CN110283934A - A kind of mycoviruses rapid detection method - Google Patents
A kind of mycoviruses rapid detection method Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of mycoviruses rapid detection method.The present invention is the following steps are included: (1) extracts fungi total serum IgE: taking 0.1g hypha,hyphae in 1.5mL centrifuge tube, 1 × TE of 100uL solution is added, is smashed to pieces with sterile pipette tips, 12000g is centrifuged 5min, draws supernatant to obtain the final product；(2) using the fungi total serum IgE as template, virus specific primers to be detected are designed, carry out RT-PCR；(3) amplified production is taken to carry out electroresis appraisal.The step of hyphae length that mycoviruses rapid detection method of the invention needs is few, fungi Total RNAs extraction is simple, and the direct detection for being adaptable to a large amount of bacterial strains also reduces cost other than quick, accurate and sensitive, reduces the working time.All there is biggish application value to nontoxic breed of variety in the production of the foundation of mycoviruses detoxification technology, virus research and mushroom class etc..
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of mycoviruses rapid detection method.
Background technique
Mycoviruses are widely present in disease fungus, asymptomatic latent infection are presented mostly, host's phenotype is not caused to become
Change.Some mycoviruses bring about a wholesome effect to host fungi, such as cause host's pathogenicity to enhance or enhance host fungi and post
With the heat resistance of plant.A small number of mycoviruses can cause host's character mutation, including cause the decline of host's pathogenicity etc..Weak poison phase
Closing mycoviruses can be used as potential bio-control factors for plant fungal disease control.Meanwhile mycoviruses and host fungi
Interaction system to understand virus and epiphyte pathogenic provide a good model and tool.In addition to this, in nature
There are mycoviruses resource abundant, most mycoviruses have multifarious molecular characterization and biological property.So
Research mycoviruses help to improve the understanding to entire virus evolution and ecology, important to pushing virological development to have
Meaning.
It needs to carry out viral elimination when studying influence of the mycoviruses to host, generally using choosing after protoplast regeneration
Monospore is taken, then the bacterium colony of monospore growth is detected.Pass through spore in offspring's transmission efficiency in research virus, picking list
Viral diagnosis is carried out after spore.For another example in mushroom plantation, it is also desirable to be detected to virus in mushroom, the nontoxic bacterium of picking
Strain.Since traditional viral diagnosis is related to the extraction of dsRNA, or RT-PCR inspection is carried out after extracting total serum IgE using kit
It surveys.But such method usually requires more mycelia, and Total RNAs extraction step is more, and time-consuming, heavy workload at high cost.Therefore have
Necessity establish it is a kind of more quickly, easily and accurately detect viral method in fungi.
Summary of the invention
Against the above technical problems, the object of the present invention is to provide a kind of mycoviruses rapid detection methods, by direct
After the hypha,hyphae of picking culture simply extracts total serum IgE, RT-PCR detection is carried out using virus specific primers, is mycoviruses
Virus harm is reduced in research and the plantation of mushroom class, and important method is provided.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of mycoviruses rapid detection method, comprising the following steps:
(1) it extracts fungi total serum IgE: taking 0.1g hypha,hyphae in 1.5mL centrifuge tube, 1 × TE of 100uL solution is added, use
Sterile pipette tips are smashed to pieces, and 12000g is centrifuged 5min, draw supernatant to obtain the final product；
(2) using the fungi total serum IgE as template, with Primer Premier5.0 software design virus-specific to be detected
Primer carries out RT-PCR, the reaction system and condition of RT-PCR are as follows: total serum IgE 5ul, random primer 3ul and DMSO 2uL are added
In the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, it is immediately placed in l min in liquid nitrogen, rear dislocation is on ice；Again according to
Secondary addition, 5 × RT Buffer 4uL, dNTP 2u1, DTT 2uL, RNase Inhibitor 1ul, SuperScriptTM II
RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
DdH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
(3) it takes amplified production to carry out electroresis appraisal: taking the PCR product of 5uL, 1% agarose electrophoresis detects amplification, contains
There is the band being consistent with expection size in virulent bacterial strain, without amplified band in virus-free control strain.
Compared with prior art, the invention has the following beneficial effects:
Mycoviruses rapid detection method of the invention combines fungi total serum IgE and simply extracts and RT-PCR detection method,
The step of hyphae length needed is few, fungi Total RNAs extraction is simple, the direct detection of a large amount of bacterial strains is adaptable to, in addition to quick, quasi-
It is really and sensitive outer, cost is also reduced, the working time is reduced.To the foundation of mycoviruses detoxification technology, virus research and mushroom
Nontoxic breed of variety etc. all has biggish application value in class production.
Detailed description of the invention
Fig. 1 is to carry out RT-PCR testing result to viral CaPV1 in the present invention.Marker (5kbp), swimming lane 1 and 2 are distinguished
For the malicious point spore anthrax bacteria bacterial strain of band and nontoxic point spore anthrax bacteria bacterial strain.
Fig. 2 is the RT-PCR testing result in the present invention to viral MoCV1-B.Marker (5kbp), swimming lane 1 and 2 are distinguished
For the malicious Pyricularia oryzae bacterial strain of band and nontoxic Pyricularia oryzae bacterial strain.
Fig. 3 is the RT-PCR testing result in the present invention to virus N oRV2.Marker (5kbp), swimming lane 1 and 2 are respectively
The malicious rice Tuber Melanosporum strain of band and nontoxic rice Tuber Melanosporum strain.
Fig. 4 is the RT-PCR testing result in the present invention to viral AbFV1.Marker (5kbp), swimming lane 1 and 2 are respectively
The raw rod method bacterial strain of rape containing AbFV1 and avirulent strains.
Fig. 5 is the RT-PCR testing result in the present invention to viral CgCV1.Marker (5kbp), swimming lane 1 and 2 are respectively
Glue spore anthracnose bacterial strain and avirulent strains containing CgCV1.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with the embodiment of the present invention, to this hair
Technical solution in bright embodiment is clearly and completely described, it is clear that described embodiment is that a part of the invention is implemented
Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creativeness
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of labour.
The sequence of known mycoviruses CaRV1, MoCV1-B, NoRV2, AbFV1 and CgCV1 used in the following embodiment
Column can be retrieved in US National Biotechnology Information center (NCBI) and be obtained.
Total serum IgE simply extracts in 1 fungi of embodiment
Culture dish 0.1g mycelia is scraped in the centrifuge tube of 1.5mL, after 1 × TE solution of 100uL is added, is smash with pipette tips
Broken, 12000g is centrifuged 5min, takes supernatant.
Virus RT-PCR detects in 2 fungi of example
According to fungi known virus Colletotrichum acutatum RNA virus 1 in point spore anthrax bacteria
(CaRV1) (KC572132.1) sequence designs specific primer with Primer 5, and primer is
CaRV1-F:5’-TCTTCAGGATACCCGCACTAC-3’
CaRV1-R:5’-GATCACGTAAATCATCGAGCC-3’
The expected amplified production size of the primer is 496bp.
RT-PCR amplification is carried out with the total serum IgE that above-mentioned primer pair is extracted.Reverse transcription reaction system and condition are as follows: total serum IgE
5ul, random primer 3ul and DMSO 2uL are added in the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, set rapidly
The l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer 4uL, dNTP 2u1, DTT 2uL, RNase
Inhibitor 1ul, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
ddH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
Amplified production is detected, the PCR product of 5uL is taken, 1% agarose electrophoresis detects amplification, contains virus
Bacterial strain occur with the band that is consistent of expection size, without amplified band in virus-free control strain, as shown in Figure 1.To PCR product
It is sequenced, as a result sequence is compared with BLAST, is really known viruse CaRV1.
Embodiment 3
According in Pyricularia oryzae to fungi known virus Magnaporthe oryzae chrysovirus1-B (MoCV1-
B) sequence designs specific primer with Primer 5, and primer is
MoCV1-B-F:5’-GTCAACATTGGCGGCACG-3’
MoCV1-B-R:5’-GCATCGCTTTCCAACCACA-3’
The expected amplified production size of the primer is 507bp.
RT-PCR amplification is carried out with the total serum IgE that above-mentioned primer pair is extracted.Reverse transcription reaction system and condition are as follows: total serum IgE
5ul, random primer 3ul and DMSO 2uL are added in the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, set rapidly
The l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer 4uL, dNTP 2u1, DTT 2uL, RNase
Inhibitor 1ul, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
ddH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
Amplified production is detected, the PCR product of 5uL is taken, 1% agarose electrophoresis detects amplification, contains virus
Bacterial strain occur with the band that is consistent of expection size, without amplified band in virus-free control strain, as shown in Figure 2.To PCR product
It is sequenced, as a result sequence is compared with BLAST, is really known viruse MoCV1-B.
Embodiment 4
According in the black spore of rice to 2 (NoRV2) sequence of fungi known virus N igrospora oryzae victorivirus
Specific primer is designed with Primer 5, primer is
NoRV2-F:5’-GACCCTAACACCATTATCCACC-3’
NoRV2-R:5’-GCGGCACCACCACCTATT-3’
The expected amplified production size of the primer is 635bp.
RT-PCR amplification is carried out with the total serum IgE that above-mentioned primer pair is extracted.Reverse transcription reaction system and condition are as follows: total serum IgE
5ul, random primer 3ul and DMSO 2uL are added in the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, set rapidly
The l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer 4uL, dNTP 2u1, DTT 2uL, RNase
Inhibitor 1ul, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
ddH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
Amplified production is detected, the PCR product of 5uL is taken, 1% agarose electrophoresis detects amplification, contains virus
Bacterial strain occur with the band that is consistent of expection size, without amplified band in virus-free control strain, as shown in Figure 3.To PCR product
It is sequenced, as a result sequence is compared with BLAST, is really known viruse NoRV2.
Embodiment 5
It is given birth in rod method according to rape to fungi known virus Alternaria brassicicola fusarivirus 1
(AbFV1) sequence designs specific primer with Primer 5, and primer is
AbFV1-F:5’-TGACCCTGGGAATGCTGTG-3’
AbFV1-R:5’-CCGTCACAGGTTCTTCCAC-3’
The expected amplified production size of the primer is 510bp.
RT-PCR amplification is carried out with the total serum IgE that above-mentioned primer pair is extracted.Reverse transcription reaction system and condition are as follows: total serum IgE
5ul, random primer 3ul and DMSO 2uL are added in the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, set rapidly
The l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer 4uL, dNTP 2u1, DTT 2uL, RNase
Inhibitor 1ul, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
ddH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
Amplified production is detected, the PCR product of 5uL is taken, 1% agarose electrophoresis detects amplification, contains virus
Bacterial strain occur with the band that is consistent of expection size, without amplified band in virus-free control strain, as shown in Figure 4.To PCR product
It is sequenced, as a result sequence is compared with BLAST, is really known viruse AbFV1.
Embodiment 6
According to fungi known virus Colletotrichum gloeosprioides in point spore anthrax bacteria
(CgCV1) sequence of chrysovirus 1 designs specific primer with Primer 5, and primer is
CgCV1-F:5’-AACATGAGGGCTTCTATTGGC-3’
CgCV1-R:5’-CATCGTTGTAAGACTTGTGGTCT-3’
The expected amplified production size of the primer is 454bp.
RT-PCR amplification is carried out with the total serum IgE that above-mentioned primer pair is extracted.Reverse transcription reaction system and condition are as follows: total serum IgE
5ul, random primer 3ul and DMSO 2uL are added in the processed 0.5ml centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, set rapidly
The l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer4uL, dNTP 2u1, DTT 2uL, RNase
Inhibitor 1ul, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer.PCR reaction system and condition include:
ddH2O 34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA
2ul, LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40s 35 are followed totally
Ring, then 72 DEG C of extension 5min.
Amplified production is detected, the PCR product of 5uL is taken, 1% agarose electrophoresis detects amplification, contains virus
Bacterial strain occur with the band that is consistent of expection size, without amplified band in virus-free control strain, as shown in Figure 5.To PCR product
It is sequenced, as a result sequence is compared with BLAST, is really known viruse CgCV1.
Above embodiments illustrate, combine simple extraction fungi total serum IgE and corresponding RT-PCR to detect using the present invention true
Bacterium virus rapid detection method can carry out specific detection to mycoviruses, and obtain accurate result.
Claims (4)
1. a kind of mycoviruses rapid detection method, which comprises the following steps:
(1) it extracts fungi total serum IgE: taking 0.1g hypha,hyphae in 1.5mL centrifuge tube, 1 × TE of 100uL solution is added, use is sterile
Pipette tips are smashed to pieces, and 12000g is centrifuged 5min, draw supernatant to obtain the final product；
(2) using the fungi total serum IgE as template, virus specific primers to be detected are designed, carry out RT-PCR；
(3) amplified production is taken to carry out electroresis appraisal.
2. a kind of mycoviruses rapid detection method according to claim 1, which is characterized in that RT- in the step (2)
The reaction system and condition of PCR are as follows: total serum IgE 5ul, random primer 3ul and DMSO 2uL be added the processed 0.5ml of DEPC from
In heart pipe, in 99 DEG C of water-bath 3min, it is immediately placed in l min in liquid nitrogen, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer
4uL, dNTP 2u1, DTT 2uL, RNase Inhibitor 1ul, SuperScriptTM II RNase H-Reverse
Transcriptase 1uL, 42 DEG C of reaction 1h；
Taking the cDNA of synthesis is template, carries out PCR amplification using above-mentioned primer, PCR reaction system and condition include: ddH2O
34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 2uL, cDNA 2ul, LA
TaqTM 0.5uL；Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 40sec 35 are recycled, then totally
72 DEG C of extension 5min.
3. a kind of mycoviruses rapid detection method according to claim 1, which is characterized in that electric in the step (3)
Swim the method for identification are as follows: take the PCR product of 5uL, 1% agarose electrophoresis detects amplification, containing virulent bacterial strain occur with
It is expected that the band that size is consistent, without amplified band in virus-free control strain.
4. a kind of mycoviruses rapid detection method according to claim 1, which is characterized in that the mycoviruses include
1 (CaRV1), Pyricularia oryzae of fungi known virus Colletotrichum acutatum RNA virus in point spore anthrax bacteria
Fungi known virus in middle fungi known virus Magnaporthe oryzae chrysovirus1-B (MoCV1-B), the black spore of rice
Fungi known virus Alternaria in the raw rod method of Nigrospora oryzae victorivirus 2 (NoRV2), rape
Fungi known virus Colletotrichum in brassicicola fusarivirus 1 (AbFV1) and point spore anthrax bacteria
gloeosprioides chrysovirus 1(CgCV1)。
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| CN111926105A (en) * | 2020-09-09 | 2020-11-13 | 江苏省农业科学院 | Primer and method for LAMP detection of alternaria brassicae |
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| CN104593502A (en) * | 2015-01-21 | 2015-05-06 | 南京农业大学 | Loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof |
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