JP6436598B1 - Primer set for specifically amplifying nucleic acid derived from strawberry pathogenic virus and method for detecting strawberry pathogenic virus - Google Patents

Abstract

Provided is a primer set for specifically amplifying a nucleic acid derived from a strawberry pathogenic virus by the LAMP method, which makes it possible to provide a more sensitive method for assaying a strawberry pathogenic virus. A primer set for specifically amplifying a nucleic acid derived from one or more strawberry pathogenic viruses selected from SMYEV, SMoV and SVBV by the LAMP method, comprising: FIP primer, BIP primer, F3 primer , A primer set including any one or more of a set of B3 primers each of which is a polynucleotide represented by a specific sequence, and the above-described primer set, a nucleic acid extracted from a specimen using the primer set as a template by the LAMP method A method for detecting a strawberry pathogenic virus, comprising performing an amplification reaction. [Selection figure] None

Description

  The present invention relates to a primer set for specifically amplifying a nucleic acid derived from a strawberry pathogenic virus and a method for detecting a strawberry pathogenic virus.

  In Japan, strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMov), and strawberry vein banding virus (SVBV) have been confirmed as pathogenic viruses of strawberry. Even if each of these viruses is infected alone, the vigor of the seedlings is not different from that of healthy seedlings, and the disease symptoms do not appear clearly. However, co-infection reduces plant height and hatches the strain. Furthermore, since the strawberry fruit does not enlarge and becomes small and the waste fruit increases, the value of the fruit is remarkably lowered. When the parent strain is infected, the number of runners is reduced, which adversely affects productivity. Although production of virus-free strawberry seedlings has already been established by shoot tip culture technology, post-cultivation assays are required.

As a method for testing a strawberry pathogenic virus, the leaflet grafting method has been conventionally known, but there is a problem that the test takes a long time.
In Non-Patent Document 1, a genetic diagnosis method using a PCR method has been reported as an assay method for strawberry pathogenic viruses. This method has been put to practical use because it does not require skill like grafting work and the detection time is greatly shortened. In recent years, a method using the LAMP method has been reported as another genetic diagnosis method (Non-patent Documents 2 and 3).

Journal of Virological Methods, Volume 111, Issue 2, August 2003, Pages 85-93. 2008 Hokkaido Agricultural Research Results Information (http://www.naro.affrc.go.jp/org/harc/seika/h20/09.06/0105/main.htm) Scientia Agricultura Sinica, 2015, 48 (3), 613-620

  It is an object of the present invention to provide a more sensitive method as an assay method for strawberry pathogenic viruses. More specifically, an object of the present invention is to provide a primer set for specifically amplifying a nucleic acid derived from a strawberry pathogenic virus that can be used in the above method.

The LAMP method is advantageous in that it does not require expensive equipment required in the PCR method and the processing time is short. The present inventors have studied various primers for use in the LAMP method, and have found a primer set that can improve the sensitivity of strawberry pathogenic viruses as compared with a method based on the known LAMP method.
The present invention has been completed based on these findings.

That is, the present invention provides the following <1> to <10>.
<1> A nucleic acid derived from one or more strawberry pathogenic viruses selected from the group consisting of strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV), and strawberry vein banding virus (SVBV) by the LAMP method. Primer set for specific amplification, comprising one or more selected from the group consisting of the following (1) to (3):
(1) a FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 1 or a polynucleotide comprising a mutation within a range capable of specifically amplifying a nucleic acid derived from SMYEV in the polynucleotide represented by the sequence of SEQ ID NO: 1,
A BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 2 or a polynucleotide comprising a mutation within a range capable of specifically amplifying a nucleic acid derived from SMYEV in the polynucleotide represented by the sequence of SEQ ID NO: 2;
A polynucleotide represented by the sequence of SEQ ID NO: 3 or a polynucleotide represented by the sequence of SEQ ID NO: 3, and an F3 primer comprising a polynucleotide containing a mutation within a range capable of specifically amplifying a nucleic acid derived from SMYEV; and SEQ ID NO: 4 A polynucleotide represented by the sequence of SEQ ID NO: 4, or a polynucleotide represented by the sequence of SEQ ID NO: 4, a B3 primer comprising a polynucleotide containing a mutation within a range in which a nucleic acid derived from SMYEV can be specifically amplified, or
An FIP primer, a BIP primer, an F3 primer, and a B3 primer each comprising a polynucleotide indicated by a complementary sequence of a polynucleotide sequence that constitutes the FIP primer, BIP primer, F3 primer, and B3 primer, respectively;
(2) a FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 7 or a polynucleotide comprising a mutation within a range capable of specifically amplifying a nucleic acid derived from SMoV in the polynucleotide represented by the sequence of SEQ ID NO: 7,
A BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 8 or a polynucleotide comprising a mutation within a range capable of specifically amplifying an SMoV-derived nucleic acid in the polynucleotide represented by the sequence of SEQ ID NO: 8,
A polynucleotide represented by the sequence of SEQ ID NO: 9 or a polynucleotide represented by the sequence of SEQ ID NO: 9, an F3 primer comprising a polynucleotide containing a mutation within a range in which a nucleic acid derived from SMoV can be specifically amplified, and SEQ ID NO: 10 A polynucleotide represented by the sequence of SEQ ID NO: 10, or a polynucleotide represented by the sequence of SEQ ID NO: 10, a B3 primer comprising a polynucleotide containing a mutation within a range in which a nucleic acid derived from SMoV can be specifically amplified, or
An FIP primer, a BIP primer, an F3 primer, and a B3 primer each comprising a polynucleotide indicated by a complementary sequence of a polynucleotide sequence that constitutes the FIP primer, BIP primer, F3 primer, and B3 primer, respectively;
(3) a FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 13 or a polynucleotide comprising a mutation within a range capable of specifically amplifying a SVBV-derived nucleic acid in the polynucleotide represented by the sequence of SEQ ID NO: 13,
A BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 14 or a polynucleotide comprising a mutation within a range capable of specifically amplifying a nucleic acid derived from SVBV in the polynucleotide represented by the sequence of SEQ ID NO: 14,
A polynucleotide represented by the sequence of SEQ ID NO: 15, or an F3 primer comprising a polynucleotide containing a mutation within a range in which SVBV-derived nucleic acid can be specifically amplified in the polynucleotide represented by the sequence of SEQ ID NO: 15, and SEQ ID NO: A polynucleotide represented by 16 sequences or a polynucleotide represented by the sequence of SEQ ID NO: 16, or a B3 primer comprising a polynucleotide containing a mutation to the extent that SVBV-derived nucleic acid can be specifically amplified; or
The FIP primer, BIP primer, F3 primer, and B3 primer which consist of a polynucleotide respectively shown by the complementary arrangement | sequence of the arrangement | sequence of the polynucleotide which comprises said FIP primer, BIP primer, F3 primer, and B3 primer, respectively.

<2> The above (1) is further
An LF primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 5 or a polynucleotide containing a mutation within a range capable of specifically amplifying a nucleic acid derived from SMYEV in the polynucleotide represented by the sequence of SEQ ID NO: 5, and SEQ ID NO: 6 One or more selected from LB primers consisting of a polynucleotide containing a mutation within a range capable of specifically amplifying a nucleic acid derived from SMYEV in the polynucleotide shown by the sequence of SEQ ID NO: 1 or the polynucleotide shown by the sequence of SEQ ID NO: 1 Or
One or more selected from LF primers and LB primers each comprising a polynucleotide represented by a complementary sequence of a polynucleotide sequence constituting each of the LF primer and the LB primer,
(2) above further
An LF primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 11 or a polynucleotide containing a mutation within a range capable of specifically amplifying a nucleic acid derived from SMoV in the polynucleotide represented by the sequence of SEQ ID NO: 11, and SEQ ID NO: One selected from LB primers consisting of a polynucleotide represented by the sequence 12 or a polynucleotide comprising a mutation within a range capable of specifically amplifying the SMoV-derived nucleic acid in the polynucleotide represented by the sequence of SEQ ID NO: 12. Or
One or more selected from LF primers and LB primers each consisting of a polynucleotide respectively represented by complementary sequences of polynucleotide sequences constituting the LF primer and LB primer, respectively, and (3) above,
An LF primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 17 or a polynucleotide containing a mutation within a range capable of specifically amplifying an SVBV-derived nucleic acid in the polynucleotide represented by the sequence of SEQ ID NO: 17, and SEQ ID NO: One selected from LB primers comprising a polynucleotide represented by the sequence 18 or a polynucleotide comprising a mutation within a range capable of specifically amplifying the SVBV-derived nucleic acid in the polynucleotide represented by the sequence SEQ ID NO: 18. Or
<1> The primer set according to <1>, comprising at least one selected from an LF primer and an LB primer each consisting of a polynucleotide indicated by a complementary sequence of a polynucleotide sequence constituting each of the LF primer and the LB primer.

<3> The primer set according to <2>, wherein each of (1) to (3) includes both an LF primer and an LB primer.
<4> A primer set for specifically amplifying a nucleic acid derived from strawberry mild yellow edge virus (SMYEV), wherein the primer set includes (1) above. Primer set.
<5> A primer set for specifically amplifying a strawberry mottle virus (SMoV) -derived nucleic acid, wherein the primer set includes the above (2). The primer according to any one of <1> to <3> set.
<6> The primer set according to any one of <1> to <3>, which is a primer set for specifically amplifying a nucleic acid derived from strawberry vein banding virus (SVBV), wherein the primer set includes (3) above. set.
<7> The primer set according to any one of <1> to <3>, including any of the above (1) to (3).

<8> A method for detecting a strawberry pathogenic virus,
Extracting nucleic acids from the specimen,
Using the primer set according to any one of <1> to <7>, performing a DNA amplification reaction by the LAMP method using the nucleic acid as a template,
A detection method comprising determining that a strawberry pathogenic virus is present in a specimen when it is determined that there is an amplification product.
<9> A method for detecting a strawberry pathogenic virus,
Extracting nucleic acids from the specimen,
Performing a DNA amplification reaction by the LAMP method using the nucleic acid as a template using the primer set according to <7>,
A detection method comprising determining the presence or absence of a strawberry pathogenic virus in a specimen and the number of types of strawberry pathogenic virus present in a specimen by analyzing an association curve of the DNA after the amplification reaction into a double strand.
<10> A kit for detecting a strawberry pathogenic virus, comprising the primer set according to any one of <1> to <7>, a strand displacement type DNA synthase, dNTPs, and a buffer solution.

  According to the present invention, a primer set for specifically amplifying a nucleic acid derived from a strawberry pathogenic virus by the LAMP method is provided. Using this primer set, strawberry pathogenic viruses can be detected with higher sensitivity.

It is a figure which shows the positional relationship of a FIP primer, F3 primer, BIP primer, B3 primer, and a target region (example of genomic RNA). It is a photograph which shows the example which performed simultaneous detection of the virus using the primer set for specifically amplifying the nucleic acid derived from SMYEV, SMoV, and SVBV. It is a figure which shows the association curve of the amplification product which amplified by the LAMP method using the primer set for amplifying specifically the nucleic acid derived from SMYEV, SMoV, and SVBV.

Hereinafter, embodiments of the present invention will be described in more detail.
In the present specification, A, G, T, and C in the nucleotide sequence of a nucleic acid indicate adenine base, guanine base, thymine base, and cytosine base in deoxyribonucleotide. In addition, in the present specification, when a plurality of bases are described using “/”, it indicates that any of the described bases may be used. For example, when “C / T” is described. Indicates that it may be a cytosine base or a thymine base.

  The LAMP (Loop-Mediated Isometric Amplification) method was developed by Notomi et al., Japanese Patent No. 3313358, Nucleic Acids Research, 28, e63, 2000, Biochemical and Biophysical 200, Biochemical and Biophysical 95. , 47, No. 9, 1742-1743, 2001. In general, the LAMP method is superior in amplification efficiency to a method using PCR and is not easily affected by impurities in the sample. Therefore, the target nucleic acid can be amplified by simple sample pretreatment.

In the present specification, the “LAMP method” is used in a sense including a RT-LAMP (Reverse transcription-Loop-mediated thermal amplification) method. In the RT-LAMP method, nucleic acid is amplified by simultaneously performing a reverse transcription reaction and a LAMP reaction.

<Primers used in the LAMP method>
The primer and primer design in the LAMP method will be described below.
The primer set used for the LAMP method includes at least an FIP primer, an F3 primer, a BIP primer, and a B3 primer. FIG. 1 is a diagram showing the positional relationship between the FIP primer, F3 primer, BIP primer, and B3 primer and the target region of viral genomic RNA when RT-LAMP is performed.
The FIP primer and BIP primer each contain two regions. That is, FIP includes F1c and F2, and BIP includes B2 and B1c. The F3 primer contains the F3 region. The B3 primer contains the B3 region. Each primer is designed as follows.

(1) FIP primer: designed to have the F2 region of the target nucleic acid at the 3 ′ end and the same sequence as F1c of the target nucleic acid at the 5 ′ end.
(2) F3 primer: designed to have the F3 region of the target nucleic acid.
(3) BIP primer: designed to have the B2 region of the target nucleic acid at the 3 ′ end and the same sequence as B1c of the target nucleic acid at the 5 ′ end.
(4) B3 primer: designed to have a B3 region of the target nucleic acid.

  Here, the F3, F2, F1, B1c, B2c, and B3c regions are regions set in this order from the 5 ′ end side to the 3 ′ end side of RNA. The B3, B2, B1, F1c, F2c, and F3c regions are regions set in this order from the 5 ′ end side to the 3 ′ end side of the complementary strand cDNA. At this time, B3 and B3c, B2 and B2c, B1 and B1c, F1c and F1, F2c and F2, and F3c and F3 are complementary strands.

  When LAMP amplification is performed using such FIP primer, F3 primer, BIP primer, and B3 primer, a dumbbell-shaped stem-and-loop structure amplification product is obtained from the cDNA of FIG. For the amplification mechanism, see, for example, the website http: // www. eiken. co. jp / or JP-A No. 2002-186871 can be referred to.

  The primer set preferably includes a loop primer in addition to the above four types of primers. As a loop primer, it is preferable to use LF primer, LB primer, or LF primer and LB primer. The LF primer and the LB primer are loop primers having a sequence complementary to the single-stranded portion of the loop on the 5 ′ end side of the dumbbell structure that is the starting point of the amplification reaction. The LF primer can be designed by having a sequence complementary to the sequence between the F1 region and the F2 region in FIG. 1, and the LB primer is complementary to the sequence between the B1 region and the B2 region in FIG. Can be designed to have a typical array. By using the LF primer and the LB primer, it is possible to increase the starting point of DNA synthesis, increase the amplification efficiency, and shorten the time required for amplification to 1/3 to 1/2.

<Primer set>
The primer set of the present invention is a primer set for specifically amplifying a nucleic acid derived from a strawberry pathogenic virus by the LAMP method. The strawberry pathogenic virus is at least one selected from the group consisting of strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV), and strawberry vein banding virus (SVBV).
The primer set of the present invention includes one or more selected from the group consisting of the following (1) to (3), and can detect a strawberry pathogenic virus according to the included primer set.
(1) Primer set for specifically amplifying nucleic acid derived from strawberry mild yellow edge virus (SMYEV),
(2) A primer set for specifically amplifying a nucleic acid derived from strawberry mottle virus (SMoV),
(3) A primer set for specifically amplifying nucleic acid derived from strawberry vein banding virus (SVBV).

  The above (1), (2) and (3) may be used alone or in combination of any two or more. It is also preferable to use a combination of the three primer sets. As shown in the examples described later, false positives are not confirmed even when the above three primer sets are used in combination. By using a combination of primer sets that are used to specifically amplify the nucleic acids of three types of viruses, it is possible to easily and quickly detect infection of any one or more viruses in strawberries at low cost. it can.

  As described above, since symptom appears clearly when strawberry is co-infected, it is particularly required to discriminate co-infection in strawberry cultivation. In the measurement using a combination of multiple primer sets for specifically amplifying nucleic acids derived from different viruses, the pathogenic virus of strawberry detected by analysis of the association curve to the double strand of the amplification product DNA The number of types can be determined, and it can be determined whether it is a single infection or a superinfection.

  Each primer in the primer set can be synthesized by a general DNA synthesizer based on the information on the base sequence.

In addition, the primer amount ratio in the primer set is not particularly limited, and may be used in a common amount ratio in the LAMP method. For example, when visually judging with 12.5 microliters of reaction solution, F3 primer and B3 primer are 2.5 pmol each, FIP primer and BIP primer are 20 pmol each, and LF primer and LB primer are 10 pmol each per sample. It can be. Absorbance meter MyAbscope (registered trademark) with endpoint determination using Kaneka temperature control function with 25 microliters of reaction solution, endpoint turbidity measurement device LT-16 (Nippon Gene), LAMP method measurement device LF-8 Plus (Nippon Gene) In this case, F3 primer and B3 primer can each be used at 5 pmol, FIP primer and BIP primer at 40 pmol, and LF primer and LB primer at 20 pmol per sample, respectively. In addition, when monitoring using Genelyzer F (TOSHIBA MEDICAL) and isothermal amplification fluorescence measuring device Genie (registered trademark) III (Nippon Gene), the reaction solution is 25 microliters per sample, F3 primer and B3 primer are 5 pmol each, The FIP primer and BIP primer can be used at 20 pmol each, and the LF primer and LB primer can be used at 10 pmol each.
Hereinafter, the design of each primer set of (1) to (3) will be described.

[(1) Primer set for specifically amplifying nucleic acid derived from strawberry mild yellow edge virus (SMYEV)]
In the present invention, the primer set for specifically amplifying SMYEV-derived nucleic acid aligns the genomes of a plurality of SMYEV strains, and F3, F2, F1c, B1c, B2, B3 in a highly conserved region. Designed by selecting a region.
Table 1 shows an alignment of genomes (coat protein genes) of a plurality of SMYEV strains.

The regions indicated by MYEVF3, MYEVFIP (F2), MYEVFIP (F1), MYEVBIP (B1), MYEVBIP (B2), and MYEVB3 in Table 1 are primer sets for specifically amplifying SMYEV-derived nucleic acids in the present invention. In the design, the regions are F3, F2, F1, B1c, B2c, and B3c regions, respectively. The specific sequence of each region is determined in Table 1, in particular based on the sequence of strain sy03 (Bhagwat et al., 2016b) and the sequence of unpublished strains not shown in Table 1, and based on those sequences , Polynucleotides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, as FIP primers, BIP primers, F3 primers, and B3 primers for specifically amplifying nucleic acids derived from SMYEV Designed.
The corresponding F2, F1c, B2, and B1c region sequences are shown in Table 2.

  Furthermore, the regions indicated by MYEVLF2 and MYEVLB2 in Table 1 were selected, and the LF primer and the LB primer comprising the polynucleotides indicated by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, were designed.

In Table 1, the F3, F2, F1, B1c, B2c, B3c, LF, and LB regions used in the design of other primer sets, which will be described later in Examples, are also shown. Primer sets were similarly designed and prepared based on these regions, and as shown in the examples described later, the results were considered to have insufficient specificity.
Table 1 also shows each region of the primer set used in the prior literature. It can be seen that the position of the region used for designing the primer set used in the present invention is significantly different from the position of each region of the primer set used in the prior literature.

The FIP primer may contain a mutation within a range in which a nucleic acid derived from SMYEV can be specifically amplified.
Specifically, in the FIP primer, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by SEQ ID NO: 1 are substituted, or deleted and / or inserted. You may have. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
Regarding the F2 region (SEQ ID NO: 61), the first from the 5 ′ side may be A / T instead of C, the fifth may be C / T / G instead of A, and the seventh is A / T / G may be used instead of C, 10th may be G / C / T instead of A, 11th may be T instead of C, and 13th C may be substituted for T and T may be T instead of C;
Regarding the F1c region (SEQ ID NO: 62), the fifth from the 5 ′ side may be C / A / G instead of T, the eighth may be C / T instead of A, and the eleventh is G may be A, 14th may be C / T instead of A, 17th may be C / A / T instead of G, and 20th is G A may be used instead.

In addition, the polynucleotide represented by SEQ ID NO: 1 is obtained by directly binding the F2 region represented by SEQ ID NO: 61 and the F1c region represented by SEQ ID NO: 61. The FIP primer has the sequence of SEQ ID NO: 1. Between the F2 region and the F1c region of the indicated polynucleotide, a sequence of 1 to 6 nucleotides (eg a sequence used as a spacer, for example A, T, TT, TTT, TTTT, TTTTA, TTTTT, TTTTTTT or restriction An enzyme recognition sequence (GAATTC; restriction enzyme EcoRI recognition site, GGATTC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site) may be included. However, in the FIP primer, the F2 region and the F1c region are It is preferable that the contact bond.
Furthermore, the mutation is preferably a mutation in a range such that the GC content in the FIP primer is 50 to 60%.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

The BIP primer may contain a mutation within a range in which a nucleic acid derived from SMYEV can be specifically amplified.
Specifically, the BIP primer is substituted with 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence of SEQ ID NO: 2, or a deletion and / or Or you may have insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
For the B2 region (SEQ ID NO: 63)
The first from the 5 ′ side may be A / G instead of C, the fourth may be G instead of A, and the seventh may be T / A instead of G. The tenth may be G / A / C instead of T, and the thirteenth may be G instead of A;
Regarding the B1c region (SEQ ID NO: 64), the third from the 5 ′ side may be T instead of C, the sixth may be T instead of C, and the seventh is A instead of G. The ninth may be T instead of C, the eleventh may be C instead of T, the seventh may be A instead of G, and 12, 13 The th may be CC, TA, TC instead of GA, and the fifteenth may be G instead of A.

  In addition, the polynucleotide represented by SEQ ID NO: 2 is one in which the B2 region represented by SEQ ID NO: 63 and the B1c region represented by SEQ ID NO: 64 are directly bound. Between the sequence of the B1c region, a sequence of 1 to 6 nucleotides (for example, a sequence used as a spacer, for example, A, T, TT, TTT, TTTT, TTTTA, TTTTT, TTTTTTT or a restriction enzyme recognition sequence (GAATTC; A restriction enzyme EcoRI recognition site, GGATC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site), but in the BIP primer, the B2 and B1c regions are directly Bonding is preferred.

Furthermore, the mutation is preferably a mutation in a range such that the GC content in the BIP primer is 50 to 60%.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

The F3 primer, the B3 primer, the LF primer, and the LB primer may each contain a mutation within a range in which a nucleic acid derived from SMYEV can be specifically amplified.
Specifically, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by each of SEQ ID NOs: 3 to 6 are substituted, or deleted and / or May have an insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
For the F3 primer (SEQ ID NO: 3), the second from the 5 'side may be G / A instead of T, the eighth may be T instead of C, the eleventh may be C instead of T, and the 14th May be G instead of A, and the 17th may be C instead of T;
Regarding the B3 primer (SEQ ID NO: 4), the third from the 5 ′ side may be T instead of C, the fourth may be T instead of C, the fifth may be T instead of G, and the sixth is G May be A instead of A, the ninth may be A instead of T, and the twelfth may be T instead of C;
Regarding the LF primer (SEQ ID NO: 5), the third from the 5 ′ side may be A instead of G, the sixth may be A instead of G, and the ninth is A instead of G. The 18th may be C instead of T, and the 20th may be G instead of A;
Regarding the LB primer (SEQ ID NO: 6), the fourth from the 5 ′ end may be G / T instead of A, the seventh may be C instead of T, and the 19th may be C instead of T.

Furthermore, the mutation is preferably a mutation in a range such that the GC content in each of the above primers is 50 to 60%.
The primer may be prepared by mixing the polynucleotides in the above ranges as mixed primers.

  The FIP primer, the BIP primer, the F3 primer, the B3 primer, the LF primer, and the LB primer may be composed of polynucleotides respectively indicated by the polynucleotides described above that are all complementary sequences at the same time.

[(2) Primer set for specifically amplifying nucleic acid derived from strawberry mottle virus (SMoV)]
In the present invention, the primer set for specifically amplifying SMoV-derived nucleic acid aligns the genomes of a plurality of SMoV strains, and F3, F2, F1c, B1c, B2, B3 in a highly conserved region. Designed by selecting a region.
Table 3 shows the genome alignment of multiple SMoV strains.

The regions indicated by SMoVF3GC, SMoVFIPGC (F2), SMoVFIPGC (F1), SMoVBIPGC (B1), SMoVBIPGC (B2), and SMoVB3GC in Table 3 are primer sets for specifically amplifying SMoV-derived nucleic acids in the present invention. In the design, the regions are F3, F2, F1, B1c, B2c, and B3c regions, respectively. The specific sequence of each region was determined based on the nucleotide sequence of the RNA2 gene of NSper51 strain shown in Table 3 (Accession No. KU200461, Bhagwat et al., 2016a). Polynucleotides represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, were designed as FIP primers, BIP primers, F3 primers, and B3 primers for specifically amplifying nucleic acids.
Table 4 shows the sequences of the corresponding F2, F1, B1c, and B2c regions.

  Furthermore, in Table 3, regions represented by SMoVLLFGC and SMoVLBGC were selected, and primers including LF primers and LB primers respectively composed of polynucleotides represented by SEQ ID NO: 11 and SEQ ID NO: 12 were designed.

In Table 3, the F3, F2, F1, B1c, B2c, B3c, LF, and LB regions used in the design of other primer sets, which will be described later in Examples, are also shown. Primer sets were similarly designed and prepared based on these regions, and as shown in the examples described later, the results were considered to have insufficient specificity.
Table 3 also shows each region of the primer set used in the prior literature. It can be seen that the position of the region used for designing the primer set used in the present invention is significantly different from the position of each region of the primer set used in the prior literature.

The FIP primer may contain a mutation within a range where SMoV-derived nucleic acid can be specifically amplified.
Specifically, the FIP primer is substituted with 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence of SEQ ID NO: 7, or a deletion and / or Or you may have insertion. In particular, one to several nucleotides may be substituted. Examples of such polynucleotides include those in which the 15th T from the 5 ′ side is A in the F2 region (SEQ ID NO: 65).

  In addition, the polynucleotide represented by SEQ ID NO: 7 is obtained by directly binding the F2 region represented by SEQ ID NO: 65 and the F1c region represented by SEQ ID NO: 66, but the FIP primer comprises F2 region, F1c region and Between 1 and 6 nucleotide sequences (for example, sequences used as spacers, for example A, T, TT, TTT, TTTT, TTTTA, TTTTT, TTTTTTT or restriction enzyme recognition sequence (GAATTC; restriction enzyme EcoRI (Recognition site, GGATC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site) In the FIP primer, the F2 region and the F1c region are directly bound to each other. Preferably it is.

Furthermore, the mutation is preferably a mutation in a range such that the GC content in the FIP primer is 50 to 60%.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

The BIP primer may contain a mutation as long as it can specifically amplify SMoV-derived nucleic acid.
Specifically, the BIP primer is substituted with 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence of SEQ ID NO: 8, or deleted and / or Or you may have insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
Regarding the B2 region (SEQ ID NO: 67), the second from the 5 ′ side may be T instead of A, the fourth may be T instead of C, and the fifth is G instead of A. The sixth may be C instead of T, the 19th may be A instead of G, and the 20th may be G instead of A;
Regarding the B1c region (SEQ ID NO: 68), the fourth from the 5 ′ side may be A instead of G, the ninth may be A / T instead of C, and the tenth instead of T C, 11th may be A / G / C instead of T, 12th may be A instead of G, and 20th is T instead of C. The 21st may be T instead of C, and the 22nd may be G instead of A.
Furthermore, it is preferable that a variation | mutation is a variation | mutation of the range from which GC content will be 50 to 60% about any area | region.

In addition, the polynucleotide represented by SEQ ID NO: 8 is obtained by directly binding the B2 region represented by SEQ ID NO: 67 and the B1c region represented by SEQ ID NO: 68, and the BIP primer corresponds to the B2 region. 1 to 6 nucleotide sequences between the sequence and the sequence corresponding to the B1c region (for example, A, T, TT, TTT, TTTT, TTTTA, TTTTT, TTTTTT or restriction enzyme recognition as examples of sequences used as spacers) It may contain a sequence (GAATTC; restriction enzyme EcoRI recognition site, GGATTC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site) However, in the BIP primer, the B2 region and The B1c region is preferably directly bonded.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

The F3 primer, the B3 primer, the LF primer, and the LB primer may contain a mutation as long as the SMoV-derived nucleic acid can be specifically amplified.
Specifically, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by each of SEQ ID NOs: 9 to 12 are substituted, or deleted and / or May have an insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
Regarding F3 primer (SEQ ID NO: 9), 4th from 5 ′ side may be T instead of G, 9th may be A instead of G, and 13th is G instead of A May be;
For the B3 primer (SEQ ID NO: 10), the sixth from the 5 ′ side may be C instead of T, the eighth may be G instead of A, and the tenth is A instead of G. And the 13th may be T instead of A;
For the LF primer (SEQ ID NO: 11), 14th from the 5 ′ side may be A instead of T;
For the LB primer (SEQ ID NO: 12), the 12th from the 5 ′ side may be A instead of G, G may be substituted for the 13th A, and G may be substituted for the 22nd A. There may be.

Furthermore, the mutation is preferably a mutation in a range where the GC content is 50 to 60% for any primer.
The primer may be prepared by mixing the polynucleotides in the above ranges as mixed primers.

  The FIP primer, the BIP primer, the F3 primer, the B3 primer, the LF primer, and the LB primer may be composed of polynucleotides that are respectively indicated by using the polynucleotides described above as complementary sequences all at the same time.

[(3) Primer set for specifically amplifying nucleic acid derived from strawberry vein banding virus (SVBV)]
In the present invention, the primer set for specifically amplifying SVBV-derived nucleic acids aligns the genomes of a plurality of SVBV strains, and F3, F2, F1c, B1c, B2, B3 in a highly conserved region. Designed by selecting a region.
Table 5 shows the alignment of genomes (coat protein genes) of multiple SVBV strains.

  As shown in Table 5, F3, F2, F1, B1c, B2c, and B3c regions were selected. The specific sequence of each region was determined based on the partial sequence of the SVBV Chugoku strain (Accession No. AY862389, Zhou et al., 2005), and based on these sequences, the SVBV-derived nucleic acid was specifically identified. As FIP primers, BIP primers, F3 primers, and B3 primers for amplification, the polynucleotides represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, were designed.

  The sequences of the F2, F1c, B1c, and B2 regions are as follows.

  Furthermore, as shown in Table 5, the LF and LB regions were selected, and primers including the LF primer and the LB primer composed of the polynucleotides shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, were designed.

  Table 7 shows each region of the primer set used in the prior document (Non-Patent Document 2). It can be seen that the position of the region used for designing the primer set used in the present invention is significantly different from the position of each region of the primer set used in the prior literature.

The FIP primer may contain a mutation as long as SVBV-derived nucleic acid can be specifically amplified.
Specifically, the FIP primer is substituted with 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence of SEQ ID NO: 13, or a deletion and / or Or you may have insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
Regarding the F2 region (SEQ ID NO: 69), the second from the 5 ′ side may be T instead of A, the fifth may be G instead of A, and the 17th is C instead of T. The 18th may be C instead of T and the 20th may be A instead of G;
For the F1c region (SEQ ID NO: 70), the seventh from the 5 ′ side may be C instead of T, the tenth may be C instead of A, and the twelfth is A instead of G. The 13th may be G instead of A, and the 22nd may be T instead of A.

  In addition, the polynucleotide represented by SEQ ID NO: 13 is obtained by directly binding the F2 region represented by SEQ ID NO: 69 and the F1c region represented by SEQ ID NO: 70, but the FIP primer is composed of the F2 region of the polynucleotide. Between the F1c region, a sequence of 1 to 6 nucleotides (for example, a sequence used as a spacer, for example, A, T, TT, TTT, TTTT, TTTTA, TTTTT, TTTTTTT or a restriction enzyme recognition sequence (GAATTC; restriction enzyme EcoRI recognition site, GGATC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site) However, in the FIP primer, the F2 region and the F1c region are directly bound. It is preferable.

Furthermore, the mutation is preferably a mutation in a range where the GC content of the FIP primer is about 40 to 65%.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

The BIP primer may contain a mutation as long as SVBV-derived nucleic acid can be specifically amplified.
Specifically, the BIP primer is substituted with 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence of SEQ ID NO: 14, or a deletion and / or Or you may have insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
For the B2c region (SEQ ID NO: 71), the 18th from the 5 ′ side may be G instead of A, the 16th may be G instead of T, and the 13th is G instead of A. There may be 9th and A may be substituted for G;
Regarding the B1 region (SEQ ID NO: 72), the second from the 5 ′ side may be C instead of T, the fifth may be A instead of T, and the 11th is A instead of G. The 3 ′ end may be T instead of G.

  In addition, the polynucleotide represented by SEQ ID NO: 14 is the one in which the sequence of the B2 region represented by SEQ ID NO: 71 and the sequence of the B1c region represented by SEQ ID NO: 72 are directly bound, A sequence of 1 to 6 nucleotides (for example, a sequence used as a spacer, for example, A, T, TT, TTT, TTTT, between the B2 region and the sequence corresponding to the B1c region of the polynucleotide represented by 14 sequences , TTTTA, TTTTT, TTTTTTT or a restriction enzyme recognition sequence (GAATTC; restriction enzyme EcoRI recognition site, GGATTC; restriction enzyme BamHI recognition site, CTGCAC; restriction enzyme PstI recognition site, GATATC; restriction enzyme EcoRV recognition site) However, in the BIP primer, the B2 region and B1 Region is preferably bonded directly.

Furthermore, the mutation is preferably a mutation in a range where the GC content of the BIP primer is about 40 to 65%.
The primer may be prepared by mixing the polynucleotide in the above range as a mixed primer.

  The F3 primer, the B3 primer, the LF primer, and the LB primer may contain a mutation within a range in which the SVBV-derived nucleic acid can be specifically amplified. Specifically, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by each of SEQ ID NOs: 15 to 18 are substituted, deleted and / or May have an insertion. In particular, one to several nucleotides may be substituted.

Examples of such polynucleotides include the following:
Regarding the F3 primer (SEQ ID NO: 15), the sixth from the 5 ′ side may be A / C instead of T, the ninth may be T instead of C, and the twelfth is T instead of C. May be;
For the B3 primer (SEQ ID NO: 16), the seventh from the 5 ′ side may be A instead of T, and the 14th may be A instead of G;
Regarding the LF region (SEQ ID NO: 17), the fifth from the 5 ′ side may be A instead of G, the seventh may be A instead of G, and the 16th is C instead of T. There may be, 25th may be T instead of C;
Regarding the LB primer (SEQ ID NO: 18), the 9th from the 5 ′ side may be A instead of T, and the 22nd may be A instead of G.

Furthermore, the mutation is preferably a mutation in a range where the GC content of each of the above primers is about 40 to 65%.
The primer may be prepared by mixing the polynucleotides in the above ranges as mixed primers.

  The FIP primer, the BIP primer, the F3 primer, the B3 primer, the LF primer, and the LB primer may be composed of polynucleotides respectively shown by using the polynucleotides described above as complementary sequences at the same time.

<LAMP method: amplification of nucleic acid>
In a DNA amplification reaction by the LAMP method, a reaction solution containing a nucleic acid extracted from a specimen, a primer set, a strand displacement DNA synthase, dNTPs (dATP, dTTP, dGTP, and dCTP), and a buffer solution is used. It may be left at a constant temperature for a certain time. In the amplification reaction of cDNA by RT-LAMP method, reverse transcriptase may be further added to the reaction solution.

  The temperature is preferably 50 ° C. or higher and 75 ° C. or lower, more preferably 60 ° C. or higher and 65 ° C. or lower, and still more preferably 63 ° C. When the standing time is 15 minutes or longer, cDNA amplification can be detected, but it is preferably 15 minutes or longer and within 1 hour, more preferably 20 minutes or longer and within 40 minutes.

  As a reverse transcriptase, a strand displacement type DNA synthase, dNTPs, and a buffer solution, for example, each reagent included in a Loopamp (registered trademark) RNA amplification reagent kit (manufactured by Eiken Chemical Co., Ltd.) can be used. An RNA amplification reagent (Nippon Gene) dedicated to LAMP primer sets for plant disease testing can also be used.

<Virus detection>
By performing nucleic acid amplification using the above primer set, it is possible to detect a specific virus simply, inexpensively and at high speed. Specifically, the nucleic acid contained in the specimen is subjected to the LAMP amplification reaction, and the presence or absence of the amplification product resulting from the amplification reaction is determined, whereby the virus in the specimen can be specifically detected.

Here, the strawberry as the “specimen” may be any part such as a leaf, a fruit, a stem, or a root, but it is preferable to use a leaf.
Nucleic acid is extracted from the sample and amplified by the LAMP method . For example, DNA can be extracted and a DNA amplification reaction can be performed by the LAMP method , while RNA can be extracted and a DNA amplification reaction can be performed by the RT-LAMP method .
Nucleic acid extraction may be performed by a known method, and can be performed by the following procedure as an example.
The collected strawberry seedling leaves are put in a tube, and an extraction buffer is added to crush the leaves in this buffer. Optionally, a heat treatment can be performed, after which the tube can be centrifuged and the supernatant used. When extracting RNA, the extraction buffer preferably contains mercaptoethanol.

  The determination of the presence or absence of the amplification product can be determined, for example, by observing the reaction solution after performing the cDNA amplification reaction by the RT-LAMP method with the naked eye and confirming that the reaction solution is clouded, and that the virus is present. Further, when a fluorescent intercalator such as SYBR Green I is added to this reaction solution and luminescence showing amplification of cDNA is confirmed under UV irradiation, it can be determined that the virus is present. This determination can also be made visually.

  Absorbance meter with temperature control function (MyAbsscope (registered trademark)) manufactured by Kaneka Co., Ltd., endpoint turbidity measuring device LT-16 (registered trademark) manufactured by Nippon Gene Co., Ltd., and measuring device LF-8 Plus manufactured by Nippon Gene Co., Ltd. The change in absorbance according to the amount of amplification product may be measured using a registered trademark. In addition to the presence or absence of an amplification product, the amount of amplification product can be determined.

  In addition, it is possible to detect the amplified nucleic acid by monitoring the association of the single-stranded nucleic acid obtained by the LAMP reaction to the double strand and analyzing the association curve. Such an analysis of the association can be performed using, for example, an isothermal amplification fluorescence measurement device Genelyzer F and an isothermal amplification fluorescence measurement device Generie (registered trademark) III (Nippon Gene) manufactured by Toshiba Medical Systems Corporation. In the measurement, a dedicated reagent for the measurement apparatus may be used.

<Virus detection kit>
Various reagents necessary for using the virus detection method of the present invention can be packaged in advance to form a kit. Specifically, the above primer set, four types of dNTPs (dATP, dCTP, dGTP, and dTTP) serving as a substrate for nucleic acid synthesis, a strand displacement type DNA synthase having strand displacement activity, and a buffer that provides conditions suitable for the enzymatic reaction Solutions, salts as cofactors (magnesium salt or manganese salt, etc.), protective agents that stabilize enzymes and templates, reverse transcriptase that synthesizes cDNA from RNA, if necessary, reagents necessary for detection of reaction products Combinations can be provided as a kit.

  The kit may include a positive control (positive control) for confirming that the LAMP reaction proceeds normally with the primer set of the present invention. As a positive control, for example, DNA containing a region amplified by the primer set of the present invention can be mentioned.

The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.
In the examples, all the base sequence descriptions are 5′-3 ′.

<Design of primer set>
A primer set was designed for each of strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV), and strawberry vein banding virus (SVBV), and the primer sets shown in the following table were prepared.
Based on known sequence information for each virus, alignment analysis was performed using MEGA6, which is DNA analysis software, to search for regions with high conservation. In the searched region, the F3c, F2c, F1c, B1, B2, and B3 regions were selected as described above (see Table 1, Table 3, and Table 5 above), and each primer set was designed. When designing, a plurality of primer sets were designed as follows using Primer Explorer V.5 as appropriate.
As the SMoV primer set, a primer set including a loop primer was designed so as to have a high GC content. The designed primer set (primer) was chemically synthesized. After purification with desalting grade and synthesis at 10 nmol, the concentration was adjusted to 100 μM with sterile distilled water. This was stored frozen at −20 ° C. as a stock solution. Each primer was diluted to a predetermined concentration to ensure a certain amount. The diluted primer was also stored frozen at -20 ° C.

<Nucleic acid extraction>
The leaves of the strawberry seedlings were collected with a 1.5 ml tube and its lid. A sample and 500 μL of extraction buffer (50 mM Tris-HCl 500 μL + mercaptoethanol 10 μL) were placed in a 1.5 ml tube and crushed with the tip of a pipette tip. The tube was centrifuged (5,000 rpm × 1 min), the supernatant was placed in a new 1.5 ml tube, Polycral VT was added to the earpick and vortexed. After incubation (100 ° C., 10 minutes), the mixture was quenched with ice water and centrifuged (15,000 rpm × 5 minutes). The supernatant was transferred to a new tube and used as an extracted sample.

<Virus detection test (single detection)>
1) Visual judgment (procedure)
LOOPAMP (registered trademark) RNA amplification reagent kit (RT-LAMP) (Eiken Chemical https://genome.e-mp.jp/products/rna.html) and Loopamp fluorescence / visual detection reagent with each virus primer set (Eiken Chemical https://genome.e-mp.jp/products/mokusi.html) was used. For each sample, 1.5 μL of the above extracted sample, 6.25 μL of 2 × Reaction mixture, 0.5 μL of Enzyme mix, 0.5 μL of fluorescent reagent, 0.5 μL of F3 primer (5 μM), 0.5 μL of B3 Primer (5 μM), 0.5 μL FIP primer (40 μM), 0.5 μL BIP primer (40 μM), 0.5 μL LF primer (20 μM), 0.5 μL LB primer (20 μM), 0.75 μL sterile A total of 12.5 μL was prepared by adding distilled water. Alternatively, 3.0 μL of the above extracted sample and 12.5 μL of 2 × Reaction mixture, 1.0 μL of Enzyme mix, 1.0 μL of fluorescent reagent, 1.0 μL of F3 primer (5 μM), 1.0 μL of B3 primer (5 μM) ), 1.0 μL FIP primer (40 μM), 1.0 μL BIP primer (40 μM), 1.0 μL LF primer (20 μM), 1.0 μL LB primer (20 μM), 1.5 μL sterile distilled water. A total of 25.0 μL was prepared by adding. These preparations were performed on ice. The reaction was carried out at 63 ° C. for 1 hour using a LifeECO thermal cycler (Bioer Technology Co., Ltd.), the reaction was stopped by treatment at 95 ° C. for 2 minutes, and then visually judged. As a negative control, 1.5 μL of sterilized distilled water was added instead of RNA.

(Confirmation of primer set function)
Regarding the primer sets for each of the viruses described above, first, the above visual determination was performed using three specimens of SMYEV, SMoV, SVBV co-infected seedling, healthy seedling, and water. However, the reaction in the LifeECO thermal cycler was performed at 63 ° C. for 90 minutes.
About the sample containing a SMYEV infection strain, the said visual determination was performed using the SMYEV primer sets 1-5, respectively. The results are as follows:
SMYEV primer set 1 was clearly positive only for infected seedlings.
SMYEV primer sets 2, 4, and 5 were all confirmed to be false positives (positive for healthy seedlings or water samples).
The detection sensitivity of SMYEV primer set 3 was poor.

Visual determination was similarly performed about the specimen containing a SMoV infection strain using SMoV primer sets 1-7, respectively. The results are as follows:
SMoV primer set 1 was clearly positive only for infected seedlings.
SMoV primer sets 2 and 4 were also negative for infected seedlings.
SMoV primer sets 3, 5, and 7 were all confirmed to be false positives.
The detection sensitivity of the SMoV primer set 6 was poor.

  As a result of the above detection test using SVBV primer set 1 for specimens containing SVBV-infected strains, the SVBV primer set was clearly positive only for infected seedlings.

  Based on the above results, SMYEV primer set 1, SMoV primer set 1, and SVBV primer set 1 were used for further testing.

(Comparison with RT-PCR and RT-LAMP method using previously reported primer set)
A detection test by RT-LAMP method using RT-PCR and various primer sets was performed on 9 runner seedlings and 1 healthy (non-infected) seedling generated from each virus-infected seedling, and the results were compared.
RT-PCR was performed by the method described in Thompson et al., 2003 (Journal of Virological Method, Vol. 111, Issue 2, 2003, Page 85-93).
In the SMYEV detection test, SMYEV primer set 1 as a primer set, a set of four primers for SMYEV described in Table 3 of Non-Patent Document 2 (2008 Hokkaido Agricultural Research Result Information), and Non-Patent Document 3 (Scientia) 4 primers (FIP primer, BIP primer, F3 primer, B3 primer) described in Table 1 of Agricultura Sinica, 2015, 48 (3), 613-620) were used. The results are shown in Table 11.

SMYEV primer set 1 also showed a positive reaction for infected seedlings that were not detected by RT-PCR or the previously reported RT-LAMP method.
In the SMoV detection test, a set of four primers for SMoV described in Table 3 of SMoV primer set 1 and non-patent document 2 (2008 Hokkaido Agricultural Research Result Information) was used as a primer set. The results are shown in Table 12.

SMYEV primer set 1 also showed a positive reaction for infected seedlings that were not detected by RT-PCR or the previously reported RT-LAMP method.
In the SVBV detection test, a set of four primers for SVBV described in Table 3 of SVBV primer set 1 and non-patent document 2 (2008 Hokkaido Agricultural Research Result Information) was used as a primer set. The results are shown in Table 13.

  The detection using SVBV primer set 1 showed the same results as the detection obtained by PCR, unlike the previously reported LAMP method.

  SMYEV and SMoV are RNA viruses, while SVBV is a DNA virus. However, clear results were obtained with the method described above using the Loopamp® RNA amplification reagent kit. SVBV detection was carried out in the same manner using a Loopamp (registered trademark) DNA amplification reagent kit (Eiken Chemical), but no clear detection results were obtained as in the example using the RNA amplification reagent kit. It is thought that mercaptoethanol used in the extraction process has an influence.

2) Determination using an absorptiometer A determination using an absorptiometer was also performed as detection of each virus using a primer set for each virus described above. A positive determination can be made when an increase in absorbance is observed after a certain reaction time.
A Loopamp (registered trademark) RNA amplification reagent kit (RT-LAMP) (Eiken Chemical) and a primer set are used. Per sample, 3.0 μL of nucleic acid extraction sample, 12.5 μL of 2 × Reaction mixture, 1.0 μL of Enzyme mix, 1.0 μL of fluorescent reagent, 1.0 μL of F3 primer (5 μM), 1.0 μL of B3 Primer (5 μM), 1.0 μL FIP primer (40 μM), 1.0 μL BIP primer (40 μM), 1.0 μL LF primer (20 μM), 1.0 μL LB primer (20 μM), 2.5 μL sterile Distilled water was added to prepare a total of 25 μL (using a D-QUICK-containing tube (KANEKA) or a normal tube). These adjustments were made on ice. The sample was set in MyAbscope (registered trademark) (absorbance meter with temperature control function, Kaneka), amplified at 63 ° C. for 1 hour, and stopped at 85 ° C. for 10 minutes. Absorbance change was measured in real time with a G sensor, and it was judged positive when an increase in absorbance was observed after a certain reaction time. Using the SMYEV primer set 1, the SMoV primer set 1, and the SVBV primer set 1, a determination was made on the same 9 infected seedlings and 1 healthy (non-infected) seedling as described above. Obtained.

3) Determination by association curve analysis 3.0 μL nucleic acid extraction sample, 1.0 μL F3 primer (5 μM), 1.0 μL B3 primer (5 μM), 0.5 μL FIP primer (40 μM), 0.5 μL BIP Primer (40 μM), 0.5 μL LF primer (20 μM), 0.5 μL LB primer (20 μM), 15 μL ISO-004, 0.25 μL AMV Reverse Transcriptase (RT-001), 2.75 μL sterile distillation A total of 25 μL was prepared by adding water. These adjustments were made on ice. After setting to Genelyzer F (TOSHIBA MEDICAL), the LAMP reaction was stopped by treatment at 63 ° C. for 1 hour and then at 95 ° C. for 2 minutes, and then gradually lowered to 80 ° C. Using SMYEV primer set 1 and SMoV primer set 1 and SVBV primer set 1, determination was performed on the same 9 infected seedlings and 1 healthy (non-infected) seedling as described above. In all cases where the visual judgment was positive, the peak of the fluorescence value due to the association was confirmed.

<Virus detection test (multiple simultaneous detection)>
1) Visual determination A Loopamp (registered trademark) RNA amplification reagent kit (RT-LAMP) (Eiken Chemical) and a Loopamp fluorescence / visual detection reagent (Eiken Chemical) were used. 1.5 μl of nucleic acid extraction sample, 6.25 μL of 2 × Reaction mixture, 0.5 μL of Loopamp fluorescence / visual detection reagent, 0.5 μL of Enzyme mix, 0.125 μL of MYEVF3 primer (20 μM, F3 primer), 125 μL MYEVB3 primer (20 μM, B3 primer), 0.25 μL MYEVFIP primer (80 μM, FIP primer), 0.25 μL MYEVBIP primer (80 μM, BIP primer), 0.25 μL MYEVLF primer (40 μM, LF primer), 0.25 μL MYEVLB primer (40 μM, LB primer), 0.125 μL SMoVF3GC primer (20 μM, F3 primer), 0.125 μL SMoVB3GC primer (20 μM, B3 primer), 0.25 μL SMoVFIPGC primer (80 μM, FIP primer), 0.25 μL SMoVBIPGC primer (80 μM, BIP primer), 0.25 μL SMoVLFGC primer (40 μM, LF primer), 0.25 μL SMoVLGBC primer (40 μM, LB primer), 0.125 μL SVBVF3 primer (20 μM, F3 primer), 0.125 μL SVBVB3 primer (20 μM, B3 primer), 0.25 μL SVBVFIPGC primer (80 μM, FIP primer), 25 μL SVBVBIPGC primer (80 μM, BIP primer), 0.25 μL SVBVLFGC primer (40 μM, LF primer), 0.2 A total of 12.5 μL was prepared by adding 5 μL of SVBVLLBGC primer (40 μM, LB primer) and 0.55 μL of sterile distilled water. These adjustments were made on ice. In order to confirm false positives, the reaction was stopped at 95 ° C. for 2 minutes after treatment at 63 ° C. for 90 minutes. It was visually confirmed that only a positive control showed a positive reaction, and the negative control was confirmed to be a negative reaction. Separately, FIG. 2 shows an example in which the reaction was stopped by treatment at 95 ° C. for 2 minutes after treatment at 63 ° C. for 60 minutes. The results shown in FIG. 2 (simultaneous detection) are shown in Table 14 together with the results obtained when SMYEV primer set 1, SMoV primer set 1 and SVBV primer set 1 were used alone.

  The results shown in FIG. 2 and Table 14 show that it is possible to detect infected seedlings without false positives even when a primer set for specifically amplifying nucleic acids derived from a plurality of strawberry pathogenic viruses is used in combination. By using a primer set together, a sample infected with any virus can be detected quickly and inexpensively.

2) Determination by association curve analysis 3.0 μL of nucleic acid extraction sample, 15 μL of ISO-004, 0.25 μL of AMV Reverse Transscriptase (RT-001), 0.25 μL of MYEVF3 primer (20 μM, F3 primer), 0.25 μL MYEVB3 primer (20 μM, B3 primer), 0.5 μL MYEVFIP primer (40 μM, FIP primer), 0.5 μL MYEVBIP primer (40 μM, BIP primer), 0.25 μL MYEVLF primer (40 μM, LF primer), 0 25 μL MYEVLB primer (40 μM, LB primer), 0.25 μL SMoVF3GC primer (20 μM, F3 primer), 0.25 μL SMoVB3GC primer (20 M, B3 primer), 0.5 μL SMoVFIPGC primer (40 μM, FIP primer), 0.5 μL SMoVBIPGC primer (40 μM, BIP primer), 0.25 μL SMoVLFGC primer (40 μM, LF primer), 0.25 μL SMoVLBGC Primer (40 μM, LB primer), 0.25 μL SVBVF3 primer (20 μM, F3 primer), 0.25 μL SVBVB3 primer (20 μM, B3 primer), 0.25 μL SVBVFIPGC primer (40 μM, FIP primer), 0.25 μL SVBVBIPGC primer (40 μM, BIP primer), 0.25 μL SVBVLFGC primer (40 μM, LF primer), 0.25 μL SVBV BGC primers (40 [mu] M, LB primer) were prepared in a total of 25μL by adding sterile distilled water 0.75MyuL. These adjustments were made on ice. After setting to Genelyzer F and treating at 63 ° C. for 1 hour, the LAMP reaction was stopped by treatment at 95 ° C. for 2 minutes, and then gradually lowered to 80 ° C. Meanwhile, an association curve analysis was performed. The results are shown in FIG. The single infected strain showed one peak, while the superinfected strain showed two peaks.

Cited references
Bhagwat, B., Dickison, V., Ding, X., Walker, M., Bernardy, M., Bouthillier, M., Creelman, A., DeYoung, R., Li, Y., Nie, X., Wang, A., Xiang, Y. and Sanfacon, H. "Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate." Archives of virology 161.6 (2016a): 1657-1663.

Bhagwat, B., Dickison, V., Su, L., Bernardy, M., Wiersma, PA, Nie, X., & Xiang, Y. Molecular characterization of divergent strawberry mild yellow edge virus isolates from eastern Canada. Journal of Phytopathology, 164.9, (2016b): 691-696.

ZHOU, HC, LI, SY, HE, ST, GUO, AG, ZHAO, X., & HAO, SH (2005). Detection of strawberry vein banding virus by PCR and sequence analysis of specific fragment [J]. Journal of Fruit Science, 3, 024.

Claims (9)

In order to amplify nucleic acid derived from one or more strawberry pathogenic viruses selected from the group consisting of strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV) and strawberry vein banding virus (SVBV) by the LAMP method. A primer set comprising at least one selected from the group consisting of the following (1) to (3):
(1) The polynucleotide represented by the sequence of SEQ ID NO: 1 or the polynucleotide represented by the sequence of SEQ ID NO: 1 contains a mutation to the extent that a nucleic acid derived from SMYEV can be amplified, and the mutation is an F2 region (SEQ ID NO: 61 ) And the F1c region (SEQ ID NO: 62), a FIP primer consisting of a polynucleotide that is an insertion of a sequence of 1-6 nucleotides ,
The polynucleotide represented by the sequence of SEQ ID NO: 2 or the polynucleotide represented by the sequence of SEQ ID NO: 2 contains a mutation to the extent that a nucleic acid derived from SMYEV can be amplified, and the mutation includes the B2 region (SEQ ID NO: 63) and B1c A BIP primer consisting of a polynucleotide which is an insertion of a sequence of 1 to 6 nucleotides between the region (SEQ ID NO: 64) ,
An F3 primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 3, a B3 primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 4,
An LF primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 5, and
A primer set comprising an LB primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 6 ,
(2) The polynucleotide represented by the sequence of SEQ ID NO: 7 or the polynucleotide represented by the sequence of SEQ ID NO: 7 contains a mutation to the extent that an SMoV-derived nucleic acid can be amplified, and the mutation is an F2 region (SEQ ID NO: 65 ) And the F1c region (SEQ ID NO: 66), a FIP primer consisting of a polynucleotide that is an insertion of a sequence of 1-6 nucleotides ,
The polynucleotide represented by the sequence of SEQ ID NO: 8 or the polynucleotide represented by the sequence of SEQ ID NO: 8 contains a mutation to the extent that an SMoV-derived nucleic acid can be amplified, and the mutation includes the B2 region (SEQ ID NO: 67) and B1c A BIP primer consisting of a polynucleotide which is an insertion of a sequence of 1-6 nucleotides between the region (SEQ ID NO: 68) ,
An F3 primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 9,
A B3 primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 10,
An LF primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 11, and
LB primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 12
Including primer set,
(3) The polynucleotide represented by the sequence of SEQ ID NO: 13 or the polynucleotide represented by the sequence of SEQ ID NO: 13 contains a mutation to the extent that an SVBV-derived nucleic acid can be amplified, and the mutation is an F2 region (SEQ ID NO: 69 ) And the F1c region (SEQ ID NO: 70), a FIP primer consisting of a polynucleotide that is an insertion of a sequence of 1-6 nucleotides ,
The polynucleotide represented by the sequence of SEQ ID NO: 14, or the polynucleotide represented by the sequence of SEQ ID NO: 14, contains a mutation to the extent that an SVBV-derived nucleic acid can be amplified, and the mutation includes the B2 region (SEQ ID NO: 71) and B1c A BIP primer consisting of a polynucleotide which is an insertion of a sequence of 1-6 nucleotides between the region (SEQ ID NO: 72) ,
An F3 primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 15,
A B3 primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 16,
An LF primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 17, and
A primer set comprising an LB primer consisting of a polynucleotide represented by the sequence of SEQ ID NO: 18 . (1) is a primer set comprising an FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 1 and a BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 2,
(2) is a primer set comprising an FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 7 and a BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 8,
The primer set according to claim 1, wherein (3) is a primer set comprising an FIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 13 and a BIP primer comprising a polynucleotide represented by the sequence of SEQ ID NO: 14. . The primer set according to claim 1 or 2, which is a primer set for amplifying a nucleic acid derived from strawberry mild yellow edge virus (SMYEV), wherein the primer set comprises (1). It is a primer set for amplifying the nucleic acid derived from a strawberry mottle virus (SMoV), The primer set of Claim 1 or 2 in which the said primer set contains said (2). The primer set according to claim 1 or 2, which is a primer set for amplifying a nucleic acid derived from strawberry vein banding virus (SVBV), wherein the primer set comprises (3). The primer set according to claim 1 or 2, comprising any of (1) to (3). A method for detecting a strawberry pathogenic virus,
Extracting nucleic acids from the specimen,
Performing a DNA amplification reaction by the LAMP method using the nucleic acid and the primer set according to any one of claims 1 to 6 ,
The specimen if an amplification product is determined to be comprises determining a strawberry pathogenic virus is present, the detection method. A method for detecting a strawberry pathogenic virus,
Extracting nucleic acids from the specimen,
Performing a DNA amplification reaction by the LAMP method using the nucleic acid and the primer set according to claim 6 ;
Wherein the amplifying association curve analysis to double-stranded DNA after the reaction, includes determining the number of types of pathogenic viruses strawberries for presence and presence of the presence of pathogenic viruses of strawberry in the specimen, the detection method. A kit for detecting a strawberry pathogenic virus, comprising the primer set according to any one of claims 1 to 6 , a strand displacement type DNA synthase, dNTPs, and a buffer.