CN114703323B - Lily virus LAV-1 specific detection target sequence, kit and detection method - Google Patents
Lily virus LAV-1 specific detection target sequence, kit and detection method Download PDFInfo
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Abstract
The invention discloses a lily virus LAV-1 specific detection target sequence, a kit and a detection method, wherein the base sequence of the target sequence is shown as SEQ ID NO. 1, the detection kit comprises a primer pair with the base sequence shown as SEQ ID NO. 2 and SEQ ID NO. 3, and the detection method comprises the following steps: extracting RNA from a sample to be detected by using a modified Trizol method, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using a primer pair for gel electrophoresis detection analysis. The invention can realize rapid and specific detection of the lily virus LAV-1 and provides technical support for further research of LAV-1 virus.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a lily virus LAV-1 specific detection target sequence, a kit and a detection method.
Background
Lily plants become internationally popular ornamental flowers due to their beautiful flower colors and flower fragrances, and cultivate over 4500 cultivars. However, lily often infects viruses to cause lily leaves to appear flowers and leaves, yellow, curly deformation and even no flowering when serious, which seriously affects the economic development of lily market. More than 20 viruses have been reported to infect lily, and common viruses are Cucumber Mosaic Virus (CMV), lily mottle virus (LMoV), lily asymptomatic virus (LSV), lily Virus X (LVX), plantain mosaic virus (PlAMV), and the like. Most of the reported lily viruses are identified by an indicator plant method, an electron microscope observation method, a serological technology and the like, but the reported lily viruses are generally limited by time consumption, difficulty in extracting virus particles and the like, and potential viruses or new viruses are difficult to efficiently and comprehensively find. High throughput technology (high-throughput sequencing, HTS) allows the simultaneous sequence identification of hundreds of thousands to millions of DNA molecules, enabling a careful comprehensive analysis of the transcriptome and genome of a species. HTS is widely used in the discovery of new viruses, the identification of known pathogens, and the analysis and comparison of genomic genetic diversity and evolution, and its use in lily has become increasingly widespread with advances in technology.
Amalgaviridae is a recently discovered taxonomic group, the Amalgaviridae family includes two genera, amalgavirus and Zybavirus, where the Amalgavirus genus reports some plant viruses, such as blueberry latent virus, azalea virus A, southern tomato virus, and the like. These plant viruses have small dsRNA genomes and have not been shown to form true virions. Instead, they propagate vertically through the seed, and are considered unlikely to be effectively transmitted extracellularly, unless possible through the vector. The positive strand of the genome of a plant Amalgavirus genus virus comprises two partially overlapping long Open Reading Frames (ORFs), with downstream ORF2 overlapping with ORF1 in the +1 frame. Thus, they are believed to encode only two proteins, one ORF1 encoding product of unknown specific function and a fusion protein encoded by ORF1+2, which is translated after +1 Procedural Ribosome Frameshift (PRF). The ORF2 encoding part of the fusion protein is indicated by a conserved sequence motif as viral RNA-dependent RNA polymerase (RdRp).
The invention discovers a Lily new virus Lily Amalgavirus 1 (LAV-1) through high-throughput sequencing, and considers that the virus is probably a new member of Amalgavirus genus of Amalgaviridae family through a phylogenetic tree and multiple comparison peer-to-peer method; and the detection rate of LAV-1 is higher as seen from the field sample detection result. However, the symptoms caused by the viruses are not clear, but the lily is bulb propagation material, and the long-term accumulated viruses have a great influence on the growth of the lily, so that the establishment of a rapid diagnosis technology has a great influence on the follow-up research and test.
Disclosure of Invention
In view of the above, the present invention aims to realize rapid, specific and low-content detection of the lily virus LAV 1.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the first aspect of the invention provides a target sequence suitable for lily virus LAV1 specificity detection, and the base sequence of the target sequence is shown as SEQ ID NO. 1.
The second aspect of the invention provides a lily virus LAV1 specificity detection kit aiming at the target sequence, wherein the kit comprises a primer pair with base sequences shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The third aspect of the invention provides a method for detecting lily virus LAV1, which specifically comprises the following steps:
s1, extracting total RNA of a lily sample;
s2, taking total RNA and synthesizing cDNA by reverse transcriptase;
s3, using cDNA as a template, performing PCR amplification by using the primer pair, and detecting the product by agarose gel electrophoresis.
Preferably, step S1 is specifically: grinding lily leaves, uniformly mixing with Trizol, centrifuging to obtain a supernatant, and adding chloroform for extraction; centrifuging to obtain supernatant, adding isopropanol, mixing, settling, centrifuging, and discarding supernatant; washing the precipitate with ethanol, and adding DEPC water into the precipitate to dissolve the precipitate to obtain the total RNA of the sample.
Preferably, step S2 is specifically: taking total RNA prepared in the step S1, adding random primer pd (N) 6 and DEPC water, boiling in water bath, rapidly cooling, adding reverse transcription mixed solution, sucking, beating, mixing uniformly, and instantaneously separating; after water bath at 37 ℃ for 1-1.5 h, the water bath is firstly carried out, then the water bath is cooled rapidly, and the water bath is obtained after instantaneous separation.
More preferably, the reverse transcription mixture includes: 4. Mu.L of 5 XM-MLV reaction buffer, 1. Mu.L of 2.5mM dNTPs, 0.5. Mu.L of 40U/. Mu.L of RNase inhibitor, 0.5. Mu.L of 200U/. Mu. L M-MLV and 4. Mu.L of LDEPC water, based on a total volume of 10. Mu.L.
Preferably, the PCR amplification system in step S3 is: a total of 15. Mu.L, 7.5. Mu.L of 2 XTaq Master Mix, 0.2. Mu.M primer pair, 1.0. Mu.L of cDNA, ddH 2 And the balance of O.
Preferably, the PCR amplification procedure of step S3 is: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 54℃and elongation at 72℃for 20s for 30 cycles; the extension was completed at 72℃for 10min.
Preferably, the products are electrophoretically detected on a 1.2% agarose gel electrophoresis, wherein a positive sample can amplify a fragment of interest of about 650bp in size.
The beneficial effects of the invention are as follows: screening to obtain a specific target sequence capable of being used for virus detection by analyzing the sequence of the lily new virus LAV-1; the specific primer is designed according to the target sequence, so that the LAV-1 virus can be rapidly and accurately detected, the detection sensitivity is high, and the support is provided for the deep research of the LAV-1 virus. Compared with ELISA detection, electron microscope detection and other methods, the detection method provided by the invention is simpler, more convenient and faster, does not need expensive instruments and reagents, and greatly saves detection cost.
Drawings
FIG. 1 is a phylogenetic tree of the lily novel virus LAV-1, wherein, A is a phylogenetic tree analysis based on the amino acid sequence of the putative protein (p 44) encoded by ORF1, and B is a phylogenetic tree analysis based on RdRp; repeating 1000 times by adopting an NJ method;
FIG. 2 is a diagram of an alignment of the RdRp conserved domain encoded by the lily novel virus LAV-1 and the conserved domain encoded by Amalgavirus genus members;
FIG. 3 shows the result of gel electrophoresis in example 1.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The invention carries out high-throughput sequencing on lily leaf samples, and the number is Hzau1: leaf mix samples of 6 lily (No. XBLY-10, XBLY-13, SEB, MM-10, MM-9, MM-1) from Wuhan, hubei province, including 2 "Siberian" variety lily, 1 "Sorbon" lily, three "wood door" lily, wherein XBLY10, XBLY13 and SEB leaf samples have no obvious symptoms, MM10, MM9 leaf samples have obvious chlorosis, and MM1 leaf pulse has slight chlorosis symptoms. . All samples were flash frozen with liquid nitrogen and sent to the Shanghai Paenode company for high throughput sequencing. The spliced contigs are aligned with BlastX and BlastN in NCBI database, and plant viruses identified from the samples are identified as Lily amalgavirus 1 (LAV-1) which is not yet reported and can infect Lily besides reported common viruses.
Construction of phylogenetic trees with Amalgaviridae, bifidae and Bifidae partial members (as shown in FIG. 1) revealed that LAV-1 is a new member of the genus Amalgaviruses of Amalgaviridae.
The RdRp amino acid sequence was aligned with Amalgavirus members to find 7 conserved sequences, as shown in FIG. 2, motif I-VII, respectively.
The detection method provided by the invention is specifically shown in the following examples aiming at the new virus.
Example 1
Firstly, screening to obtain a specific target sequence for detecting LAV-1 virus, wherein the base sequence of the target sequence is shown as SEQ ID NO. 1. Then, specific primers were designed, the sequences of which were as follows:
LAV1-F：5’-AGAGGAGGATGTGGAGGATT-3’(SEQ ID NO:2)；
LAV1-R：5’-GGAGACATACCCCTCGTAGC-3’(SEQ ID NO:3)。
the method for detecting lily viruses by using the primer pair comprises the following specific steps:
(1) Total RNA in lily leaf samples was extracted using a modified Trizol method.
Taking 0.1g of lily leaves, grinding with liquid nitrogen, rapidly adding into a tube, adding 800 mu L of Trizol, and mixing uniformly upside down; centrifuging at 12000rpm and 4 ℃ for 15min; carefully sucking the supernatant, adding an equal volume of chloroform, extracting twice, shaking vigorously, standing on ice for 5min, and centrifuging at 12000rpm and 4 ℃ for 15min; carefully sucking the supernatant, adding equal volume of pre-cooled isopropanol, gently mixing upside down, and settling at-20deg.C for 30min; centrifuging at 12000rpm and 4 ℃ for 15min; discarding supernatant, adding 75% ethanol, reversing upside down, washing the precipitate for 2-3 times, and centrifuging at 12000rpm at 4deg.C for 1min; air-separating at 12000rpm and 4 ℃ for 1min; residual ethanol is sucked by a gun head, the sediment is dried in an ultra-clean workbench for about 5min, 25 mu LDEPC water is added, the sediment is gently sucked and beaten until the sediment is completely dissolved, and the total RNA of the sample is obtained and is preserved at the temperature of minus 80 ℃ for standby.
And extracting macromolecular proteins in the extraction process for 2-3 times by using chloroform to obtain RNA with high purity, high quality and good integrity.
(2) The cDNA synthesis is specifically as follows: about 6-8. Mu.L (1. Mu.g) total RNA or dsRNA was taken and 1. Mu.L of the random primer pd (N) 6 (80 nmol) DEPC H was added 2 O was made up to 10. Mu.L and added to a 0.5mL centrifuge tube without enzyme; boiling water for 10min, and rapidly cooling on ice for 3-5 min; mu.L of the prepared reverse transcription mixed solution (containing 4. Mu.L of 5 XM-MLV reaction buffer, 1. Mu.L dNTPs (2.5 mM), 0.5. Mu.L LRNA enzyme inhibitor (RRI) (40U/. Mu.L), 0.5. Mu.L M-MLV (200U/. Mu.L) and 4. Mu.L LDEPC H) was added 2 O), sucking and beating, mixing uniformly, instantaneously separating, carrying out water bath at 37 ℃ for 1-1.5 h, and carrying out boiling water bath at 85 ℃ for 5min; rapidly placing on ice, instantly leaving, and preserving at-20deg.C.
(3) PCR was performed using cDNA as a template. The PCR reaction system (15. Mu.L) was: 7.5. Mu.L of 2 XTaq Master Mix, upstream and downstream primers LAV 1-F/R0.2. Mu.M, 1.0. Mu.L of cDNA, ddH 2 O makes up 15. Mu.L. The PCR amplification procedure was: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 54℃for 20s at 72℃for 30 cycles, and complete extension at 72℃for 10min.
The product was electrophoretically detected on a 1.2% agarose gel, stained in 0.5. Mu.g/mL Ethidium Bromide (EB), and visualized in a gel imaging system.
Determination criteria: lily amalgavirus 1 positive samples amplified a fragment of interest of approximately 650bp in size (see FIG. 3). The sequence is constructed on a pMD18-T vector, and sequencing results are aligned on NCBI, so that the homoplasmic viruses can be obtained by alignment. Therefore, whether LAV-1 is identified in the lily sample can be rapidly and specifically determined according to the existence and the size difference of the target fragment in the sample. The band amplified by the specific primer designed by the invention is clear, and the content of LAV-1 virus in the lily sample can be judged according to the brightness of the band.
While the invention has been described in terms of what are presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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ggcctcggtg gagaggagga tgtggaggat tccgctgcct cagagggaag tatgggcgga 120
cctgattccg atagggcctc tagtgacaag gaggacgagg aaccaaaacc gaactctgag 180
ggaactcctc cccccgaacc tgttcagcct gaagtgccaa gtggggaaca agaaaggtcc 240
acctctggca aaaggacccg aatcaagaag agactgcgcc ctcgccctgc tcgagaaccc 300
aagaatccaa gtgctaagaa ggtcccgaag gttaaaaaat cccgtaattc cgaccgcgcg 360
aagtaaattc gaggcggggg ttcggcgggt gattggaggt ggcgagttgc ggacatgggc 420
gagtgattcg aataagtacc gtggcggggg ttgccttagc gatgcacttc tgttgcttgc 480
tacggcttcc tatacaccgc ctgaggaatt cttgtcggat tacttttctg ttgatgctgc 540
taggaacctc ctctctttgc ctgccggaat ccccgtcccg atgggcccgg atgctgtcgt 600
catgtcgaat ttcaataacg acgccacatc cggtcctttt ctgcgtgctt ttggtattaa 660
ggataagtat ggaataaagg cggatctcga acgattcgcg tggaagtgct acgaggggta 720
tgtctcctca ggttgtgatg gaaggttcct acccttcatc ctggcgagag ttggttaccg 780
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Claims (3)
1. The method for detecting the lily virus LAV-1 is characterized by comprising the following steps:
s1, extracting total RNA of a lily sample;
s2, taking total RNA and synthesizing cDNA by reverse transcriptase;
s3, performing PCR amplification by using cDNA as a template and using primer pairs shown as SEQ ID NO. 2 and SEQ ID NO. 3, and performing agarose gel electrophoresis detection on the product;
the primer pair specifically amplifies a target sequence shown as SEQ ID NO. 1;
the step S2 specifically comprises the following steps: taking total RNA prepared in the step S1, adding random primer pd (N) 6 and DEPC water, boiling in water bath, rapidly cooling, adding reverse transcription mixed solution, sucking, beating, mixing uniformly, and instantaneously separating; after water bath at 37 ℃ for 1-1.5 h, carrying out boiling water bath and then rapid cooling, and carrying out instantaneous separation to obtain the water-cooling type water-cooling device;
the reverse transcription mixed solution comprises: 4. Mu.L of 5 XM-MLV reaction buffer, 1. Mu.L of 2.5mM dNTPs, 0.5. Mu.L of 40U/. Mu.L of RNase inhibitor, 0.5. Mu.L of 200U/. Mu. L M-MLV and 4. Mu.L of DEPC water, based on a total volume of 10. Mu.L; the PCR amplification system in the step S3 is as follows: a total of 15. Mu.L, 7.5. Mu.L of 2 XTaqMastermix, 0.2. Mu.M primer pair, 1.0. Mu.L cDNA, ddH 2 The balance of O;
the PCR amplification procedure in step S3 is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 54℃and elongation at 72℃for 20s for 30 cycles; the extension was completed at 72℃for 10min.
2. The method for detecting lily virus LAV-1 according to claim 1, wherein the step S1 is specifically: grinding lily leaves, uniformly mixing with Trizol, centrifuging to obtain a supernatant, and adding chloroform for extraction; centrifuging to obtain supernatant, adding isopropanol, mixing, settling, centrifuging, and discarding supernatant; washing the precipitate with ethanol, and adding DEPC water into the precipitate to dissolve the precipitate to obtain the total RNA of the sample.
3. The method for detecting lily virus LAV-1 according to claim 1, wherein the product is electrophoretically detected on 1.2% agarose gel electrophoresis.
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