JP6895168B2 - Virus diagnostic method - Google Patents

Description

The present invention relates to, for example, a virus diagnostic method for detecting a plant virus belonging to the genus Emaravirus.

Prompt identification of pathogens is essential for effective response to crop disease outbreaks. Among pathogens, plant viruses cannot be diagnosed by culture or optical microscopy, so genetic diagnosis is important for rapid detection and diagnosis.

By the way, it has become clear that the virus of the genus Emaravirus, which was recently established as a taxon of plant viruses by the International Committee on Taxonomy of Viruses, causes enormous damage to fruit trees, flowers, grains, etc. all over the world. ..

To date, 6 species have been set as Emaravirus spp., But there are 12 species including the provisional species that are considered to be Emaravirus discovered in recent years, and it is expected that they will be discovered one after another. In Japan, in addition to fig mosaic virus (FMV), the present inventors have found that the causative agent of mosaic disease, which impedes the production of perilla (perilla), is a new type of emaravirus. It was clarified that it is a perilla mosaic virus (PMoV).

Emaravirus causes enormous damage exceeding 30 billion yen a year in India (Non-Patent Document 1), but once it occurs even in countries where it has not occurred, it is thought that it will cause agricultural damage and hinder trade. Therefore, strict regulations are imposed on plant quarantine and the like (Non-Patent Documents 2 to 6).

In addition, there is an increasing need for means to prove that agricultural products are not infected with the emala virus in order to prevent the invasion of the emara virus into the country and for the "aggressive agriculture, forestry and fisheries industry" promoted by the government. ing.

On the other hand, in Non-Patent Document 7, five types of Emara virus (European mountain ash ringspot-associated virus (EMARaV), FMV, Rose rosette virus (RRV), Raspberry leaf blotch virus (RLBV), Redbud yellow ringspot virus (RYRV)) ), Using 2 types of primer sets designed based on the base sequence of), 4 types of viruses tested (EMARaV, FMV, Pigeonpea sterility mosaic virus (PPSMV), High Plains wheat mosaic virus (HPWMoV) [Maize red stripe virus) (MRSV)]) was successfully detected.

In Non-Patent Document 8, 6 types of emaraviruses (EMARaV, FMV, PPSMV, PPSMV2, RRV, HPWMoV) were tried to be detected, and 4 types were successfully detected, but PPSMV and PPSMV2 could not be detected.

However, these conventional detection methods are not sufficient for comprehensive and rapid detection of Emaravirus spp., And the presence of Emaravirus may be overlooked.

Patil and Kumar, 2015. Pigeonpea sterility mosaic virus: a legume-infecting Emaravirus from South Asia. Mol. Plant Pathol. 16: 775-786. Irwin et al. 1999. Pathogen risks associated with bulk maize imports to Australia from the United States of America. Coutts et al. 2014. First report of Wheat mosaic virus infecting wheat in Western Australia. Plant Dis. 98: 285. Lebas et al. 2005. Development of an RT-PCR for High Plains virus indexing scheme in New Zealand post-entry quarantine. Plant Dis. 89: 1103-1108. Ochoa-Corona et al. 2015. New Zealand stresses that it is High Plains virus free, and the virus struggles with an identity crisis. J. Virol. 89: 7439-7440. Botelho et al. 2016. Interception of Wheat mosaic virus (WMoV) in Brazil in maize seeds from the United States. Pesq. Agropec. Bras., Brasilia. 51: 688-691. Elbeaino et al. (2013). Emaravirus-specific degenerate RT-PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus. J. Virol. Methods. 188: 37-40. Olmedo-Velarde et al. (2016) Towards a broad detection of emaraviruses: Endpoint RT-PCR reaction. American Phytopathological Society Meeting, August, 2016, Tampa, Florida, USA.

In view of the above circumstances, an object of the present invention is to provide a genetic diagnostic method for comprehensively and rapidly detecting a virus of the genus Emaravirus.

As a result of diligent research to solve the above problems, RT-PCR using a newly designed primer set based on the sequence information of the Emaravirus genus virus shows that the Emaravirus genus virus is comprehensive and rapid. We have found that it can be detected in the above, and have completed the present invention.

That is, the present invention includes the following.
(1) A primer set for amplifying a base sequence specific to an Emaravirus virus, which comprises any two of the following primers (A) to (C).
(A) Primer for the conserved region consisting of the nucleotide sequence shown in SEQ ID NO: 1 in RNA1 of Emaravirus virus;
(B) Primer for the conserved region consisting of the nucleotide sequence shown in SEQ ID NO: 2 in RNA1 of Emaravirus virus;
(C) Primer for the conserved region consisting of the nucleotide sequence shown in SEQ ID NO: 3 in RNA1 of Emaravirus genus virus (2) Primer set according to any one of (a) to (e) below, (1). Primer set.
(a) A primer set consisting of a primer containing the nucleotide sequence shown in SEQ ID NO: 4 and a primer containing the nucleotide sequence shown in SEQ ID NO: 5;
(b) A primer set consisting of a primer containing the nucleotide sequence shown in SEQ ID NO: 6 and a primer containing the nucleotide sequence shown in SEQ ID NO: 7;
(c) A primer set consisting of a primer containing the nucleotide sequence of SEQ ID NO: 8 and a primer containing the nucleotide sequence of SEQ ID NO: 9;
(d) A primer set consisting of a primer containing the nucleotide sequence of SEQ ID NO: 10 and a primer containing the nucleotide sequence of SEQ ID NO: 9;
(e) Primer set including the primer containing the nucleotide sequence of SEQ ID NO: 11 and the primer containing the nucleotide sequence of SEQ ID NO: 12 (3) The genus Emaravirus, which comprises the primer set of (2). Kit for virus detection or Emaravirus virus infection diagnosis.
(4) Detection of Emaravirus virus, which comprises a step of performing an amplification reaction of a target nucleic acid region of Emaravirus virus using the primer set described in (1) or (2) or the kit described in (3). Emaravirus virus infection diagnosis method.

According to the present invention, it is possible to quickly and comprehensively detect a known or unknown Emaravirus genus virus that infects fruit trees, flowers, grains, vegetables and the like in the agricultural field.

The alignment of each motif of the base sequence of emaravirus RNA1 is shown. The base sequence is represented by DNA. Bases stored in all sequences were displayed without background color. The region and extension direction in which each degenerate primer is annealed are indicated by a solid line arrow for the new primer and a broken line arrow for the previously reported primer (Non-Patent Document 7). It is a continuation of FIG. 1-1. The target position of the degenerate primer in emaravirus RNA1 is shown. The upper bar shows RNA1 of the emaravirus (the region of nt 3000 to 4000 out of the total length of about 7.2 kb), and the dashed and solid rectangles indicate the previously reported and newly discovered conservation motifs, respectively. The positions where the degenerate primers shown in Tables 3 and 4 are annealed are indicated by arrows. It is a gel electrophoresis image of RT-PCR performed using various primer sets. # 1 to # 6 are newly designed degenerate primers for detecting emala virus, and # 7 and # 8 are previously reported universal primers (Non-Patent Document 7). The estimated size of the amplification product with each primer is shown below. The sample names written above each lane are H: healthy perilla leaves, P: PMoV-infected perilla leaves, and F: FMV-infected fig leaves. It is a gel electrophoresis image of one-step RT-PCR using a new primer set. A one-step RT-PCR reaction was performed using a novel primer set of # 1 or # 3. The samples marked above each lane are H: healthy perilla leaves, P: PMoV-infected perilla leaves, and F: FMV-infected fig leaves. The estimated size of the amplification product with each primer is shown below. It is a gel electrophoresis image of PCR regarding the narrowing down of a novel primer set using artificially synthesized DNA. A PCR reaction was performed using a new primer set excluding # 2. Samples are healthy perilla leaves (lane 1), PMoV-infected perilla leaves (lane 2), FMV-infected fig leaves (lane 3), EMARaV (lane 4), RLBV (lane 5), HPWMoV (lane 6), PPSMV (lane 7). ), RYRV (lane 8), PPSMV2 (lane 9). Lanes 1 to 3 were templated from plant RNA-derived cDNA, and lanes 4 to 9 were templated from artificially synthesized DNA (plasmid). Primer sets # 1 and # 3 were selected. It is a gel electrophoresis image of RT-PCR showing the comparison of the detection sensitivities of 10 emaravirus species by the degenerate primer set. # 1 and # 3 are new primer sets in the present application, and # 7 and # 8 are previously reported primer sets. Lane M is a 100 bp ladder, followed by AcCRaV, EMARaV, FMV, PMoV, PPSMV, PPSMV2, RLBV, RRV, RYRV, and HPWMoV. In each lane,-is only healthy host plant RNA, and + is an amplification product from samples with artificially synthesized viral RNA (where FMV and PMoV are actual infected leaf RNA). It is a gel electrophoresis image of RT-PCR showing the detection of a novel emaravirus-like virus by a degenerate primer set. # 1 and # 3 are new primer sets in the present application, and # 7 and # 8 are previously reported primer sets. The upper row shows the results of RT-PCR with shikimi, and the lower row shows the results of RT-PCR with total RNA extracted from pear leaves. H in each lane is a symptomatological shikimi or pear leaf, and M is a leaf showing a ring pattern symptom (shikimi) and a mosaic symptom (pear). The amplification product indicated by the arrowhead showed sequence similarity to RNA1 of the emaravirus, suggesting that a new species of emaravirus may have been detected. In # 7 and # 8, amplification products of the expected size could not be obtained. Specific primers are specific primers designed later based on the emaravirus-like sequences found in Illicium anisatum and pear, and amplification products were obtained from symptomatic samples. In pears, there is a thin band in the disease-free leaves, so it may be infected at a low concentration. The results of phylogenetic tree analysis of emaravirus-like sequences detected in pears and shikimi are shown. MEGA7.0 (Kumar et al. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0) is a sequence of about 500 bases from Motif 2 to Motif 3 from the base sequences obtained from known emaravirus, pear, and shikimi. For bigger datasets. Mol. Biol. Evol. 33: 1870-1874.), Alignment was performed, and a phylogenetic tree was prepared using the neighbor binding method. The number on each branch indicates the bootstrap value that was tried 1000 times. The sequence obtained from pear (Nashivirus) and the sequence obtained from Shikimi (Shikimi) showed high similarity to the sequences of known emalaviruses, suggesting that a new species of emalavirus may have been detected. A comparison of the nucleotide sequences of each degenerate primer and virus in PreMotif A, Motif 2, Motif A, Motif 3 and Motif C is shown. The base sequence is represented by DNA. Align a total of 14 known and novel emala-like virus sequences (12 in the PreMotif A and Motif C regions) and compare the sequences with the degenerate primers of primer sets # 1, # 3, # 7, and # 8. did. Of the primer base sequences, the bases that do not cause a mismatch for all virus sequences are underlined, and the bases that can cause a mismatch for any of the virus sequences are underlined. In # 1 and # 3, the antisense primer of # 3 had a mismatch of only one base in the Shikimi virus, but the others were completely covered. In # 7 and # 8, 2 strains of FMV (AB697826, AB697827) in PreMotif A-sense, 1 base each in PPSMV and PPSMV2, 5 bases in PMoV, PPSMV and Nashivirus in Motif A-sense and Motif A-antisense. 5 bases each, HPWMoV (denoted as WMoV in the figure) and Shikimi 2 bases each, PPSMV2 1 base each, Motif C-antisense 1 base each for FMV (AB697826) and RYRV, 2 bases each for PMoV and WMoV , There was a mismatch. It is a continuation of FIG. 9-1.

Hereinafter, the present invention will be described in detail.
The primer set according to the present invention contains any two of the following primers (A) to (C) that selectively hybridize with a nucleotide sequence specific to the Emaravirus genus virus. It is a primer set for amplifying a base sequence specific to virus.
(A) Primer for the conserved region (GAYMGDGARAUHUAYACWGGHAAY: Motif 2) consisting of the nucleotide sequence shown in SEQ ID NO: 1 in RNA1 of Emaravirus virus;
(B) Primer for the conserved region (GAUGCHWSNA ARUGGUCWGCWMGRGAU: Motif A) consisting of the nucleotide sequence shown in SEQ ID NO: 2 in RNA1 of Emaravirus virus;
(C) Primer for the conserved region (AAYUGGYUDCARGGNAAYYUNAAY: Motif 3) consisting of the nucleotide sequence shown in SEQ ID NO: 3 in RNA1 of Emaravirus genus virus.

In the present invention, the nucleotide sequences of RNA1 encoding RNA-dependent RNA polymerase (RdRP) of perilla mosaic virus and Emaravirus genus virus on the database are compared and analyzed, and regions with high sequence commonality (conservation) among virus species are compared and analyzed. Region) was searched, and as a more common conservative region different from the regions reported so far, it consists of a conserved region (Motif 2) consisting of the nucleotide sequence described in SEQ ID NO: 1 and a nucleotide sequence described in SEQ ID NO: 3. A new storage area (Motif 3) was found (Figs. 1 and 2). According to RT-PCR using a degenerate primer prepared based on the newly found sequence of the conserved region (Motif A) consisting of the conserved region and the known nucleotide sequence shown in SEQ ID NO: 2, it is known. And unknown viruses of the genus Emaravirus can be comprehensively detected, and also emaravirus species that have not been detected by the conventional primers described in Non-Patent Documents 7 and 8 and the like can be detected.

The virus of the genus Emaravirus to be detected in the present invention means a plant virus that is generally transmitted by Fushidani and whose genome is a positive-strand (-) RNA of a multi-segment (4 to 8 segment). Specific examples of the Emaravirus genus virus include European mountain ash ringspot-associated virus (EMARaV), fig mosaic virus (FMV), Pigeonpea sterility mosaic virus (PPSMV), Raspberry leaf blotch virus (RLBV), and Rose rosette virus (Rose rosette virus). RRV), High Plains wheat mosaic virus (HPWMoV) (also known as Maize red stripe virus (MRSV), High Plains virus (HPV) or Wheat mosaic virus (WMoV)), Actinidia chlorotic ringspot-associated virus (AcCRaV), perilla mosaic virus (PMoV), Pigeonpea sterility mosaic virus 2 (PPSMV2), Redbud yellow ringspot virus (RYRV) and the like. Further, in the present invention, an unknown virus of the genus Emaravirus can be detected.

The "primers for the conserved region" in (A) to (C) above are conserved so that they can be specifically annealed to the entire conserved region or a part thereof (sense sequence) or their complementary sequence (antisense sequence). It means a primer having the same or substantially the same base sequence as the whole region or a part thereof or their complementary sequences. Here, the "substantially the same base sequence" means that one or several bases (for example, 1 to 3 bases, preferably 1 or 2 bases) are different from the entire storage region or a part thereof or their complementary sequences. However, it means a base sequence that can be specifically annealed to the entire conserved region or a part thereof or a complementary sequence thereof.

The primer (A) is a primer having the same or substantially the same base sequence as the entire storage region or a part thereof as a forward primer in combination with the primer (reverse primer) of (B) or (C). is there. The primer (B) is a primer having the same or substantially the same base sequence as the complementary sequence of the entire storage region or a part thereof as a reverse primer in combination with the primer (forward primer) of (A). is there. Alternatively, the primer (B) is a primer having the same or substantially the same base sequence as the entire storage region or a part thereof as a forward primer in combination with the primer (reverse primer) of (C). The primer (C) has the same or substantially the same base sequence as the complementary sequence of the entire storage region or a part thereof as a reverse primer in combination with the primer (forward primer) of (A) or (B). It is a primer having.

Primers (A) to (C) take into consideration the severity (1,000 or less, preferably 200 or less) and Tm value (35 to 65 ° C, preferably 45 to 55 ° C) suitable for detection by, for example, RT-PCR. Can be designed. In addition, inosine residues (I) that do not bind to the target sequence but do not inhibit double-strand formation are added to the primers (A) to (C) so that the primer does not become too degenerate. As the base of, for example, 1 to 3 bases (preferably 1 or 2 bases) may be contained. Further, the lengths of the primers (A) to (C) are not particularly limited as long as they can be specifically annealed to the target sequence, but are, for example, 18 bases to the length of the entire storage region, preferably 20 bases to. The length of the entire storage area can be mentioned.

Specific primer sets according to the present invention include any of the following primer sets (a) to (e).
(a) A primer containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4 (GDGARATHTAYACWGGHAAY) (Ema-Motif 2-s-20mer: corresponding to the primer of (A) as a forward primer) and the nucleotide sequence shown in SEQ ID NO: 5. (GCWGACCAYTTNSWDGCATC) or a primer set consisting of a primer (Ema-Motif A-as-20mer: corresponding to the primer (B) as a reverse primer) (# 1 primer set in the examples);
(b) A primer containing or consisting of the nucleotide sequence shown in SEQ ID NO: 6 (GATGCHWSNAARTGGTCWGC) (Ema-Motif As-20mer: corresponding to the primer of (B) as a forward primer) and the nucleotide sequence shown in SEQ ID NO: 7 (ARRTTNCCYTGHARCCARTT) ) (Ema-Motif 3-as-20mer: equivalent to the primer of (C) as a reverse primer) and a primer set (# 3 primer set in the examples);
(c) A primer (Ema-Motif 2-s-2I-24mer: corresponding to the primer of (A) as a forward primer) containing or consisting of the nucleotide sequence (GAYMGIGARATITAYACWGGHAAY) shown in SEQ ID NO: 8 and the primer shown in SEQ ID NO: 9. Primer set (# 4 primer set in Examples) consisting of a primer containing or consisting of a base sequence (GCWGACCAYTTISWDGCATC) (Ema-Motif A-as-I-20mer: corresponding to the primer of (B) as a reverse primer) );
(d) A primer containing or consisting of the nucleotide sequence of SEQ ID NO: 10 (GAYMGDGARATHTAYACWGG) (Ema-Motif 2-sI-20mer: corresponding to the primer of (A) as a forward primer) and the nucleotide sequence of SEQ ID NO: 9. (GCWGACCAYTTISWDGCATC) or a primer set consisting of a primer (Ema-Motif A-as-I-20mer: corresponding to the primer of (B) as a reverse primer) (primer set of # 5 in Examples);
(e) A primer containing or consisting of the nucleotide sequence of SEQ ID NO: 11 (GATGCHWSIAARTGGTCWGC) (Ema-Motif AsI-20mer: corresponding to the primer of (B) as a forward primer) and the nucleotide sequence of SEQ ID NO: 12 (RTTIARRTTICCYTGHARCC) ) (Ema-Motif 3-as-2I-20mer: equivalent to the primer of (C) as a reverse primer) and a primer set (# 6 primer set in the examples).

The present invention also relates to a kit for detecting an emalavirus virus or diagnosing an emaravirus virus infection, which comprises a primer set according to the present invention. The kit includes, for example, reverse transcription enzyme used for RT-PCR, DNA polymerase; nucleic acid synthesis substrate (dNTP) used for RT-PCR, buffer solution, salts, container, etc .; reagent required for detection of RT-PCR amplification product. Classes (eg, agarose gel, ethidium bromide, etc.); instructions for use and the like can be further included.

Further, the present invention includes a step of performing an amplification reaction of a target nucleic acid region of an emaravirus virus using the primer set or kit according to the present invention described above, and comprises detecting an emaravirus virus or emara. It relates to a method for diagnosing a virus infection of a virus genus (hereinafter referred to as "this method"). The present method includes a step of performing a nucleic acid amplification reaction using the primer set according to the present invention, and uses, for example, RT-PCR method, quantitative RT-PCR method, RT-nested PCR method, one-step RT-PCR method and the like. can do.

For example, when the RT-PCR method is used in this method, RNA is first extracted, purified, and purified from a plant-derived sample to be detected for the presence or absence of Emaravirus virus infection according to a conventionally known method. Using RNA as a template, cDNA is prepared using reverse transcriptase and primers such as random hexamer. Here, as a plant-derived sample, for example, the whole plant body, plant organs (for example, leaves, petals, stems, roots, seeds, etc.), plant tissues (for example, epidermis, phloem, parenchyma, xylem, vascular bundle, etc.) , Plant cultured cells and the like.

Next, the prepared cDNA is used as a template for PCR using the primer set according to the present invention. For the PCR reaction solution, for example, per 25 μL of the reaction solution, each primer contained in the primer set according to the present invention has a final concentration of 0.1 to 10 μM (preferably 1.0 to 3.0 μM), cDNA 0.1 to 2 μL (preferably 0.2 to 1 μL), and DNA. Prepare the polymerase 0.1 to 2 U (preferably 0.5 to 1 U) and dNTP to be contained at a final concentration of 0.1 to 1 mM (preferably 0.1 to 0.3 mM). The thermal cycling conditions for PCR can be appropriately determined according to the primer set used, and for example, denaturation: 90 to 99 ° C (preferably 94 to 98 ° C) for 1 to 60 seconds (preferably 5 to 10). Seconds), annealing: 35-65 ° C (preferably 42-48 ° C) 10-60 seconds (preferably 15-30 seconds), elongation: 65-75 ° C (preferably 68-72 ° C) 10-60 seconds (preferably 68-72 ° C) 25 to 50 cycles (eg 35 to 45 cycles) of 15 to 30 seconds.

Then, after the PCR reaction, the reaction solution is subjected to agarose gel electrophoresis and stained with ethidium bromide to confirm the presence or absence of an amplification product of a size expected from the primer set used. Emaravirus virus infection can be diagnosed (or tested or evaluated) by the presence or absence of amplification products.

When the quantitative RT-PCR method is used in this method, for example, in addition to the reagents used in the RT-PCR method, a fluorescent probe (labeled at the 5'end with a reporter) that complementarily binds to the amplified region. In addition, PCR can be performed using a fluorescent probe labeled with Quenche at the 3'end, and the amplification product can be quantified using the fluorescent signal as an index.

When the RT-nested PCR method is used in this method, a primer set is used to anneal to a position outside the target region to be amplified by the primer set according to the present invention, and RNA from a plant-derived sample is used as a template. PCR amplification of cDNA. Then, as in the RT-PCR method, the cDNA is used as a template for PCR using the primer set according to the present invention (Nested PCR).

When the one-step RT-PCR method is used in this method, the primer set itself according to the present invention is used to prepare cDNA using RNA from a plant-derived sample as a template, and the prepared cDNA as a template. Perform PCR.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.

The genus Emaravirus is a major obstacle to agricultural production as a plant virus transmitted by Eriophyidae. It is also speculated that there are many unidentified new virus species (Mielke-Ehret and Muehlbach (2012) Emaravirus: A Novel Genus of Multipartite, Negative Strand RNA Plant Viruses. Viruses 4: 1515-1536.). As a method for detecting and diagnosing emaravirus, Non-Patent Document 7 reports a degenerate primer based on the base sequences of five types of emaravirus (EMARaV, FMV, RLBV, RRV, RYRV) and a method by RT-PCR method. doing.

In this example, in addition to these five species, a wider range of emara is based on the sequence information of PPSMV, PPSMV2, HPWMoV, and a new species of perilla mosaic virus (PMoV) discovered by the present inventors. We designed a degenerate primer that can detect virus species.

1. Design of degenerate primer for detection of emaravirus In order to obtain the amino acid sequence of RNA-dependent RNA polymerase (RdRP) encoded by RNA1 of emaravirus, 9 kinds of emaravirus shown in Table 1 (in order to obtain the amino acid sequence of RNA dependent RNA polymerase (RdRP)) A total of 12 strains) were obtained. In addition to these, the RdRP full-length sequence of Perilla mosaic virus discovered by the present inventors is also added, and ClustalX (Thompson et al. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools . Nucleic Acids Res. 25: 4876-4882.) Aligned the amino acid sequences and searched for highly conserved regions. As a result, in addition to the conserved regions (PreMotif A, Motif A, Motif C) used as the target of the primer in Non-Patent Document 7, two highly conserved regions were found and named Motif 2 and Motif 3 (Motif 2 and Motif 3). Figure 1). The positional relationship of these Motifs is shown in FIG.

In order to compare the nucleotide sequences on the RdRP gene for each Motif, the nucleotide sequences of each emaravirus species were obtained in the same manner (Table 2). By aligning the base sequences, 3 sets of degenerate primers (6 sets in total) for amplifying Motif 2 to Motif A and Motif A to Motif 3 by the RT-PCR method were designed (Fig. 1). Inosine residues were used in 3 of these sets so that the primer did not become too debilitating (Table 3). The combinations of primers used for each primer set are shown in Table 4, and the target positions on the viral RNA are shown in FIG.

2. Examination of detectability of domestically occurring emaravirus species (PMoV and FMV) We attempted to detect PMoV and fig mosaic virus (FMV), which are domestically occurring species of emaravirus, with the designed primers. The detection sensitivity and the like were compared with the primers of Non-Patent Document 7.

First, total RNA was purified from healthy perilla leaves, PMoV-infected perilla leaves, and FMV-infected fig leaves using TRIzol Reagent (Thermo Scientific). Using this RNA as a template, cDNA was prepared using PrimeScript Reverse Transcriptase (Takara Bio Inc.). Random hexamer was used as the primer, and the procedure was in accordance with the product manual.

Using this cDNA as a template, a PCR reaction was carried out using a degenerate primer set (Table 4: # 1 to # 8) and Taq DNA Polymerase (Takara Bio Inc.). The reaction was carried out in a 25 μL system, 0.5 μL of cDNA was added, and the final concentration of primers was 2 μM each. The thermal cycling conditions were 98 ° C for 10 seconds, 45 ° C for 30 seconds, and 72 ° C for 30 seconds for 40 cycles. 5 μL of the reaction product was subjected to 2% agarose gel electrophoresis, and amplification of the reaction product was confirmed by ethidium bromide staining. The results are shown in FIG.

As shown in FIG. 3, in the newly designed primer sets other than # 2 among the newly designed # 1 to # 6, amplification products of the expected size were obtained specifically for PMoV and FMV-infected leaves. On the other hand, PMoV could not be detected with the previously reported primers # 7 and # 8, FMV was not detected with # 7, and only # 8 was detected. The results not detected in # 7 are reproducible (Fig. 6) because, as shown in Fig. 9, FMV strains (AB697826 and AB697827) in Japan have a 1-base mismatch with the Pre-motif A-sense primer. It is possible that the annealing was inadequate because of the recognition.

3. Examination of detectability by one-step RT-PCR Total RNA was purified from healthy perilla leaves, PMoV-infected perilla leaves, and FMV-infected fig leaves using TRIzol Reagent (Thermo Scientific). Using this RNA as a template, reverse transcription and PCR reaction are performed in the same tube using degenerate primer set # 1 or # 3 and PrimeScript One-Step RT-PCR Kit Ver. 2 (Takara Bio). PCR was performed. The reaction was carried out in a 20 μL system, 0.5 μL of RNA was added, and the final concentration of primers was 1 μM each. The reverse transcription reaction was carried out at 50 ° C. for 30 minutes, the reverse transcriptase was inactivated at 94 ° C. for 2 minutes, and then the PCR reaction was continued. The thermal cycling conditions were 94 ° C for 30 seconds, 48 ° C for 30 seconds, and 72 ° C for 15 seconds for 40 cycles. 5 μL of the reaction product was subjected to 2% agarose gel electrophoresis, and amplification of the reaction product was confirmed by ethidium bromide staining. The results are shown in FIG.

As shown in FIG. 4, in the newly designed primer sets # 1 and # 3, amplification products of the expected size were obtained specifically for PMoV and FMV-infected leaves.

4. Examination of detectability of domestic unoccurred Emaravirus species
In addition to FMV and PMoV, the detectability of a total of 10 species including 8 species of emaravirus that has not occurred in Japan was examined. The virus species are shown in Table 5. Of these, AcCRaV is a virus whose sequence was unknown at the stage of primer design.

Seo et al. (2014) A novel set of polyvalent primers that detect members of the genera Bromovirus and Cucumovirus. J. Virol. Methods 203: 112-115. Therefore, a pseudo sample of infected leaf RNA was prepared as follows.

First, the nucleotide sequence of RNA1 of a domestic undeveloped species was obtained from the database (Table 5), and out of the total length of about 7.2 kb, the region encoding Pre Motif A, Motif 2, Motif A, Motif 2 and Motif C and before and after it. DNA having about 1.5 kb of the sequence (nt 2500-4000) was prepared by artificial gene synthesis and cloned into a plasmid. In addition, a T7 RNA promoter is placed upstream of this sequence, and RNA is prepared by in vitro transcription with T7 RNA polymerase (Takarabio), and template DNA is degraded with DNaseI (Takarabio) to decompose the virus. RNA having a partial sequence was prepared. A mixture of 1 ng of artificially synthesized RNA of each emaravirus and 100 ng of total RNA purified from healthy leaves of each host plant shown in Table 5 was used as a pseudo-infected leaf RNA sample. ..

From # 1 to # 6, we narrowed down the highly practical primer sets. Using the cDNA from FMV and PMoV-infected leaves and the artificially synthesized DNA (plasmid) of PPSMV, PPSMV2, RYRV, EMARaV, RLBV, and HPWMoV as templates, perform the same PCR as above using the primer sets of # 1, # 3 to # 6. As a result, specific products were obtained in both cases (Fig. 5), but it was judged that the amplification efficiency of # 1 and # 3 was high, and only these two sets were used in the subsequent experiments. Since the primer sets # 1 and # 3 selected here and the previously reported primers # 7 and # 8 do not contain inosine residues, they are suitable for the following experiments as primers containing inosine, but are accurate. The more accurate Ex Taq DNA Polymerase (Takara Bio) was used instead of the lower Taq DNA polymerase.

Next, cDNA was synthesized from the pseudo-infected leaf RNA sample prepared above by reverse transcription using random hexamer, and a PCR reaction was performed. However, the PCR reaction with primer sets # 7 and # 8 followed the protocol of Non-Patent Document 7 except that Taq DNA polymerase of Takara Bio Inc. was used as the enzyme instead of Promega. The results are shown in FIG.

As shown in FIG. 6, the band of the previously reported primer # 7 was thin as a whole, and FMV and PMoV were not detected. HPWMoV could not be detected in # 8.

On the other hand, in # 1 and # 3, although the HPWMoV band was thin with the # 3 primer, it was detected, and in all other cases, a clear band was observed, and all 10 virus species tested were detected. Was possible.

5. Examination of detectability of unknown emaravirus Since the sequence was unknown at the time of designing the new degenerate primer and AcCRaV, which was not taken into consideration in the design, was also detectable, the sequence is unknown. It was considered possible that the emaravirus species could also be detected. Therefore, we attempted to detect the virus using this primer set for the ring pattern symptom of Shikimi and the mosaic symptom of pear, which were suspected to be related to the virus because the symptom occurred due to the infestation of Eriophyidae in Japan.

First, total RNA was extracted from the leaves (symptomatic and asymptomatic samples) of each plant using Fruit mate (Takara Bio Inc.) and TRIzol Reagent. Using 500 ng of this RNA, reverse transcription reaction was performed by PrimeScript Reverse Transcriptase. Random hexamer was used as the primer, and the procedure was in accordance with the product manual. Using 2 μl of this cDNA as a template, PCR reaction was performed with Ex Taq DNA Polymerase. # 1, # 3, # 7, and # 8 sets were used as the detection primers, and the PCR temperature conditions were the same as in item 2. The results are shown in FIG.

As shown in FIG. 7, as a result of PCR performed using Shikimi as a sample, amplification products of the expected size were obtained from the samples with symptoms from # 1 and # 3 (upper part of FIG. 7). On the other hand, no specific amplification products were observed from # 7 and # 8. In addition, in the PCR performed using pear as a sample, a band was confirmed at the expected position in 1 of the 2 samples showing the mosaic only in # 3 (lower part of FIG. 7). (Although there is a band of the expected size in # 1, it was excluded because it does not correlate with the symptoms. However, since there is a thin band in the pear asymptomatic leaves with specific primers, it is possible that the infection is at a low concentration. is there).

These bands were excised and the nucleotide sequence of the purified DNA was determined. These sequences encode open reading frames without stop codons, and when a Blastp search was performed on the amino acid sequence, many RdRPs of emaraviruses such as FMV were hit. Furthermore, the base sequences of these DNAs and the base sequences of RNA1s of 10 known Emaravirus viruses are shown in MEGA 7.0 (Kumar et al. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol. Biol. Alignment was performed using Evol. 33: 1870-1874.), And a phylogenetic tree was prepared by the neighbor binding method (Fig. 8). The sequences obtained from Shikimi and Pear belonged to the same clade as FMV et al. And PMoV, respectively. However, from the length of each branch, it was considered likely that it was a different species from the known emaravirus species.

From the above results, it was shown that the new primer could detect two new emalaviruses.

6. Discussion The reason why the pear and shikimi viruses could not be detected by the previously reported primer sets (# 7 and # 8) is thought to be the large number of mismatches between the primer sequence and the viral RNA sequence (Fig. 9). In the primer sets (# 1 and # 3) constructed in this example, only one base mismatch was observed with the shikimi virus in the # 3 antisense primer (Ema-motif3-as-20mer), but the other primers were It was able to cover all virus sequences without mismatch. On the other hand, in the previously reported primer set, a mismatch of 5 bases in pear and 2 bases in shikimi was confirmed in # 7 antisense primer (Motif A-antisense) and # 8 sense primer (Motif A-sense). The sequences of the regions corresponding to PreMotif A and Motif C of the emaravirus-like sequences of Shikimi and Pear are not yet clear, but similar to Motif A, it is expected that there will be a mismatch between the primers of # 7 and # 8. ..

Also, for known viruses, Motif A-sense and Motif A-antisense cause 5, 2, and 2 base mismatches with PPSMV, HPWMoV, and PPSMV2, respectively, as well as Pre-motif A-sense and Motif C. Even with the -antisense primer, a mismatch of 1 to 5 bases can occur depending on the virus species (Fig. 9).

From the above results, it was judged that the primer set and the experimental method constructed in this example are superior to the method of Non-Patent Document 7 as a method for detecting known and unknown emaravirus species.

The degeneracy primers of # 1 and # 3 have a degeneracy of 192 to 432, which is considerably higher than that of 6 to 32 of # 7 and # 8 (Table 3), but in the actual detection experiment. The high degree of degeneracy did not seem to be an obstacle. In the latter half of the test, primers # 4, # 5, and # 6 containing inosine were not used, but these primers may be used depending on the plant species and virus species to be detected, the conditions of the PCR enzyme used, and the like. It is considered that there are more appropriate situations, and it does not exclude the possibility of use.

Claims (3)

A primer set according to any one of (a) to (e) below for amplifying a base sequence specific to a virus belonging to the genus Emaravirus.
(a) A primer set consisting of a primer containing the nucleotide sequence shown in SEQ ID NO: 4 and a primer containing the nucleotide sequence shown in SEQ ID NO: 5;
(b) A primer set consisting of a primer containing the nucleotide sequence shown in SEQ ID NO: 6 and a primer containing the nucleotide sequence shown in SEQ ID NO: 7;
(c) A primer set consisting of a primer containing the nucleotide sequence of SEQ ID NO: 8 and a primer containing the nucleotide sequence of SEQ ID NO: 9;
(d) A primer set consisting of a primer containing the nucleotide sequence of SEQ ID NO: 10 and a primer containing the nucleotide sequence of SEQ ID NO: 9;
(e) A primer set comprising a primer containing the nucleotide sequence of SEQ ID NO: 11 and a primer containing the nucleotide sequence of SEQ ID NO: 12. A kit for detecting an Emaravirus virus or diagnosing an Emaravirus virus infection, which comprises the primer set according to claim 1. With claim 1, wherein the primer set or claim 2, wherein the kit comprises performing an amplification reaction of a target nucleic acid region of Emma La genus virus, Emma La genus virus detection or Emma La genus infection diagnosis Method.

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