detection system only needs the dsRNA of 10ng just can clearly to detect, carry out reverse transcription with the peculiar primer of virus, strengthened specificity and improved the reliability of detected result), the advantages such as reliable, the easily judgement of detected result, good reproducibility.Gene is physical basis of heredity, is not subject to the impact of extraneous factor etc., and the detection collection of illustrative plates that obtains simultaneously has specific band: 852bp, its dominant marker judges very clear.

The present invention provides reliable detection method for Lentnus edodes provides virus-free bacterial classification, and there is larger using value the aspects such as the foundation of mushroom strain detoxification technology, the virus-free breed of variety of mushroom.

Description of drawings

Fig. 1 is for using primer L1-S and L1-A to detect the DNA collection of illustrative plates of Xianggu mushroom strain amplification, and wherein M～7 are the swimming lane numbering; The molecular weight of the numeral dna fragmentation in left side, swimming lane M is the Mark of DL2000, and swimming lane 1～7 is viruliferous Xianggu mushroom strain, and labeled fragment is 852bp.

Fig. 2 is for using primer L1-S and L1-A to detect the DNA collection of illustrative plates of Xianggu mushroom strain amplification, and wherein M～13 are the swimming lane numbering; The molecular weight of the numeral dna fragmentation in left side, swimming lane M is the Mark of DL2000, and 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, does not detect the labeled fragment of 852bp.

Embodiment

The RT-PCR detection method specifically may further comprise the steps:

(1) extraction of mushroom dsRNA;

(2) the mushroom dsRNA that extracts is become cDNA by reverse transcription;

Holding temperature before the RT reaction and the dsRNA concentration in the RT reaction system, reverse primer concentration, dNTPs concentration, ThermoScript II M-MLV enzyme amount etc. are as parameter, reaction is optimized one by one to the RT of each parameter, 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 clearly specific band.

(3) RT-PCR detects: take the cDNA that obtains through the RT reaction as template, to cDNA addition, the Mg in the 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 the 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 clearly specific band.

In order fully to disclose the detection method of a kind of Lentinula edodes Virus of the present invention, be illustrated below in conjunction with embodiment.

Embodiment 1: a kind of RT-PCR detection method of mushroom dsRNA virus

A kind of RT-PCR detection method of mushroom dsRNA virus may further comprise the 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), uses Primer5 or other primer-design software, obtain the detection primer of Lentinula edodes Virus RT-PCR.The characteristics of this primer are that the Lentinula edodes Virus gene is had the specific amplified site, and the clip size that obtains 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

Mushroom strain (Wang Li etc., the dsRNA of Xianggu mushroom strain detects, edible mushrooms journal 2009.), the mushroom strain after detoxification to undesired mushroom strain or generation virus disease carry out 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 the 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, switching enters 250 ml triangular flasks, contains in the 100 ml PDA liquid nutrient mediums;

3, be placed on 25 ℃ of thermostat containers and leave standstill cultivation 20d-25d;

4, put on disposable glove, cultured shiitake mushroom hypha in the triangular flask, pour two-layer gauze for filtering into, choose the agar block that consumes except not with tweezers or key, aseptic water washing, filter paper blots;

5, take by weighing 0.5～1g mycelia, wrap with filter paper, directly be used for extracting viral dsRNA or-20 ℃ and save backup.

(2) dsRNA of mushroom extracts

1, puts into mortar collecting good mushroom mycelium, 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 Mushroom virus dsRNA extracting method [J] of Simple fast. experimental technique and management, 2010,27 (12): 51～53,57.)

Three, RT-PCR detects and sets up

The mushroom dsRNA that extracts becomes cDNA by reverse transcription, and take the cDNA that transcribes as template, the RT-PCR that sets up Lentinula edodes Virus detects.

(1) RT of mushroom dsRNA reaction

Best RT reaction conditions is: add 100 ng/ul dsRNA 1ul, 10 umol/L reverse primer L1-A, 1.5 ul, DEPC water 7.5 ul in the PCR pipe, then 95 ℃ of insulation 5 min place rapidly 5 min on ice.Add again 5 * RT Buffer, 4 ul, 10 mmol/L dNTPs, 1 ul, 40 U/ul RNA enzyme inhibitorss, 0.5 ul, 100 U/ul ThermoScript II M-MLV, 0.5 ul, DEPC water 5ul, place on the PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min namely obtain the cDNA of reverse transcription.

(2) pcr amplification

The PCR optimum augumentation system is: cDNA template 2 ul, 10 * PCR Buffer, 3 ul(contain 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 polymerases, 0.24 ul, complement to 30 ul with DEPC water.

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 buffer, and mixing at 1.0% sepharose, contains Goldview ^TMThe DNA dyestuff carries out electrophoresis, and 5V/cm constant voltage electrophoresis 50 min observe and take pictures by the ultraviolet gel imaging system.Perhaps, get pcr amplification product 8 ul, with 1.5 ul, 6 * tetrabromophenol sulfonphthalein sample-loading buffer mixing, 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 take a picture at the gel imaging instrument.

But being the bacterial classification specific amplified that contains mushroom dsRNA virus, the pcr amplification product detected result goes out the single fragment of 852bp, as shown in Figure 1, if mushroom strain does not go out fragment with the then amplification of this dsRNA virus, as shown in Figure 2.

Experiment results proved, when with primer L1-S 5 '-ACCGCAATCAATAACT-3 ' and primer L1-A 5 '-AGACAAGATGGAGACG-3 ' Xianggu mushroom strain with dsRNA virus being carried out the RT-PCR detection, can detect the band of a 852bp, then can't detect this band through the Xianggu mushroom strain of detoxification.The RT-PCR that utilizes of the present invention detects Xianggu mushroom strain whether with the method for dsRNA virus, and its detection time is short, highly sensitive, detected result reliable, 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 sterilization 30min;

2, PDA liquid nutrient medium: potato 200 g, glucose 20 g, water 1000 mL, pH nature, 121 ℃ of sterilization 30min;

3, one-step RT-PCR test kit (RevertiAid ^TMFirst Strand cDNA Synthesis Kit) available 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 available from the precious biotech firm in Dalian.

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉a kind of RT-PCR primer and detection method of mushroom dsRNA virus

<160> 2

<170> PatentIn version 3.5

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<212> DNA

＜213〉artificial sequence

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accgcaatca ataact 16

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<212> DNA

＜213〉artificial sequence

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agacaagatg gagacg 16

Claims (3)

1. the RT-PCR primer of a mushroom dsRNA virus, it is characterized in that: described RT-PCR primer is as follows:

Forward primer L1-S 5 '-ACCGCAATCAATAACT-3 ',

Reverse primer L1-A 5 '-AGACAAGATGGAGACG-3 '.

2. the RT-PCR detection method of a mushroom dsRNA virus, it is characterized in that: at first extract mushroom dsRNA, become cDNA by reverse transcription, then take the cDNA of reverse transcription as template, the RT-PCR that sets up mushroom dsRNA virus detects, and RT-PCR product size is 852bp.

3. the RT-PCR detection method of mushroom dsRNA virus according to claim 2, it is characterized in that: described RT-PCR detection method specifically may further comprise the steps:

(1) extraction of mushroom dsRNA;

(2) the mushroom dsRNA that extracts is become cDNA by reverse transcription: in the 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 inhibitorss, 0.5 ul, 100 U/ul ThermoScript II M-MLV, 0.3～0.5 ul, DEPC water 3～4.7ul, place on the PCR instrument, 42 ℃ of reaction 1 h, 70 ℃ of deactivation 10 min namely obtain the cDNA of reverse transcription; Wherein reverse primer L1-A is 5 '-AGACAAGATGGAGACG-3 ';

(3) RT-PCR detects: take the cDNA of reverse transcription as template, the RT-PCR that sets up mushroom dsRNA virus detects, and the pcr amplification system: cDNA template 1～3 ul, 10 * PCR Buffer, 3 ul contain 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 polymerases, 0.3～0.6 ul, complement to 30 ul with DEPC water; Wherein forward primer L1-S is 5 '-ACCGCAATCAATAACT-3 ', and reverse primer L1-A is 5 '-AGACAAGATGGAGACG-3 ';

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, then do not represent not carry dsRNA virus if amplify the specific DNA band.

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