JP2001258571A - Method for detecting apple chlorotic leaf spot virus and primer for detection - Google Patents

Abstract

(57) Abstract: A method for amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus using RNA extracted from tissue of a test plant as a template. Performing nucleic acid amplification using one homologous primer and a first complementary primer to obtain a first amplification product, and then using the first amplification product as a template,
Nucleic acid amplification using a second homologous primer and a second complementary primer capable of amplifying a second partial region contained in the first partial region of genomic RNA of the plurality of isolates of apple chlorotic leaf spot virus A method for detecting apple chlorotic leaf spot virus from the test plant by obtaining a second amplification product. According to the present invention, a plurality of ACLSV isolates can be detected simultaneously. Therefore, according to the present invention, it is possible to accurately diagnose an ACLSV infection of an apple tree at any time, and thereby, it is possible to supply an ACLSV-uninfected apple seedling.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a plant virus, and more particularly, to a method for detecting an apple chlorotic leaf spot virus.
s, hereinafter referred to as "ACLSV". A) detecting a plurality of isolates simultaneously, and an oligonucleotide that can be used in the method.

[0002]

2. Description of the Related Art ACLSV causes high apple disease 3
It is a kind of pathogenic virus that weakens the apple rootstock, Marubakaidou. It is widely distributed throughout the world and is an important disease that causes peach, plum, cherry and apricot besides apple.

[0003] ACLSV is composed of a single-stranded RNA of about 2.5 × 10 ³ kDa.
It is a virus consisting of a 22 kDa coat protein. Therefore, whether or not a test plant is infected with ACLSV is determined by a bioassay using a graft to an indicator plant, an immunoassay using an antibody against a coat protein, for example, an enzyme-linked immunosorbent assay (enzyme-linked immunosorbent). assay, hereinafter referred to as "ELISA"), a hybridization method using a DNA or RNA complementary to a virus genome as a probe, and the like.

[0004] However, although bioassays are sensitive,
It takes several months to several years of testing, and is difficult to determine because ACLSV latent strains may have weak or no symptoms. In addition, in ELISA and hybridization, ACLSV can be detected in the indicator plant, but sensitivity is not sufficient to directly detect from apple trees with low virus concentration, and only young leaves collected at the same time as petals can be detected. (Yoshiaki Sugano, Nobuyuki Yoshikawa, and Sou Takahashi, Bulletin of the Japanese Society for Plant Pathology, Vol. 57, p. 278).

Recently, methods have been developed that exponentially amplify specific sequences of genes. One is the polymerase chain reaction (PC) that amplifies a specific DNA fragment exponentially.
R) method (Saiki, RK et al., Science 239, 487-491).
(1988)). The other is a method of exponentially amplifying a specific RNA fragment, using Nucleic Acid Sequence-Based Amplification.
certification (NASBA) method (Davey and Malek, 1989,
European Patent No. EP0329822). These methods have also been applied to virus diagnosis. That is, after exponentially amplifying a partial sequence of the virus genome, the resulting amplified fragment is detected by gel electrophoresis or hybridization, whereby the virus can be detected with extremely high sensitivity. Furthermore, nested PCR and nested NASB are used as methods for detecting viruses and the like with higher sensitivity.
A (Kievits, T. et al., J. Virol. Methods 35, 273-286 (1
991)) has been developed.

[0006] ACLSV includes isolates (from latent to virulent) that have different pathogenicity to Marubakaidou. On the other hand, the entire nucleotide sequence of the ACLSV genome that apples, plums and sweet cherries have each been determined (German, S. et al.
Virology, Vol. 179, 104-112 (1990), Sato, K. et al., J.
Gen. Virol., Vol. 74, 1927-1931 (1993), German, S.
Et al., Arch. Virol., Vol. 142, 833-841 (1997)), and comparing the nucleotide sequences among them reveals sequence differences in many regions (homology: 76-82%). Therefore, it is conceivable that there is a difference in the nucleotide sequence of the genome between ACLSV isolates with different pathogenicity that are insulated by apple trees. Furthermore, it has been found that each ACLSV isolate that the apple tree infects is a heterogeneous population containing multiple nucleotide sequences (Nakahara et al., Bulletin of the Japanese Society for Plant Pathology, Vol. 64, p. 605 (1998)). Therefore, when the above-described gene amplification method is applied to the detection of ACLSV, a certain A
With one type of primer designed for the CLSV genome,
It is not possible to amplify the genomic sequence of all ACLSV isolates. Although a method using a gene amplification method as a method for detecting ACLSV that is poisoned by apple trees is very promising, as described above, the conventional method is not suitable for diagnosing all ACLSV isolates.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a high-sensitivity diagnostic method capable of simultaneously detecting a plurality of ACLSV isolates directly from a test plant.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, a plurality of sets of primers designed based on genomic RNAs of a plurality of ACLSV isolates having different pathogenicity. It has been found that all of the plurality of ACLSV isolates can be directly detected from the apple tree by the gene amplification method using, and the present invention has been completed.

That is, the present invention provides (i) amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus using RNA extracted from a test plant tissue as a template. Nucleic acid amplification is performed using the obtained first homologous primer and the first complementary primer to obtain a first amplification product;
Genomic R of said plurality of isolates of apple chlorotic leaf spot virus using the amplification product of
Performing a nucleic acid amplification using a second homologous primer and a second complementary primer capable of amplifying a second partial region included in the first partial region of NA to obtain a second amplification product; A method for detecting an apple chlorotic leaf spot virus from the test plant is provided.

Here, the plurality of isolates of the apple chlorotic leaf spot virus are P205 strain, P195 strain,
P195L, PK32, PK51, P142, P143, P202, P
The strains are preferably K5 strain, P125 strain, MK1, MK8, MK9, MO31, MO41 and B81 strains.

[0011] The first homologous primer is SEQ ID NO: 1,
The second homologous primer is an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1; and (2) the second homologous primer is an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1. Is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, 3 or 4; (ii) when the first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, Or (iii) when the first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 4
And an oligonucleotide comprising a base sequence represented by the formula:

The first complementary primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 6, 7 or 8, and the second complementary primer is
(Iv) when the first complementary primer is an oligonucleotide containing the base sequence represented by SEQ ID NO: 6, an oligonucleotide containing the base sequence represented by SEQ ID NO: 5; (v) the first complementary primer Is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 7; or an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 5 or 6; or (vi) the first complementary primer is represented by SEQ ID NO: 8. In the case of an oligonucleotide containing a base sequence represented by SEQ ID NO: 5, 6, or 7, an oligonucleotide containing a base sequence represented by SEQ ID NO: 5, 6, or 7 is preferable.

[0013] More preferably, the first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3, and the second homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4. The first complementary primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 6, and the second complementary primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 5. The test plant,
It is preferably apple, peach, plum, cherry or apricot. Preferably, the tissue is a petal, leaf or bark.

Furthermore, the present invention provides (i) a homologous primer and a complementary primer capable of amplifying the first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; and (ii) an apple chlorotic leaf Genome R of said plurality of isolates of spot virus
A primer set for detecting apple chlorotic leaf spot virus from a test plant, comprising: a homologous primer and a complementary primer capable of amplifying a second partial region included in the first partial region of NA.

Here, the plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain,
P195L, PK32, PK51, P142, P143, P202, P
The strains are preferably K5 strain, P125 strain, MK1, MK8, MK9, MO31, MO41 and B81 strains.

The above-mentioned primer set preferably comprises two types of homologous primers selected from the group consisting of oligonucleotides comprising the nucleotide sequences represented by SEQ ID NOs: 1 to 4, and bases represented by SEQ ID NOs: 5 to 8 It contains two types of complementary primers selected from the group consisting of oligonucleotides containing sequences, and more preferably contains oligonucleotides containing the base sequences represented by SEQ ID NOS: 3 to 6.

The present invention further provides (i) first and second partial regions of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus using RNA extracted from a test plant tissue as a template. Performing a nucleic acid amplification using the first and second homologous primers and the first and second complementary primers capable of amplifying E. coli to obtain a first amplification product; and (ii) using the first amplification product as a template And
Nucleic acid amplification using third and fourth homologous primers and third and fourth complementary primers capable of amplifying third and fourth partial regions of genomic RNA of the plurality of isolates of apple chlorotic leaf spot virus Go second
A method for detecting apple chlorotic leaf spot virus from the test plant.

Here, regarding the mutual positional relationship between the first to fourth partial regions, the first partial region and the second partial region include the third partial region and the fourth partial region, respectively. Preferably, the fourth partial region is included in the second partial region, the second partial region is included in the third partial region, and the third partial region is included in the first partial region. To be included.

Here, the plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain,
P195L, PK32, PK51, P142, P143, P202, P
The strains are preferably K5 strain, P125 strain, MK1, MK8, MK9, MO31, MO41 and B81 strains.

The first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 1,
The second homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3, the third homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, and the fourth homologous primer is Is an oligonucleotide containing the base sequence represented by SEQ ID NO: 4, and the first complementary primer is SEQ ID NO: 8
Wherein the second complementary primer is an oligonucleotide comprising a base sequence represented by SEQ ID NO: 6, and the third complementary primer is an oligonucleotide comprising a base sequence represented by SEQ ID NO: 6.
Is preferably an oligonucleotide containing the base sequence represented by SEQ ID NO: 7, and the fourth complementary primer is preferably an oligonucleotide containing the base sequence represented by SEQ ID NO: 5.

The test plants are apples, peaches, plums,
It is preferably sweet cherry or apricot. Preferably, the tissue is a petal, leaf or bark. Furthermore, the present invention provides (i) a homologous primer and a complementary primer capable of amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; A homologous primer and a complementary primer capable of amplifying a second partial region of genomic RNA of the isolate of (i); (iii)
A homologous primer and a complementary primer capable of amplifying a third partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; and (iv) a genomic RNA of said plurality of isolates of apple chlorotic leaf spot virus; A primer set for detecting an apple chlorotic leaf spot virus from a test plant, comprising a homologous primer and a complementary primer capable of amplifying the fourth partial region.

Here, with respect to the mutual positional relationship between the first to fourth partial regions, the first partial region and the second partial region include the third partial region and the fourth partial region, respectively. Preferably, the fourth partial region is included in the second partial region, the second partial region is included in the third partial region, and the third partial region is included in the first partial region. To be included.

Here, the plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain,
P195L, PK32, PK51, P142, P143, P202, P
The strains are preferably K5 strain, P125 strain, MK1, MK8, MK9, MO31, MO41 and B81 strains. Further, it is preferable that the primer set includes an oligonucleotide containing the base sequence represented by SEQ ID NOS: 1 to 8.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Virus Detection Method of the Present Invention The virus detection method of the present invention includes extraction of RNA from a test plant, a gene amplification reaction using a synthetic oligonucleotide primer for ACLSV detection, and detection of an amplification product obtained by the amplification reaction. Hereinafter, each of these steps will be described in detail.

(1) Extraction of RNA from Test Plant Tissue In the virus detection method of the present invention, total RNA is extracted from apple tree and other test plant tissues and used as a template for amplification. In addition, the genomic RNA of ACLSV has a continuous sequence of adenine (polyA) added to the 3 ′ end similarly to the mRNA of the host plant.
A can be extracted and used as a template. Furthermore, after separating virus particles, virus genomic RNA can be extracted and used as a template.

The test plant is not particularly limited as long as it can be infected with any isolate of ACLSV, and is preferably apple, peach, plum, sweet cherry or apricot, and more preferably apple. The above-mentioned tissue is not particularly limited as long as it can be infected with any isolate of ACLSV, and is preferably a petal, leaf or bark.

The extraction of total RNA from the test plant tissue is performed, for example, by freezing the tissue with liquid nitrogen, grinding the cell tissue, and extracting the total RNA with guanidium thiocyanate or a phenol / chloroform solution. It can be carried out later by ethanol precipitation or the like. MRNA from a test plant tissue can be purified from the above-mentioned total RNA or a triturated solution of the tissue using oligo dT cellulose or the like. A specific operation method is described in Molecular Cloning (Maniatis et al., AL
aboratory Manual, Cold Spring Harbor Laboratory Pr
ess, 1982). Such total RNA
Alternatively, mRNA extraction / purification can also be performed using a commercially available RNA extraction kit. Extraction of the viral genomic RNA from the test plant tissue is carried out by an immunocapture method (Jansen et al., Proc. Natl. Acad. Sci.
67-2871 (1990)) and the like can be applied to ACLSV.

(2) Gene amplification reaction using synthetic oligonucleotide primer for ACLSV detection In the virus detection method of the present invention, the RNA sample obtained in the above (1) is used as a template, and the gene using the synthetic oligonucleotide primer for ACLSV detection is used. Perform amplification reaction.

As the gene amplification reaction, nested PC
A nested amplification reaction such as the R method and the nested NASBA method is used. In particular, dual nested PC
The R method, the dual nested NASBA method, and the like are useful. Details of these will be described later, but ordinary nested P
When using the CR method or the nested NASBA method, at least two kinds of homologous primers and at least two kinds of complementary primers are required. When using the dual nested PCR method or the dual nested NASBA method, at least four kinds of homologous primers are used. And at least four complementary primers are required.

The primers used in the virus detection method of the present invention are genomic RNAs of a plurality of ACLSV isolates.
Can be amplified based on a highly conserved region (conserved region) among the genomic RNAs of the plurality of isolates. Here, the plurality of isolates are isolates that are to be simultaneously detected by the virus detection method of the present invention. However, the isolate to be detected is not limited to the one used for primer design, and any isolate that can be detected by the designed primer, that is, any isolate having the above conserved region, can be detected. Can be targeted. AC like this
LSV isolates may be those known to those skilled in the art, and are not particularly limited, for example, P205 strain, P195 strain, P195L strain, PK3
2, PK51, P142, P143, P202, PK5, P125
Strains, MK1, MK8, MK9, MO31, MO41, B81 and the like. The 16 isolates listed here are ACLSV isolates with different pathogenicity against Marubakaidou, which are preserved at the Apple Branch Disease Laboratory, Fruit Tree Experimental Station of the Ministry of Agriculture, Forestry and Fisheries (92 Nabeyashiki, Shimokitagawa, Morioka City, Iwate Prefecture). is there.

The conserved region can be specified by comparing the base sequences of genomic RNA or cDNA of the plurality of isolates. Such a base sequence can be obtained from a nucleotide sequence database known to those skilled in the art, for example, Ge.
It can be easily obtained by searching nBank or the like, or a newly sequenced one can be used. For example, as the plurality of isolates, P205 strain, P195
Strains, P195L, PK32, PK51, P142, P143, P202
Strains, PK5, P125, MK1, MK8, MK9, MO31, MO
When the 41 strain and the B81 strain are used, SEQ ID NO: 11 which is the cDNA base sequence of the 3 ′ terminal in these genomes
Base sequences represented by -42 (these sequences are shown separately in the first half and the second half because there are undetermined regions in these cDNA sequences); SEQ ID NO: 11 (first half of P205 strain) and SEQ ID NO: 12 (second half of P205 strain) ): SEQ ID NO: 13 (first half of P195 strain)
And SEQ ID NO: 14 (the latter half of the P195 strain): SEQ ID NO: 15 (P195
SEQ ID NO: 17 (first half of PK32 strain) and SEQ ID NO: 18 (second half of PK32 strain):
SEQ ID NO: 19 (first half of PK51 strain) and SEQ ID NO: 20 (second half of PK51 strain): SEQ ID NO: 21 (first half of P142 strain) and SEQ ID NO: 22
(P142 strain second half): SEQ ID NO: 23 (first half of P143 strain) and SEQ ID NO: 24 (second half of P143 strain): SEQ ID NO: 25 (first half of MK8 strain)
And SEQ ID NO: 26 (second half of MK8 strain): SEQ ID NO: 27 (first half of B81 strain) and SEQ ID NO: 28 (second half of B81 strain): SEQ ID NO: 29
(First half of P202 strain) and SEQ ID NO: 30 (second half of P202 strain): SEQ ID NO: 31 (first half of PK5 strain) and SEQ ID NO: 32 (second half of PK5 strain): SEQ ID NO: 33 (first half of PI25 strain) and SEQ ID NO: 34
(Second half of PI25 strain): SEQ ID NO: 35 (first half of MK1 strain) and SEQ ID NO: 36 (second half of MK1 strain): SEQ ID NO: 37 (first half of MO31 strain)
And SEQ ID NO: 38 (second half of MO31 strain): SEQ ID NO: 39 (MO41
Strain first half) and SEQ ID NO: 40 (MO41 strain second half): SEQ ID NO: 4
1 (first half of the MK9 strain) and SEQ ID NO: 42 (second half of the MK9 strain). Among them, the cDNA base sequence of the P205 strain is registered in GenBank (registration number: D14
996).

Further, c derived from the genome of an isolate whose sequence is unknown
The DNA base sequence can be determined using a method similar to the method for determining SEQ ID NOS: 11 to 42 above. That is, first, several synthetic oligonucleotide primers are designed based on portions showing homologous sequences in three ACLSV isolates isolated from apple, plum, and cherry, for which the entire base sequence of genomic RNA has already been determined. Next, the petals or the young leaves at the same time that the ACLSV concentration is relatively high are collected from the apple tree that insulates each isolate, and the total RNA is extracted. Using this total RNA as a template, reverse transcription and PCR are carried out using the above-mentioned synthetic oligonucleotide primers, and an amplification product that has been well amplified is subjected to sequencing.

For example, the cD at the 3 'end of the genome
To determine the NA base sequence, the homologous primer: 5 '
-GAAGATCGCAGAAGGGGATATTC-3 '(SEQ ID NO: 9) and the complementary primer: 5'-GTCTACAGGCTATTTATTATAAG-3' (SEQ ID NO: 10) can be used as primers, thereby providing the 3 'end portion in the genome of the isolate. A cDNA of about 1800 bases can be amplified.

The nucleotide sequence of the cDNA fragment obtained in this manner is obtained by
It can be determined by performing a sequencing reaction using the DNA fragment as a template, and further, a new primer can be synthesized based on the determined sequence, and the nucleotide sequence can be similarly determined. By repeating this, almost the entire nucleotide sequence of the cDNA fragment can be determined.

The above-mentioned conserved region can be specified by aligning the nucleotide sequences obtained as described above. For alignment of a plurality of sequences, a method known to those skilled in the art can be used. For example, commercially available software can also be used. In order to identify a conserved region that is the basis for primer design based on such an alignment (sequence comparison), it is not necessary that all nucleotide sequences of the identified conserved region are completely homologous. In particular, in the vicinity of the 3 'end portion, the preservability is particularly increased. This is because the complementarity between the primer and the target ACLSV genome is very important, especially if the 3 'end of the base sequence of the primer does not complementarily bind to the target, the amplification efficiency is significantly reduced. It is. The number of bases of each primer is not particularly limited as long as it is the number of bases usually used as a primer, but is preferably 16 bases or more, more preferably 2 bases or more.
0 bases or more. The number of bases of the fragment amplified by the designed primer may be any number of bases that can be separated by a normal DNA fragment separation method, for example, electrophoresis,
Although not particularly limited, preferably 100 bases to 2000 bases
Bases, more preferably 150 bases to 1000 bases.

As an example, the isolate to be detected is P205
Strains, P195, P195L, PK32, PK51, P142, P143
Strains, P202, PK5, P125, MK1, MK8, MK9, MO
FIGS. 1 to 5 show alignments of these sequences in the case of 31 strains, MO41 strain and B81 strain. The conserved region was identified based on these sequence comparison diagrams, and the cDN of all isolates was determined.
An oligonucleotide consisting of a plurality of base sequences can be designed so that A is amplified. The specific nucleotide sequence of the designed oligonucleotide is not particularly limited, and examples thereof include a nucleotide sequence represented by SEQ ID NOS: 1 to 4 (homologous primer) and a nucleotide sequence represented by SEQ ID NOs: 5 to 8 (complementary primer). The conserved regions on which these designs are based are shown at the top of the comparative sequence in FIGS. 1-5 (numbers shown correspond to SEQ ID NOs). here,
SEQ ID NOs: 1 to 4 are homologous primers designed in order from the 5 'upstream region to the downstream region on the ACLSV genome. Similarly, SEQ ID NOS: 5 to 8 are complementary primers similarly designed from the 5 'upstream region to the downstream region.

The homologous primer and the complementary primer designed as described above are synthesized according to a conventional method, and a gene amplification method is performed using the same. At this time, the RNA sample obtained in the above (1) is used as a template.

As the gene amplification method, any method known as a nested-type gene amplification method can be used as described above, and is not particularly limited.
Preferably, a nested PCR method is used. Such a method has an advantage that the detection sensitivity is higher than a normal gene amplification method. Hereinafter, a method using nested PCR will be described in detail.

When amplifying the base sequence of ACLSV genomic RNA by PCR, the cD
It is necessary to perform NA synthesis. As the reverse transcriptase, those known to those skilled in the art can be used and are not particularly limited. Usually, a reverse transcriptase such as a retrovirus, for example, a commercially available AMV reverse transcriptase XL (Life Sciences) is used. be able to. Primers used for the reverse transcription reaction include:
Primers known to be used for the first strand cDNA synthesis, for example, random primers and the like can be used, but primers specific to the sequence to be amplified by PCR performed later can also be used. As such a specific primer, a complementary primer designed as described above, preferably a primer having a base sequence represented by SEQ ID NOS: 5 to 8, can be used. However, a primer designed in the 5'-upstream region in the ACLSV genome cannot be used as compared with the complementary primer used in the second PCR performed later. CD as above
NA synthesis is the first strand cDN in normal RT-PCR
A person skilled in the art can easily carry out the method according to the method used for the synthesis of A. A kit capable of performing reverse transcription and PCR in the same reaction solution, for example, an RNA PCR kit (Gene Am
The operation can be simplified by using pEZrTth RNA PCR kit (PerkinElmer).

Next, nested PCR is performed using the cDNA synthesized by reverse transcription as a template. In the PCR method, DNA of a sense strand is synthesized by annealing of a homologous primer to a template (here, the above-mentioned cDNA) and an extension reaction by a heat-resistant DNA polymerase, and thereafter, heat denaturation is performed by changing the temperature of the reaction solution. The cycle reaction of annealing the complementary and homologous primers to the sense strand and antisense strand DNA, and elongation with the heat-resistant DNA polymerase in this order is repeated about 30 to 50 times, and as a result, the double-stranded DNA of the target sequence is converted into an exponential function. It is a method of amplifying (about ^105-8 times). For detailed principles, see, for example, Molecular Cloning (Maniatis
A Laboratory Manual, Cold Spring Harbor Labora
tory Press, 1982). In PCR, to amplify a target gene sequence, 20 to 30
A primer set of a synthetic oligonucleotide having a base sequence is used. Nested PCR involves two consecutive PCRs to amplify the target gene sequence. At this time, the second PCR was performed using the first PCR product as a template, a homologous primer designed 3 ′ downstream from the homologous primer used in the first PCR, and a first complementary primer.
A shorter sequence is amplified by a complementary primer designed in the 5 'upstream region. By doing so, the sequence that is non-specifically amplified the first time is not amplified the second time, and the specificity of the amplified sequence is improved, and as a result, the detection sensitivity is improved.

In the present invention, for detecting ACLSV,
Nested PCR is performed using the homologous primer and the complementary primer designed as described above. As the homologous primer, an oligonucleotide containing the base sequence represented by SEQ ID NOs: 1 to 4 is preferably used. As the complementary primer, an oligonucleotide containing a base sequence represented by SEQ ID NOS: 5 to 8 is preferably used. Oligonucleotides containing the nucleotide sequences represented by SEQ ID NOs: 1 to 4 are homologous primers designed in order from the 5 'upstream region to the downstream region on the ACLSV genome as described above. Oligonucleotides containing the nucleotide sequences represented by SEQ ID NOS: 5 to 8 are complementary primers designed in order from the 5 'upstream region to the downstream region. Therefore, for example, two are selected from SEQ ID NOs: 1 to 4, and the smaller one of the SEQ ID NOs is used as a homologous primer in the first PCR and the rest in the second PCR. Similarly, two of SEQ ID NOs: 5 to 8 may be selected, and the larger SEQ ID NO may be used for the first PCR and the rest may be used for the second PCR.

Nested PCR using the above-mentioned template and primer can be easily performed by those skilled in the art by a known method, but can also be performed using a commercially available kit. For example, the above reverse transcription reaction and the first PC
R to an RNA PCR kit (Gene Amp EZ rTth RNA PCR kit,
The second PCR can be performed using a heat-resistant DNA polymerase (TaKaRa Ex Taq, manufactured by Takara Shuzo).

The gene amplification method used in the virus detection method of the present invention may be a normal nested PCR as described above, or a dual nested PCR developed by the present inventors.

Generally, the amplification efficiency of PCR is lower than the theoretical value. This is because there are limitations due to several factors, one of which is the primer used. That is, the amplification efficiency of PCR is
It will be limited by the lower efficiency of accurate annealing with the target. Therefore, the present inventors have proposed a nested PC
R in each PCR reaction, two primer pairs (4
It has been found that, by adding two primers, DNA synthesis from the primer side, which is a limiting factor, can be supplemented by DNA synthesis from another primer, thereby improving amplification efficiency. This method is the above-described dual nested PCR.

In order to utilize dual nested PCR in the virus detection method of the present invention, the above-mentioned nested P
In CR, two primer pairs are used for each of the first PCR and the second PCR. In this case, the first partial region and the second partial region to be amplified by the two primer pairs used for the first PCR are respectively amplified by the two primer pairs used for the second PCR. It is necessary to include the third partial region and the fourth partial region to be
Is included in the second partial area, the second partial area is included in the third partial area, and the third partial area is included in the first partial area.
To be included in the partial area of

For example, in the above-mentioned dual nested PCR, when an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 to 8 is used as a primer,
A second PCR was performed using the four primers SEQ ID NOs: 1, 3, 6, and 8, and a second PCR was performed using SEQ ID NOs: 2,
4, 5 and 7 can be used. As a result, a stable improvement in the amplification efficiency can be seen as compared with normal nested PCR. In the above case, it is considered that the primers of SEQ ID NOs: 1, 2, 7, and 8 may be used in the first PCR, and the primers of SEQ ID NOs: 3, 4, 5, and 6 may be used in the second PCR. On the contrary, the amplification efficiency is reduced. Although the reason for this is unknown, it is thought that it may be due to the 5'-3 'exonuclease activity of DNA polymerase.

[0047] Further, since ACLSV the virus detection method of the present invention is a detection target is a virus having an RNA genome, in place of the above-mentioned PCR, NASBA method (N ucleic A cid
S equence B ased A mplification, Davey and Malek, 198
9, European Patent No. EP0329822). The NASBA method is an RNA-specific amplification method for amplifying an RNA (antisense strand) complementary to the RNA using the RNA as a template, and its principle is, for example, as follows. The complementary primer used here is the 5 ′
A T7 RNA polymerase promoter sequence is added to the end.

(A) Anneal the complementary primer to the target RNA (sense strand). (B) By extension reaction using AMV-RT (reverse transcriptase),
First strand cDNA is synthesized. (C) Target RNA / first strand c obtained by (b) above
The target RNA of the DNA hybrid is degraded by RNaseH, and the first-strand cDNA (antisense strand) becomes single-stranded. At this time, the single-stranded RNA is not decomposed because it does not serve as a substrate for RNaseH.

(D) Single chain c obtained by the above (c)
The homologous primer anneals to the DNA. (E) The homologous primer annealed to the single-stranded cDNA
It is extended by the DNA polymerase activity of AMV-RT, whereby the template for the subsequent RNA amplification and the promoter of T7 RNA polymerase become double-stranded DNA. (F) T7 RNA polymerase recognizes this promoter sequence and synthesizes many copies of the antisense strand RNA of the target RNA up to the 5 'end of the homologous primer.

(G) Newly synthesized antisense RN
The homologous primer anneals to A and becomes the first by AMV-RT.
A strand cDNA (sense strand) is synthesized. The resulting RNA of the RNA / cDNA hybrid is degraded by RNaseH, and the first-strand cDNA becomes single-stranded. A complementary primer anneals to the 3 ′ end of this single-stranded cDNA, and the complementary primer is extended by AMV-RT, and the next R
The template for NA amplification and the promoter of T7 RNA polymerase become double-stranded DNA, which induces the production of many copies of RNA (antisense strand). By performing such steps continuously at a constant temperature, the antisense strand of the target RNA is exponentially amplified.

The above-mentioned NASBA method can be easily implemented by those skilled in the art by referring to the above-mentioned literature and the explanation of the above-mentioned principle. In addition, the above-mentioned NASBA method
By using a commercially available kit such as lification Kit (ORGANON TEKNIKA), it can be easily carried out. When the above-mentioned NASBA is used in the virus detection method of the present invention, the nested NASBA method (Kievi
ts, T. et al., J. Virol. Methods 35, 273-286 (1991)) or the dual nested NASBA method.

When the normal nested NASBA method is performed, two NASBA operations are performed. The first NASBA is the above (1)
Nested PC using the RNA sample obtained in
This is performed using the same homologous primer and complementary primer as in the first PCR in R. Thereby, an RNA corresponding to the complementary strand of the target RNA is obtained as an amplification product. Next, a second NASBA is performed using the amplification product as a template and the same homologous and complementary primers as in the second PCR in the nested PCR. Here, the relationship with the template in the second NASBA is that the one used as the homologous primer in the second PCR is used as the complementary primer, and the one used as the complementary primer is used as the homologous primer. You should be careful. The complementary primer used here (this is a complementary primer in relation to the template (antisense strand) in the second NASBA, and a homologous primer in relation to the genomic RNA (sense strand) of ACLSV) Are T7R at the 5 'end.
A promoter sequence for NA polymerase has been added.

When the dual nested NASBA method is performed, two NASBAs are used in each of the first and second NASBAs.
The procedure can be carried out in the same manner as in the above-mentioned ordinary nested NASBA method, except that the homologous primer and the complementary primer are used for each book. As the homologous primer and the complementary primer to be used, those described for the above dual nested PCR can be similarly used. Here, in relation to the template in the second NASBA, the second PCR
It should be noted that in, the one used as a homologous primer is used as a complementary primer, and conversely, the one used as a complementary primer is used as a homologous primer. The complementary primer used here (this is a complementary primer in relation to the template (antisense strand) in the second NASBA, and a homologous primer in relation to the genomic RNA (sense strand) of ACLSV) All have a promoter sequence of T7 RNA polymerase added to the 5 'end.

(3) Detection of Amplification Product The amplification product can be detected by ethidium bromide staining or the like after the amplification reaction solution is separated by agarose or polyacrylamide gel electrophoresis. In this case, it can be determined from the molecular weight of the amplified fragment expected by electrophoresis whether or not the amplified fragment is derived from the ACLSV genome. However, it is preferable to confirm the specificity not only by the molecular weight of the amplified fragment but also by hybridization with the cDNA probe to the amplified product. Examples of the method include microplate hybridization, in addition to ordinary dot blot hybridization. Furthermore, PCR / RT-P, which can perform specificity test by hybridization simultaneously with PCR reaction
The CR tack man method (Perkin Elmer) can be used as a known technique.

2. Primer set of the present invention The present invention further relates to a primer set that can be used in the above-described virus detection method of the present invention. Each primer included in the primer set can be designed as described above.

When the above-mentioned primer set is to be used for an ordinary nested amplification method, for example, nested PCR, nested NASBA, etc., it contains two homologous primers and two complementary primers. Such a primer set is preferably SEQ ID NO: 1
Two kinds of homologous primers selected from the group consisting of oligonucleotides containing the base sequence represented by No. 4 and two kinds of complementary primers selected from the group consisting of oligonucleotides containing the base sequence represented by SEQ ID Nos. 5 to 8 And more preferably an oligonucleotide containing the base sequence represented by SEQ ID NOS: 3 to 6.

When the above primer set is used for a dual nested amplification method, for example, dual nested PCR, dual nested NASBA, etc., four homologous primers and four complementary primers are used. It contains a primer, and preferably contains an oligonucleotide containing the base sequence represented by SEQ ID NOS: 1 to 8.

When the above-mentioned primer set is for use in NASBA, a complementary primer for use in the first NASBA and a homologous primer for use in the second NASBA (this is a second primer) The primers are complementary primers in relation to the NASBA template (antisense strand), but are homologous primers in relation to the ACLSV genomic RNA (sense strand).
It is preferable that a promoter sequence of NA polymerase is added.

Further, reagents other than the primers used in the virus detection method of the present invention can be added to the primer set of the present invention to prepare a kit for detecting ACLSV. Such reagents include, but are not limited to, RNA extraction reagents, PCR reagents or NASBA reagents, and electrophoresis reagents. Examples of the RNA extraction reagent include a buffer for grinding, guanidine thiocyanate, phenol, β-mercaptoethanol, and S
DS, chloroform and the like. As the PCR reagent, for example, Tris-HCl, KCl, MgCl2, various dNT
P, Taq DNA polymerase, water and the like. The NAS
As the reagent for BA, for example, AMV-RT, RNaseH, T7RN
A polymerase, BSA (bovine serum albumin), nucleotide mixture, DTT, MgCl2, 72% DMSO, 2M KCl, water and the like. Examples of the electrophoresis reagent include agarose or polyacrylamide gel, a loading buffer, and an ethidium bromide staining reagent.

[0060]

The present invention will be described more specifically with reference to the following examples. However, these examples are for explanation, and do not limit the technical scope of the present invention. [Preparation Example 1] Preparation of RNA sample from apple tree Young leaves of May and bark of February were collected from five strains of ACLSV-poisoning apple trees (stored at the apple branch disease laboratory in the Fruit Tree Experimental Station, Ministry of Agriculture, Forestry and Fisheries). And stored in a refrigerator at -80 ° C. 100 mg of each of these stored samples was frozen in liquid nitrogen and RNeasy Pl
Total RNA was extracted with ant Mini Kit (Qiagen). The obtained extract was used as an RNA sample.

Example 1 Design and Synthesis of Primers ACLSV Isolates with Different Pathogenicity to Marubakaidou
Strains (preserved in the Department of Agriculture, Forestry and Fisheries Fruit Tree Research Station Apple Branch Disease Laboratory), ie, P195, P195L, PK32, PK51, P51
142, P143, P202, PK5, P125, MK1, MK8
The nucleotide sequences of the genomic RNAs of the strains, MK9 strain, MO31 strain, MO41 strain and B81 strain were analyzed.

It was isolated from apples, plums and sweet cherries, for which the entire base sequence of the genomic RNA had already been determined.
In the three ACLSV isolates, the part showing the homologous sequence was AC
Considering that LSV is highly conserved, several oligonucleotide primers were designed based on this part and synthesized according to a conventional method. Petals or young leaves at the same time with a relatively high ACLSV concentration were collected from apple trees poisoning the respective isolates, and the total RN was determined according to the method described in Preparation Example 1.
A was extracted. When reverse transcription and PCR were performed using the above synthetic oligonucleotide primers, a homologous primer: 5′-GAAGATCGCAGAAGGGGATATTC-3 ′ (SEQ ID NO: 9) and a complementary primer: 5′-GTCTACAGGCTATTTATTATA
In the combination of AG-3 ′ (SEQ ID NO: 10), a cDNA fragment of about 1800 bases at the 3 ′ end in the genome of all isolates was able to be amplified.

Next, the nucleotide sequence of this cDNA fragment was analyzed. First, a sequencing reaction was performed using the above-mentioned primers used for amplification and the cDNA fragment as a template to determine the nucleotide sequence. Further, a new primer was synthesized based on the determined sequence, and the nucleotide sequence was determined by the same sequencing reaction as described above. This was repeated to determine almost the entire base sequence of the cDNA fragment.
Since there is an undetermined region inside these cDNA fragments, the base sequence of each cDNA fragment is divided into the first half and the second half, and SEQ ID NO: 13 (first half of P195 strain) and SEQ ID NO: 14 (second half of P195 strain) , SEQ ID NO: 15 (first half of P195L strain),
SEQ ID NO: 16 (second half of P195L strain), SEQ ID NO: 17 (first half of PK32 strain), SEQ ID NO: 18 (second half of PK32 strain), SEQ ID NO: 19
(First half of PK51 strain), SEQ ID NO: 20 (second half of PK51 strain), SEQ ID NO: 21 (first half of P142 strain), SEQ ID NO: 22 (second half of P142 strain),
SEQ ID NO: 23 (first half of P143 strain), SEQ ID NO: 24 (second half of P143 strain), SEQ ID NO: 25 (first half of MK8 strain), SEQ ID NO: 26 (MK8
Strain late), SEQ ID NO: 27 (first half of B81 strain), SEQ ID NO: 28
(The latter half of B81 strain), SEQ ID NO: 29 (first half of P202 strain), SEQ ID NO: 30 (second half of P202 strain), SEQ ID NO: 31 (first half of PK5 strain),
SEQ ID NO: 32 (second half of PK5 strain), SEQ ID NO: 33 (first half of PI25 strain), SEQ ID NO: 34 (second half of PI25 strain), SEQ ID NO: 35 (MK
The first half of one strain), SEQ ID NO: 36 (second half of MK1 strain), SEQ ID NO: 37
(First half of MO31 strain), SEQ ID NO: 38 (second half of MO31 strain), SEQ ID NO: 39 (first half of MO41 strain), SEQ ID NO: 40 (second half of MO41 strain),
This is shown in SEQ ID NO: 41 (first half of MK9 strain) and SEQ ID NO: 42 (second half of MK9 strain).

The nucleotide sequence of the cDNA fragment derived from the above 15 isolates and the nucleotide sequence of the cDNA fragment derived from the P205 strain (first half: SEQ ID NO: 11; second half: SEQ ID NO: 12, GenBank registration number: D
14996), which is shown in FIGS. In the upper part of FIG. 1, the name of each isolate is shown on the left side of each base sequence. Based on these figures, the cDN of all isolates
An oligonucleotide having a base sequence represented by SEQ ID NOS: 1 to 8 that can be used for amplification of A is designed,
It was synthesized according to a conventional method. SEQ ID NOS: 1 to 4 are ACLS
These are homologous primers designed in order from the 5 'upstream region to the downstream region on the V genome. Similarly, SEQ ID NOS: 5 to 8 are complementary primers similarly designed in order from the 5 'upstream region to the downstream region.

Example 2 Detection by Nested PCR cDNA was synthesized from the RNA sample obtained in Preparation Example 1,
The cDNA was amplified by nested PCR. Nested PCR was performed as follows. First, reverse transcription and first PCR were performed using an RNA PCR kit (Gene Amp EZ rTth RNA PCR kit, PerkinElmer). The PCR reaction solution was prepared by adding about 50 ng of an RNA sample as a template in a 5 μl system, and using oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 3 and 6 as primers to give final concentrations, respectively.
It was prepared by adding to 10 μM. Subsequent reactions were performed according to the kit instructions. The temperature condition of the amplification reaction is 6
30 minutes at 0 ° C., 1 minute at 94 ° C., followed by 1 minute at 94 ° C.
40 cycles of 2 minutes at -60 ° C for 5 seconds, then 60 cycles
C. for 7 minutes. The second PCR was performed using a heat-resistant DNA polymerase (TaKaRaEx Taq, manufactured by Takara Shuzo). Next, according to the instruction manual, a reaction solution 9 containing oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 4 and 5 as primers to a final concentration of 10 μM, respectively.
5 μl was added to the solution subjected to the reverse transcription and the first PCR, and the second PCR was carried out by a conventional method. The temperature condition of the amplification reaction was 94 ° C. for 2 minutes, followed by 94 ° C. for 30 minutes.
35 cycles of −60 ° C. for 30 seconds and −72 ° C. for 2 minutes for 35 seconds, followed by 72 ° C. for 7 minutes.

The cDNA of ACLSV was detected by agarose gel electrophoresis of the obtained amplification product (FIG. 6, panel B). In panel B of FIG. 6, ACLSV was detected from all infected tissues, not from healthy (MO38), and it was confirmed that ACLSV could be accurately diagnosed.

Example 3 Detection by Dual Nested PCR cDNA was synthesized from the RNA sample obtained in Preparation Example 1,
The cDNA was amplified by dual nested PCR.
Dual nested PCR was performed as follows. First, reverse transcription and PCR were performed in the same manner as in Example 2.
However, oligonucleotides having the nucleotide sequences represented by SEQ ID NOS: 1 and 3, and 6 and 8 were added to the reaction solution as primers. Next, the second PCR was performed in Example 2.
The same was done. However, SEQ ID NOs: 2 and 4, and 5
Oligonucleotides having the nucleotide sequences represented by and 7 were added to the reaction solution as primers.

The cDNA of ACLSV was detected by agarose gel electrophoresis of the obtained amplification product (FIG. 6, panel C). In panel C of FIG. 6, ACLSV was detected from all infected tissues, not from healthy (MO38), and it was confirmed that ACLSV could be diagnosed accurately.
Also, comparing this result with Example 2 (panel B),
In the dual nested PCR, the variation in the staining image of the cDNA with ethidium bromide between samples was smaller than that in the nested PCR, and it was considered that the cDNA was stably amplified.

Comparative Example 1 Detection by Normal PCR cDNA was synthesized from the RNA sample obtained in Preparation Example 1,
The cDNA was amplified by normal PCR. Normal PCR
The above amplification using the RNA PCR kit (Gene Amp EZ rTth
RNA PCR kit, PerkinElmer). P
The CR reaction solution was prepared by adding about 250 ng of an RNA sample as a template in a 20 μl system, and adding oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 4 and 5 as primers to a final concentration of 10 μM each. Subsequent reactions were performed according to the kit instructions. The temperature conditions for the amplification reaction were 60 ° C. for 30 minutes, 94 ° C. for 1 minute,
C. for 15 seconds at -60.degree. C. for 2 minutes for 40 cycles, followed by 60.degree. C. for 7 minutes.

The cDNA of ACLSV was detected by agarose gel electrophoresis of the obtained amplification product (FIG. 6, panel A). In panel A of FIG.
V was partially detected in the young leaves of May, which had a high concentration in the apple tree tissue, but could not be detected at all in the bark of February.

[0071]

According to the present invention, a plurality of ACLSV isolates can be detected simultaneously. Therefore, according to the present invention, it is possible to accurately diagnose an ACLSV infection of an apple tree at any time, and thereby, it is possible to supply an ACLSV-uninfected apple seedling.

[0072]

[Sequence List] SEQUENCE LISTING <110> Director-General of National Institute of Fruit Tree Science, Ministry of Agriculture, Forestry and Fisheries <120> Methods for Detecting Apple Chlorotic Leaf Spot Virus and Primers used therein <130> P00-0008 <160 > 42 <170> PatentIn Ver. 2.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <220> <221> degenerate <222> (18 ) <223> n is a, t, c or g <220> <221> degenerate <222> (21) <223> n is a, t, c or g <400> 1 tcvaaytcva thwgbgtngc ngtvatgtt 29 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <220> <221> degenerate <222> (7) <223> n is a, t, c or g <220> <221> degenerate <222> (13) <223> n is a, t, c or g <400> 2 mgaygtnagr gtnggbmaya tgtg 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <220> <221> degenerate <222> (6) <223> n is a, t, c or g <400> 3 artccnytdh ttcagargg g tcatc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <220> <221> degenerate <222> (6) <223> n is a, t, c or g <400> 4 thtctncaag rgartttcar tttgc 25 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 5 atcgctatgt tcgcgaagat gg 22 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <220> <221> degenerate <222> (3) <223> n is a , t, c or g <220> <221> degenerate <222> (9) <223> n is a, t, c or g <400> 6 ganyybayna cyttykgvtc ctccat 26 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 7 gytdatrtty ggrtcyraag adgtagt 27 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 8 gcytcacava cytgrcggaa rgtcat 26 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artific ial Sequence: Primer <400> 9 gaagatcgca gaaggggata ttc 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 10 gtctacaggc tatttattat aag 23 < 210> 11 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <400> 11 gagagagtac caactacgtg cattggggag cactatcaat atccattgac gcactgttca 60 gaaagaatgc cggggtttca gggtggtgtt atgtgtacga caacagatgg gaaacttttg 120 agcaggcgat gctacaaaag tttcggttca acctggatag tggctcggcc acattggtga 180 cctcaccgaa ttttccagtt tcattggatg atcctggttt gtccaattcg atcagtgtag 240 cggtgatgtt tgagaatctg aacttcaagc ttgaaagcta ccctataagt gttagggtag 300 ggaacatgtg tagattcttc gacagcttct tgagttgcgt caagaataaa gtggactcca 360 actttttgct tgaggccgct aatgcagatc cgcttggcgc gggtgctttc gggtttgaac 420 aggacgatca ggt 433 <210> 12 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 12 ggcaaaggaa aagctggaac tgaatccatt acttcagaag ggtcatcctt cgataacatt 60 tctgcaagag agtttcagtt tgctagacaa gatcagaagg cgaaggatgg cggcagtgct 12 0 gaacctccaa ttaaaagtgg acgcagatct gaaagcgttc ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caattctgga acagacactg gaggccatct tcgcgaacat 240 agcgatacag gggacctcgg aacaaacaga gttcctggac gtgctggtgg aggtgaaatc 300 catggaggat cagaaggtgg tggggtcatt caatctgaag gaggtagtcg gtttgatcaa 360 aatattcagg actacatctt cggacccgaa tataagcagc atgaccttcc gccaagtgtg 420 tgaggcattt gcccc 435 <210> 13 <211> 434 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (11) <220> <221> unsure <222> (44) <220> <221> unsure <222> (53) <220 > <221> unsure <222> (381) <400> 13 grgaragcac naaytaygtg caytggggrg crytgtcaat atcnatwgay gcnytgttya 60 graaraaygc wggrgtbtcm gghtggtgyt aygtgtayga yawyagrtgg gagacdtttg 120 agcargcmat gctrcagaaa ttycgdttca ayytvgayag tggytcrgch acrttggtga 180 chtcwccaaa ytttccdgtt tcaytrgatg atccaggyct gtcvaattca atcagygtrg 240 cagtvatgtt ygagaaycts aayttcaarc twgagagyta cccaatcagt gtsagrgtrg 300 gbaacatgtg cagrttctty gayagcttct tgagcagtgt gaagaayaar gttgattcma 360 ayttyactry tdgargcygc naaygcdgwy cckytkgghg ydggwgcytt yggatttgag 420 caggaygacc aggt 434 <210> 14 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 14 ggaaaaggda argctggamc haaatccvtt rcttcagarg ggtcatccrt kgataacatw 60 tctscaagag artttcagtt tgctmgacar grtmaggmga aggagratgg cagcagtkct 120 gaayytwcag ctaaargtgg acgcagatct gaargcgttc ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caatcctgga acagacactg gagtccatct tcgcgaacat 240 agcratmcag ggaacgtcgg agcagacgga rttcctggay ctrrtsgtgg aggtgaartc 300 gatggaggay cagaaggtca tmgggtcctw caatytgaag gaggtggtca ayatgatcaa 360 agcwttcaar actacctctt cggatccgaa catcagcaac atgactttcc gccaggtgtg 420 tgaggctttc gcacc 435 <210> 15 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (11) <220> <221> unsure <222> (44) <220> <221> unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <222> (398) <400> 15 grgadagcac naaytaygtg cattggggrg cdytgtcaat atcnatwgay gcnytgttya 60 graaramygc hggrgtbtch ggntggtgyt acgtgt ayga yaayagrtgg garacktttg 120 agcargchat gctrcagaaa ttycgdttca ayytsgayag yggytcrgcm acrttggtga 180 chtchccraa ytttccdgtt tcaytrgayg atccaggyct gtcvaattcr atyagygtgg 240 cagtsatgtt ygagaayctb aayttcaarc thgagagyta cccaatcagt gtsagrgtrg 300 gbaacatgtg cagrttctty gayagcttct tgagywgtgt gaagaayaar gttgattcma 360 ayttthtrct kgargcygcb aatgcagayc cdttgggngc dggmgcytty ggatttgagc 420 aggaygayca ggt 433 <210> 16 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 16 ggmaaaggra argctggamc maartccrtt rmttcagarg ggtcatccdt cgataacatw 60 tctscaagag artttcagtt tgctmgacaa rrtmaggmga aggagratgg crgcrrttct 120 gaayytwcag ctaaargtgg acgcagatct gaargcrtty ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caaycytgga acagacactg gagtccatct tcgcgaacat 240 agcratycag ggracrtcgg agcaracgga rttcctggay ctrrtggtgg argtgaartc 300 vatggaggay cagaaggtca tvgggtcmta caatytgaag gaggtggtca ayatgatcaa 360 agcattcaar actacctctt crgatccgaa catcagcarc atgactttcc gycaggtgtg 420 tgaggcttty gcacc 435 <210> 17 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <222> ( 128) <220> <221> unsure <222> (146) <220> <221> unsure <222> (185) <220> <221> unsure <222> (299) <220> <221> unsure <222 > (302) <400> 17 grgarrgyac saaytaygtg cattggggrg cwytgtcrat atcmattgay gcnytkttya 60 graaraatgc yggdgtbtcw ggntggtgyt aygtstayga yaayagrtgg garacdtttg 120 agcargcnat gytrcaraar ttymgnttca ayytggayag tggbtcrgcc acrttggtga 180 cytcnccraa ytttccdgtb tcrytrgatg ayccdggyyt ktcmaattcr atyagygtrg 240 cvgtvatgtt tgagaayyts aayttyaagc twgaragyta cccmatmagt gtbagrgtng 300 gnaayatgtg ymgrttctty gayagcttct tgagcagygt cargaayaar gttgaytcca 360 acttyctgct tgargctgch aatgcwgayc crytggghgc aggggcyttc ggrtttgarc 420 aggatgayca rgt 433 <210> 18 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 18 ggvaaargmr argctggavc haaatccatt gatcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tg saayctdcar ctaaargtrg acgcagatyt gaargcrttc ctggccgcrg aaggcagacc 180 ccttcatgga aagacagggg caatcctgga acagayrctg gagkccatct tcgcgaacat 240 agcgattcag ggaacstcag agcagacgga gtttctggay ctavtggtgg aggtgaartc 300 matggaggab cagaaggtgr tcgggtcctw caatctgaag gaggtggtca aywtgrtcaa 360 rgccttcaag actacmtctt cggayccraa catmagcaac atgacyttcc gccaggtgtg 420 tgaggccttt ygcac 435 <210> 19 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <222> (140) <220> <221> unsure <222> (146) <220> <221> unsure <222> (242) <220> <221> unsure <222> (302) <220> <221> unsure <222> (380) < 220> <221> unsure <222> (392) <400> 19 grgarrgyac saaytaygtg cattggggrg caytgtcrat atcmattgay gcnytkttya 60 graagaatgc yggrgtytch ggntggtgyt aygtrtayga caacagatgg garacdtttg 120 arcargcvat gytrcaraan ttycgnttca ayytdgayag yggbtcrgcc acrttggtga 180 cytcwccraa ytttccdgtb tcrytrgatg ayccdggyyt ktcmaaytcr atyagygtrg 240 cngtratgtt ygagaayctb aayttc aagc twgaragyta ccchatmagt gtbagrgtdg 300 gnaayatgtg yagrttctty gacagyttyt tgagyagygt cargaayaar gtkgaytcca 360 ayttyytgct kgargcygcn aatgcwgayc cnytggghgc aggdgcttty ggrt220 <gt> <br> <br> 420 <gt> 222> (285) <220> <221> unsure <222> (323) <400> 20 ggaaaaggca argytggacc aaaatccvtt gcttcagagg ggtcatccrt ggayaacatm 60 tctgcaagag agtttcagtt tgctcgacaa gatcaggmga argaggatgg cagcrgttct 120 saatctacag ctaaargtgg acgcagatct gaaagcgttc ctgcccgcag aaggcagacc 180 ccttcatgga aagacagggg caathctgga acagayactg gaggccatct tcgcgaacat 240 agcratwcar ggracstyrg arcaramgga gttcctggay ckkrnrgtgg argtgaartc 300 matggaggay cmvaargktg atngggtcmt wcaatctgaa ggaggtggkk caayhtratc 360 aarrymttca rgactacmtc ttyggaycc rt gg gt gtc gtc gtg gtc gtc gtg gtc gtc gtg gtc gtc gtg gtc gtc gtg gtc gtg gtc gtc gtg gtc gtc gtc 420g unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <22 2> (200) <220> <221> unsure <222> (284) <220> <221> unsure <222> (299) <220> <221> unsure <222> (302) <220> <221> unsure <222> (380) <400> 21 grgagrgyac vaaytaygtg cattggggrg cwytgtcrat atcmattgay gcnytkttya 60 graaraatgc hggrgtbtcw ggntggtgyt aygtrtayga yaacagrtgg garacdtttg 120 arcargcvat gytrcaraar ttycggttca ayytggayag tggbtcrgcc acrttggtga 180 cytcwccraa ytttccrgtn tcrytrgatg ayccdggyyt ktcmaattcr atyagygtrg 240 cvgtgatgtt tgagaaycts aacttcaagc twgaaagyta cccnatmagt gtbagrgtng 300 gnaayatgtg yagrttctty gacagcttct tgagcagygt caagaayaaa gtkgaytcma 360 ayttyctgyt ggargcygcn aatgcwgayc crytggghgc agggagcttt cggatttgar 420 caggaygayc argt 434 <210> 22 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 22 ggmaaaggma argctggacc haaatccatt rcttcagarg ggtcatccrt kgataacatw 60 tctgcaagag artttcagtt tgctmgacar raymaggmga mgragratgg cagcagttct 120 gaayytrcar ctaaargtgg acgcagatct gaargcrtyc ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caatyctgga acagacactg gagkccatct tcgcgaaca t 240 agcgatmcag gggacctcgg agcagacsga gttcctggav ctgatggtgg aggtgaartc 300 catggaggay cagaaggtka tcgggtcmtw caatctgaag raggtggtca ayatgatcaa 360 agycttcaag actacmtctt tggayc220 gatacctca tggaytccag tcagtcag tcagtcag gtcag tcagtcag 221> unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <222> (128) <220> <221> unsure <222> (215) <220 > <221> unsure <222> (284) <220> <221> unsure <222> (293) <220> <221> unsure <222> (299) <220> <221> unsure <222> (302) <220> <221> unsure <222> (380) <220> <221> unsure <222> (392) <400> 180 cbtcwccraa ytttccdgtb tcrytrgatg ayccnggyyt gtcvaattcr atyagygtrg 240 cvgtratgtt ygagaayctb aayttyaagc twgaragyta yccnatmagy gtnagrgtng 300 gnaayatgtg yagrttctty gayagctt yy tgagyagygt cargaayaar gtkgaytcca 360 ayttyytgct dgargcygcn aatgcdgayc cnytgtgghg caggrtgctt tyggrtttga 420 rcaggaygay cargt 435 <210> 24 <211> 436 <212> DNA <213> Apple chlorotic leaf spot virus <400> 24 ggvaaaggca argctggacc haaatccatt rcttcagagg ggtcatccrt kgayaacath 60 tctgcaagag agtttcagtt tgctcgacaa rrtcaggmga aggagratgg crgcagttct 120 saayytdcar ctaaargtgg acgcagatyt gaaagcgttc ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caathctgga acagayaytg gagkccatct tcgcgaacat 240 agcratmcar gggacstcrg arcagackga gttcctggay gtgayggtgg aggtcaagtc 300 aatggaggac cagaaggtga tmggstcwtw caatctgaag gaggtggtcv ayhtratmaa 360 rrymttcarr gactacmtct tyggayccga ayatharcav catgacyttc cgccargtgt 420 gtgaggcwtt ygcmcc 436 <210> 25 <211> 434 < 212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (53) <220> <221> unsure <222> (83) <220> <221> unsure <222> (128) <220> <221> unsure <222> (303) <400> 25 gagarrgyac saaytaygtg cattggggrg cwytgtcrat atcmattgay gcnytgttya 60 graaraaygc hggr gtbtcw ggntggtgyt aygtrtayga caacagatgg garacdtttg 120 agcargcnat gytrcaraar ttycgrttca aycyggayag yggbtcrgcc acrttggtga 180 cbtcwccraa ytttccdgtb tcdhtrgatg aycmdggyyt tgtcvaaytc ratyagygtr 240 gcvgtvatgt ttgagaayct saacttcaag ctwgaragyt accchatmag tgtbagrgtr 300 ggnaayatgt gyagrttctt ygacagctty ttgagyagtg tcaagaayaa rgttgaytcc 360 aacttyctgc tkgargctgc haatgcwgay ccryttgghg cdggdgcytt yggrtttgar 420 caggatgayc argt 434 <210> 26 <211 > 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 26 ggaaaaggca aagctggacc haaatcchtt gmttcagarg ggtcatccgt ggayaacata 60 tctgcaagag agtttcagtt tgctcgacaa aatcaggaga aggagaatgg cggcagtrct 120 caatcttcag ctaaaggtgg acgcagatct gaaagcrttc ctggccgyag aaggcagacc 180 ccttcatgga aagacagggg caathctgga acagacactg gaggccatct tcgcgaacat 240 agcgatccaa gggacctcgg arcagacgga gttyctggac ctrayggtgg argtsaagtc 300 matggaggay cmgaargkva tvggvtcmtw caatctgaag gaggtrgtca atatratcaa 360 rrymttcarg actacatctt cggatccgaa yataarcaac atgacyttcc gccargtktg 420 tgaggcttt t gcccc 435 <210> 27 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <400> 27 gagaaagcac aaattacgtg cactggggag cattgtccat atcaattgac gctcttttca 60 gaaagaacgc aggagtttcc ggctggtgct acgtgtatga caacaggtgg gaaacttttg 120 agcaggccat gctgcagaaa tttcgtttca atttagacag tggttcagcc acattggtga 180 cttctccaaa ctttcctgtt tcactggatg acccaggtct ttcaaattca ataagcgtag 240 cggtaatgtt cgaaaatctc aatttcaagc ttgagagtta cccaattagt gtgagagtgg 300 gcaatatgtg cagattcttt gatagtttct tgagttgtgt gaagaataaa gtggactcaa 360 atttcctact tgaggccgcc aacgcagatc ctcttggtgc aggagctttt gggtttgaac 420 aggatgatca ggt 433 <210> 28 <211> 442 <212> DNA <213> Apple chlorotic leaf spot virus < 220> <221> unsure <222> (64) <400> 28 aggaaaagaa aagctggaac cgaatccatt kcttcagagg ggtcatccat cgataacatt 60 tctncaagag aatttcagtt tgctagacag gatcagaagg cgaaggagga tggcrrcrgt 120 kctraacytb carytaaarg tggacgcaga tctgaaagcg ttcctggccg cagaaggcag 180 accccttcat ggaaagacag gggcaatcct ggaacagaya ctggagkcca tcttcgcgaa 240 catagcgatw carggrac st cggagcarac rgagttcytg gacstrstgg trgargtgaa 300 rtcmatggag gatcagaarg tgrtrggrtc stwcaatctg aargaagtgg tcaacttaat 360 ratcaaagmt attcaagact acatcttcgg ayccgaacat magcaayatg acyttccgyc 420 aggtgtgtga rgcwttcgcw cc 442 <210> 29 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <400> 29 gagagagtac caactacgtg cactggggag cactatcaat atccattgac gcactgttca 60 gaaagaatgc cggggtttca gggtggtgtt atgtgtacga camcagatgg gaaacttttg 120 agcaggcgat gctacaaaag tttcggttca acctggatag tggctcggcc acattggtga 180 cctcaccaaa ttttccagtt tcattggatg atcctggttt gtccaattcg atcagtgtas 240 cggtgatgtt tgagaatctg aacttcaagc ttgaaagcta ccctataagt gttagggtag 300 ggaacatgtg tagattcttc gacagcttct tgagttgcgt caagaataaa gtggactcca 360 actttttgct tgaggccgct aatgcagatc cgcttggcgc gggtgctttc gggtttgaac 420 aggacgatca ggt 433 <210> 30 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 30 ggcaaaggaa aagctggaac tgaatccatt acttcagaag ggtcatcctt cgataacatt 60 tctgcaagag agtttcagtt tgctagacaa gatcagaagg cgaagga tgg cggcagtgct 120 gaacctccaa ctaaaagtgg acgcagatct gaaagcgttc ctggccgcag aaggcagacc 180 ccttcatgga aagacagggg caattctgga acagacactg gaggccatct tcgcgaacat 240 agcgatacag gggacctcgg aacaaacaga gttcctggac gtgctggtgg aggtgaaatc 300 catggaggat cagaaggtgg tggggtcatt caatctgaag gaggtagtcg gtttgatcaa 360 aatattcagg actacatctt cggacccgaa tataagcagc atgaccttcc gccaagtgtg 420 tgaggcattt gcccc 435 <210> 31 <211> 433 < 212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (164) <220> <221> unsure <222> (197) <220> <221> unsure <222> (299) <400> 31 grgagagcac waaytaygtg caytggggrg caytrtcwat atcmatwgay gcdttgttca 60 graaraatgc wggdgtttcm ggwtggtgyt aygtrtmyga caacagrkgg gagacrttyg 120 agcargcvat gctrcagaar ttymrwttya ayctkgacag tggntcvgcv acattggkra 180 cktcwccaaa yttyccngtt tcattggayg atccaggycy ytcsaattca atyagbgtkg 240 cwgtsatgtt tgagaaycts aayttcaagc tkgaragtta ycchatmagt gtdagggtng 300 gyaacatgtg caggttctty gayagyttcy tgagyagtgt baagaayarr gtdgattcma 360 acttyytgyt rgaagctgcm aatgcrgacc ctttaggagy aggagctttt gggtttgagc 420 aggaygatca rgt 433 <210> 32 <211> 437 <212> DNA <213> Apple chlorotic leaf spot virus <400> 32 ggsaaaggva argctggamc haaatccvtt rcttcagrgg ggtcatccrt kcgataacat 60 wtctscaagg agaatttcag tttgctcgac arartmaggv gaargagrat ggcagcgrtw 120 ctgaayytwc agctaaargt ggacgcagat ctgaaggcrt tcctggccgc agaaggcaga 180 ccccttcatg gaaagacagg ggcaatcctg gaacagacac tggagkccat cttcgcgaac 240 atagcgatcc aaggracstc rgagcagacg garttyctgg acctrrtggt ggaggtgaar 300 tcvatggagg aycagaaggt sataggstcm twcaatctga aggaggtggt caatatgatc 360 aarrymttca agactacmtc ttcggatccg aayatmagca acatgacytt ccgycaggtg 420 tgtgaggcyt tcgcacc 437 <210> 33 <211> 434 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (11) <220> <221> unsure <222> (83) <220> <221> unsure <222> (84) <400> 33 gggaaagtac naattatgts cattggggag cattgtcgat atccattgac gcdttgttca 60 ggaaaaatgc tggggtatcg ggnnggtgtt acgtgtatga taacaggtgg gagacrtttg 120 agcaggccat gctgcaaaaa tttcgattca atttrga cag tggytcrgcm ackttggtga 180 cwtcwccaaa ttttccggts kcattggatg atccaggttt gtccaattca atyagcgtgg 240 ccgtratgtt tgagaayctc aatttcaagc ttgagagtta ccctataagc gtcaggggtg 300 ggtaacatgt gyaggttctt tgacagcttc cttagcagtg tgaaaaacaa ggttgaytcw 360 aaytttctgc twgaggctgc aaatgcagac cctttgggag cmggggcttt tggrtttgar 420 cargatgacc aggt 434 <210> 34 <211> 437 <212> DNA <213> Apple chlorotic leaf spot virus <220> <221> unsure <222> (86) <400> 34 ggaaaaggwa ragctggacc maaaatccmt tgyttcagar ggvtcaycyr tkgataacat 60 htctscaaga gartttcagt wtgctngmca wgmwcaggmg aaggaggatg gcagcagkbc 120 tvaacytgca rctaaaagtg gacgcagatc tgaaagcgty cctggccgca gaaggcagac 180 cccttcatgg aaagacaggg gcaathctgg aacagayact ggagkccatc ttcgcgaaca 240 tagcgatwca rggracstcg garcaracrg arttcctsga ybtgvyrgtg gaggtsaart 300 cmatggagga ycagaaggtg rtvggstcmt wcaatctgaa ggaggtrgtc rrywtgatsa 360 arryhttcar gaactacmtc <ttcggaycg aayatmarcagcc aayatmarcagcg aayatmarcagcg ttcggayccg aayatmarcagcc ag eaf spot virus <400> 35 gagagaacac taattacaty cattgggggg ctctttcrat atcaatagat gcattgttca 60 agaaaaatgc tggtgtttcw ggrtggtgyt atgtrtatga caaccggtgg garacttttg 120 agcargcsag gytrcaaaar ttymgkttya ayttggayag tggwtcmgcc acwttggtga 180 cytcwccraa cttccctgtt tctcttgatg atccwggact ttcaaattcc atttgygttg 240 crgtcatgtt cgagaatctr aayttcaart tggacagcta yccyattagy gtyagggtag 300 gaactatgtg caggttctty gacagctttc tvagyaahgt taagaayaag agcgattcca 360 acttccttct agaggcttcr aatgcwgayc cattgggtgc aagcgccttt gggtttgatg 420 ctgacgatca rgt 433 <210> 36 <211> 436 <212> DNA <213> Apple chlorotic leaf spot virus <400> 36 gggaaaggra argctggacc vaagtccatt gcttcagarg ggtcatcckt ggataacatt 60 tccrcaagag aatttcagtt tgctagacaa agtcaggcga aggaggatgg cggcagttct 120 aaatcttcar ytaaaggtgg acgcagatct gaaagcgttc ctrgccgcak aaggcagacc 180 ccttcatgga aagacagggg caatcctgga acagacaytt ggagtccatc ttcgcgaaca 240 tagcaatcca rgggacctcg garcagacgg agttcctgga yrtgayggtg gaagtgaagtagagtagagtagagtagagtcaagag 300cag atcggtrgtg gatcttatca 360 aggcattcaa gactacatct tcggacccga atataaacgg ratgaccttc cgccaggttt 420 gtgaggcctt tgcycc 436 <210> 37 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <400> 37 gagaaagcac aaattatgtg cattggggag ctttgtcaat atcaatagat gctttattca 60 ggaaaaatgc aggggtttcc ggttggtgct atgtatatga caacaggtgg gaaacatttg 120 agcaggcaat gctmcagaaa tttcgtttca acttggatag tggatcagcc acattggtga 180 cctccccaaa ctttcctgtc tcgttggacg atccaggcct ctcgaattca atcagcgtgg 240 cggtgatgtt tgagaatctg aacttcaaat tggaaagtta tccaattagt gtgagagtag 300 gcaacatgtg caggttcttt gacagcttct tgagcagtgt taagaacaaa gttgattcta 360 attttctact tgaagccgca aatgcagacc ctctgggagc aggagctttt gggtttgaac 420 aggatgatca agt 433 <210> 38 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 38 ggaaaaggaa aagctggaac caaatccatt gcttcagagg ggtcatccat tgataacatt 60 tctccaagag aatttcagtt tgctcgacaa gatcaggcga aggagaatgg cagcagttag 120c gaatttacag ctaaaagtgagcgagcagcagcgagcagcagcagcagcgagcagcagcgagcagcgagccagcagcgagcagcgagcgagcagcgagcagcgagcagcagcgagcagcagagcgagcagcagagcagcagagcagcagcgagcagcgagcagcagcagcagcgagcgagcagcagcagcgag agacagggg caatcctgga acagatactg gagtccatct tcgcgaacat 240 agcgatwcag ggcacgtcag agcagacaga attcctggat ctartggtgg aggtgaartc 300 matggaggat cagaaggtaa tcgggtctta caacctgaag gaggtggtca acatgatcaa 360 agchttcaag actacctctt cggatccgaa catcagcaac atgactttcc gycaggtgtg 420 tgaggcwtty gcmcc 435 <210> 39 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 39 grgagagyac maaytaygtg cactgkgggg agcaytrtcm atatcmattg aygcmctttt 60 caggaagaat gctggagttt cgggttggtg ttacgtatac gacaatagat gggaaacttt 120 cgagcaggcc atgctgcaaa agtttcgttt taatttggac agtggttcag ctacgttggt 180 gacctcccca aattttccag tttcattgga cgatccaggt ctttcaaatt ccataagcgt 240 ggcggtgatg ttcgagaacc tcaatttcaa gctagagagt tacccaatca gtgtgagggt 300 gggcaatatg tgcagrttct ttgacagctt cctaagtagt gtgaagaaca aagtggattc 360 caactttcta cttgaggctg ctaatgcwga tccgcttgga gcaggggtct ttggttttga 420 gcaggacgat caagt 435 <210> 40 <211> 438 <212> DNA <213> Apple chlorotic leaf spot virus <400> 40 ggcaagagca agactggagc aaaatccact tcttcagagg ggtc atcctt tgataacatt 60 tctacaagag agtttcagtt tgctagacaa gatcagaagg cgaaggcatg gcggcagtat 120 tgaatcttca attaaaggtg gacgcagtat ctgaaggcgt tcctggccgc agaaggcaga 180 ccccttcatg gaaagacagg ggcaattctg gaacagacac tggaggccat ctthgcgaac 240 atagcgatac aagggacctc ggagcaaacg gagttcctgg acgtactggt ggaggtgaaa 300 tccatggagg accagmaggt ggtggggtca ttcaattctg aaggaggtag tcaacttgat 360 caaaatattc aggactactt cttcggaccc gaacataagc aatatgacct tccgccaggt 420 gtgtgaagca tttgctcc 438 <210 > 41 <211> 433 <212> DNA <213> Apple chlorotic leaf spot virus <400> 41 gagagagtac gaactatgtg cattgggggg ctttatctat atcgattgac gccctcttca 60 ggaaaaatgc tggtgtgtct ggttggtgct acgtttatga caataggtgg gaaacttttg 120 aacaagctat gttgcagaag ttcaggttca atttggatag cggatccgcg acattggtca 180 cttcaccaaa ctttccagtg tcattggatg acccaggcct ttcaaattca ataagtgtgg 240 ccgtgatgtt cgagaactta aatttcaagc ttgagagcta tccgattagc gttagagtcg 300 gcaacatgtg ccggttcttt gatagtttct tgagcagtgt caagaacagg gtggattcaa 360 actttctgct ggaggcatca aatgctgaac cccttgg ggc tggagctttt ggatttgaac 420 aggacgatca agt 433 <210> 42 <211> 435 <212> DNA <213> Apple chlorotic leaf spot virus <400> 42 ggaaaaggga aagctggacc aaaatccatt acttcagagg ggtcatccgt tgataacatt 60 tctgcaagag agtttcagtt tgctcgacaa aatcaggcga aggaggatgg cagcagttct 120 gaatctgcaa ctaaaggtag acgcagattt gaaggctttc ctggccgcgg aaggcagacc 180 ccttcatgga aagacagggg caatcctgga acagatgttg gagtccatct tcgcgaacat 240 agcgatacag gggacgtcgg agcaaacgga gttcctggat ctggtggtgg aagtgaagtc 300 aatggaggac caaaaggtga tcgggtcata caacctgagg gaggtggtca atatgatcaa 360 ggccttcaag actacatctt cggacccaaa catcagcaac atgactttcc gccaggtgtg 420 tgaggccttc gcacc 435

[0073]

[Sequence List Free Text] [SEQ ID NO: 1] Primer.
N of the 18th base and the 21st base is a, t, c, or g. [SEQ ID NO: 2] Primer. N of base 7 and base 13
Is a, t, c, or g. [SEQ ID NO: 3] Primer. N of the sixth base is a, t,
c or g.

[SEQ ID NO: 4] Primer. 6th base n
Is a, t, c, or g. [SEQ ID NO: 5] Primer. [SEQ ID NO: 6] Primer. N of base 3 and base 9
Is a, t, c, or g. [SEQ ID NOS: 7 to 10] Primers.

[Brief description of the drawings]

FIG. 1 is a view showing alignment of cDNA fragments derived from various ACLSV isolates.

FIG. 2 is a view showing alignment of cDNA fragments derived from various ACLSV isolates.

FIG. 3 shows an alignment of cDNA fragments derived from various ACLSV isolates.

FIG. 4 is a view showing an alignment of cDNA fragments derived from various ACLSV isolates.

FIG. 5 is a view showing alignment of cDNA fragments derived from various ACLSV isolates.

FIG. 6 is an electrophoresis photograph showing detection of ACLSV by ordinary PCR, nested PCR, and dual nested PCR.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/569 C12R 1:92) // (C12N 15/09 ZNA (C12Q 1/68 A C12R 1:92) ) C12R 1:92) (C12Q 1/68 C12N 15/00 ZNAA C12R 1:92) C12R 1:92) (72) Inventor Koichi Suzaki No. 4, Akahira, Shimogurikawa-shi, Morioka-shi, Iwate Pref. No. F-term (Reference) 4B024 AA07 AA14 CA01 HA14 4B063 QA01 QA18 QA19 QQ04 QQ10 QQ52 QR32 QR38 QR62 QS14 QS25

Claims (19)

[Claims] (1) RN extracted from tissue of a test plant
Using A as a template and performing nucleic acid amplification using a first homologous primer and a first complementary primer capable of amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus, (Ii) using said first amplification product as a template, wherein said first amplification product is contained in said first partial region of genomic RNA of said plurality of isolates of apple chlorotic leaf spot virus; Performing a nucleic acid amplification using a second homologous primer capable of amplifying the partial region of No. 2 and a second complementary primer to obtain a second amplification product; whereby apple chlorotic leaf spot virus is obtained from the test plant. How to detect. 2. The method according to claim 2, wherein the plurality of isolates of the apple chlorotic leaf spot virus are P205, P195, P195L.
Strains, PK32, PK51, P142, P143, P202, PK5
The virus detection method according to claim 1, which is a strain, P125 strain, MK1, MK8, MK9, MO31, MO41 or B81 strain. 3. The first homologous primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 1, 2, or 3, and the second homologous primer is:
(I) when the first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 1, an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, 3 or 4; When the one homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3 or 4; or (ii)
i) when the first homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3,
An oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4. 4. The first complementary primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 6, 7, or 8, and the second complementary primer is (i.
v) when the first complementary primer is an oligonucleotide containing the base sequence represented by SEQ ID NO: 6,
An oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5; (v) when the first complementary primer is an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7, the nucleotide represented by SEQ ID NO: 5 or 6 An oligonucleotide comprising a base sequence represented by SEQ ID NO: 5, 6 or 7 when the first complementary primer is an oligonucleotide comprising a base sequence represented by SEQ ID NO: 8 nucleotide;
The virus detection method according to claim 1, wherein 5. The method according to claim 1, wherein the first homologous primer is SEQ ID NO: 3.
Wherein the second homologous primer is an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4, and the first homologous primer is an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4.
The virus detection method according to claim 1, wherein the complementary primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 6, and the second complementary primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 5. 6. The method for detecting a virus according to claim 1, wherein the test plant is an apple, peach, plum, sweet cherry or apricot. 7. The method according to claim 1, wherein the tissue is a petal, a leaf, or a bark. 8. A homologous primer and a complementary primer capable of amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; and (ii) a homologous primer of apple chlorotic leaf spot virus. The first of the genomic RNAs of a plurality of isolates
A primer set for detecting apple chlorotic leaf spot virus from a test plant, comprising: a homologous primer and a complementary primer capable of amplifying a second partial region contained in the partial region of 9. The plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain, P195L
Strains, PK32, PK51, P142, P143, P202, PK5
The primer set according to claim 8, which is a strain, P125 strain, MK1, MK8, MK9, MO31, MO41 or B81 strain. 10. A member selected from the group consisting of oligonucleotides containing the nucleotide sequences represented by SEQ ID NOs: 1 to 4.
9. The primer set according to claim 8, comprising two kinds of complementary primers selected from the group consisting of one kind of homologous primer and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NOS: 5 to 8. 11. The primer set according to claim 8, comprising an oligonucleotide containing a base sequence represented by SEQ ID NOS: 3 to 6. 12. (i) R extracted from the tissue of a test plant
Using NA as a template, the first of genomic RNA of multiple isolates of apple chlorotic leaf spot virus
And performing a nucleic acid amplification using first and second homologous primers capable of amplifying the second partial region and first and second complementary primers to obtain a first amplification product;
i) third and fourth homologous primers capable of amplifying third and fourth partial regions of genomic RNA of said plurality of isolates of apple chlorotic leaf spot virus using the first amplification product as a template; A method for detecting apple chlorotic leaf spot virus from the test plant by performing nucleic acid amplification using third and fourth complementary primers to obtain a second amplification product. 13. The plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain, P195 strain.
L, PK32, PK51, P142, P143, P202, PK5
13. The virus detection method according to claim 12, which is a strain, P125 strain, MK1, MK8, MK9, MO31, MO41 or B81 strain. 14. The first homologous primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 1, and the second homologous primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 3. Third
Is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, the fourth homologous primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4, and the first complementary primer is SEQ ID NO: 2. An oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 8, the second complementary primer is an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 6, and the third complementary primer is a nucleotide sequence represented by SEQ ID NO: 7 13. The method for detecting a virus according to claim 12, wherein the fourth complementary primer is an oligonucleotide containing a base sequence represented by SEQ ID NO: 5. 15. The virus detection method according to claim 12, wherein the test plant is an apple, peach, plum, sweet cherry or apricot. 16. The virus detection method according to claim 12, wherein the tissue is a petal, leaf, or bark. 17. (i) a homologous primer and a complementary primer capable of amplifying a first partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; (ii) a plurality of apple chlorotic leaf spot virus. A homologous primer and a complementary primer capable of amplifying a second partial region of the genomic RNA of the isolate; (iii)
A homologous primer and a complementary primer capable of amplifying a third partial region of genomic RNA of a plurality of isolates of apple chlorotic leaf spot virus; and (iv) a genomic RNA of said plurality of isolates of apple chlorotic leaf spot virus; A primer set for detecting apple chlorotic leaf spot virus from a test plant, comprising a homologous primer and a complementary primer capable of amplifying the fourth partial region. 18. The method according to claim 18, wherein the plurality of isolates of apple chlorotic leaf spot virus are P205 strain, P195 strain, P195 strain.
L, PK32, PK51, P142, P143, P202, PK5
The primer set according to claim 17, which is a strain, P125 strain, MK1, MK8, MK9, MO31, MO41 or B81 strain. 19. The primer set according to claim 17, comprising an oligonucleotide containing the base sequence represented by SEQ ID NOS: 1 to 8.