Detection method and kit for plant viruses using the RT-LAMP method

Abstract

To provide a genetic diagnosis method that detects Japanese pear chlorotic spot-associated virus (JPCSaV), which is considered to be a pathogen of the mosaic symptom, a disease of pear.SOLUTION: A JPCSaV detection or JPCSaV infection diagnostic method includes the step in which a RT-LAMP primer set to one of RNA1-RNA5 of JPCSaV is used to conduct an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a genetic diagnosis method for detecting Japanese pear chlorotic spot-associated virus (JPCSaV), which is believed to be the causative agent of the pear disease "mosaic disease." [Background technology]

[0002] Mosaicism, which causes chlorotic spots on the leaves of Japanese pears, was confirmed to have occurred in Kumamoto Prefecture in 2010 (Non-Patent Document 1). In addition to chlorotic spots, in severe cases, early leaf drop and necrosis on the branch surface and fruit stalk are observed. According to a survey by the present inventors, the occurrence of mosaicism has been confirmed in Japanese pear production fields throughout the country, including Akita Prefecture, by 2019. It has been reported that mosaicism is correlated with infestation by the Japanese pear rust mite, which causes rust symptoms in pears, but on the other hand, the symptoms have led to the possibility that infection with some kind of plant virus may be involved. The present inventors discovered a new virus thought to be of the genus Emaravirus of the family Fimoviridae from Japanese pears exhibiting mosaicism, clarified the full-length sequence of RNA1, named it Japanese pear mosaic-associated virus (JPMaV), and presented it at the 2018 Annual Meeting of the Phytopathological Society of Japan (Non-Patent Document 2) (the sequence itself has not been published). It has been reported that this sequence has been detected not only in Japanese pears exhibiting mosaic disease throughout the country, but also in European pears exhibiting symptoms similar to mosaic disease (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Sugiura, Naoyuki (2015) Mosaic symptoms (tentative name) on pears caused by the false pear rust mite and their occurrence. https: / / www.pref.kumamoto.jp / common / UploadFileOutput.ashx?c_id=3&id=9980&sub_id=2&flid=32942 [Non-Patent Document 2] Yuya Chiaki et al. (2018) Emaravirus-like sequences detected in mosaic-infected Japanese pear leaves. Annual Meeting of the Phytopathological Society of Japan 2018. [Non-Patent Document 3] Tanizawa, S. et al. (2018) Emaravirus-like sequences detected in association with pear mosaic disease in Niigata Prefecture. 2018 Kanto Branch Meeting of the Phytopathological Society of Japan Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the above-mentioned circumstances, the present invention aims to provide a method for accurately detecting and diagnosing the virus by deciphering the full-length genome sequence of JPMaV other than RNA1, designing primers based on these sequences, and creating a detection and diagnostic method using RT-LAMP.

[0005] In order to more accurately describe the symptoms on pear leaves, the name JPMaV has been changed to Japanese pear chlorotic spot-associated virus (JPCSaV), and will be referred to as "JPCSaV" hereafter. Furthermore, "JPCSaV" may also be referred to as pear chlorotic leaf spot-associated virus (PCLSaV). [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, in addition to RNA1, the base sequences of JPCSaV RNAs 2 to 5 were identified, and primers for RT-LAMP corresponding to the base sequences of the identified JPCSaV RNA segments were designed. It was found that by using these primers and the RT-LAMP technique, JPCSaV can be detected specifically and with high sensitivity, which led to the completion of the present invention.

[0007] That is, the present invention includes the following. (1) A primer set for amplifying a nucleotide sequence specific to JPCSaV by RT-LAMP, comprising F3 primer, B3 primer, FIP primer, and BIP primer for any one of RNA1 to RNA5 of JPCSaV, each of which has the nucleotide sequence set forth in SEQ ID NOs: 1 to 5, or the above four primers and a Floop primer and / or a Bloop primer. (2) For JPCSaV RNA1, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:6; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:7; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:8; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:9; The primer set according to (1), (3) The primer set according to (2), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 10 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO: 11. (4) For JPCSaV RNA1, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:12; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:13; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 14; and A BIP primer consisting of the base sequence set forth in SEQ ID NO: 15; The primer set according to (1), (5) The primer set according to (4), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 16 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO: 17. (6) For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:18; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:19; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 20; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 21; The primer set according to (1), (7) The primer set according to (6), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 22. (8) For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:23; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:24; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 25; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 26; The primer set according to (1), (9) The primer set according to (8), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 27 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO: 28. (10) For JPCSaV RNA3, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:29; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO: 30; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 31; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 32; The primer set according to (1), (11) The primer set according to (10), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 33. (12) For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO: 34; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:35; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 36; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 37; The primer set according to (1), (13) The primer set according to (12), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:38. (14) For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:39; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:40; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 41; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 42; The primer set according to (1), (15) The primer set according to (14), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 43 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO: 44. (16) For JPCSaV RNA5, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:45; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:46; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 47; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 48; The primer set according to (1), (17) The primer set according to (16), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 49. (18) A kit for detecting JPCSaV or diagnosing JPCSaV infection by RT-LAMP, comprising the primer set according to any one of (1) to (17). (19) A method for detecting JPCSaV or diagnosing JPCSaV infection, comprising a step of carrying out an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP using the primer set according to any one of (1) to (17) or the kit according to (18). (20) A primer set according to any one of (1) to (17), For the mitochondrial COI gene of Acanthurus pseudomonas, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:51; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:52; A FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 53; and A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:54; A primer set for amplifying a base sequence specific to the false pear rust mite by LAMP, comprising: A combination of primer sets for amplifying a base sequence specific to JPCSaV by RT-LAMP and amplifying a base sequence specific to Aedes ricinus by LAMP, comprising: (21) A combination of the primer sets according to (20), further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 55 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO: 56, for the mitochondrial COI gene of Aedes ricinus. (22) A kit for detecting JPCSaV or diagnosing JPCSaV infection by RT-LAMP and detecting Aedes robinia fuscata by LAMP, comprising a combination of the primer sets according to (20) or (21). (23) A method for detecting JPCSaV or diagnosing JPCSaV infection and detecting Aedes difficile, comprising the steps of: performing an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP; and performing an amplification reaction of a target nucleic acid region of Aedes difficile by LAMP, using a combination of primer sets described in (20) or (21) or a kit described in (22). Effect of the Invention

[0008] According to the present invention, JPCSaV, which is thought to be the pathogen of the disease "mosaic disease," can be easily and quickly detected from pear trees. Furthermore, according to the present invention, infected trees can be identified and removed in pear orchards to prevent the creation of new infected trees, and the presence or absence of JPCSaV infection can be quickly detected at the site of import / export of fruits, cuttings, etc. [Brief description of the drawings]

[0009] [Figure 1] A schematic diagram of the JPCSaV genomic RNA is shown. [Figure 2-1] The full-length sequence of JPCSaV genomic RNA (full-length base sequences of JPCSaV segments RNA1 to RNA5) is shown. The base sequence is written in the 5' to 3' direction. The underlined base sequence indicates the open reading frame, and because it is a minus-strand RNA virus, it is oriented from the C-terminus to the N-terminus. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 2-4] This is a continuation of Figure 2-3. [Figure 2-5] This is a continuation of Figure 2-4. [Figure 2-6] This is a continuation of Figure 2-5. [Diagram 2-7] This is a continuation of Figure 2-6. [Figure 3-1] 1 shows a primer list according to the present invention. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4] The primer map of the present invention on the JPCSaV genome is shown. The black and white arrowheads indicate the F3 and B3 primers, respectively, and the white boxes between them indicate the amplification products. The names of the primer sets are written below them. [Figure 5-1] 1 shows the positions and sequences of the primers according to the present invention on the JPCSaV genome sequence. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 5-4] This is a continuation of Figure 5-3. [Figure 5-5] This is a continuation of Figure 5-4. [Figure 5-6] Continuation of Figure 5-5. [Figure 5-7] This is a continuation of Figure 5-6. [Figure 6-1] 1 shows the results of the RT-LAMP reaction in Example 1. "CS" indicates leaves with chlorotic spots, and "H" indicates healthy pear leaves. Panel A shows a turbidity amplification curve graph, and Panel B shows the LAMP reaction time (min). "nd" indicates non-detection. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 6-3] This is a continuation of Figure 6-2. [Figure 7] Photographs showing virus sampling using the needle method. [Figure 8] This shows the results of the simple RT-LAMP method (syringe method) in Example 1. The values ​​in parentheses indicate the reaction time (minutes). "nd" indicates non-detection. [Figure 9] Photographs showing the color of reaction tubes after RT-LAMP reaction in Example 1. Panel A shows the RT-LAMP reaction solution of normal composition, and Panel B shows the RT-LAMP reaction solution with added malachite green. The samples are as follows: 1: Aichi CS-1, 2: Aichi CS-2, 3: Aichi H, 4: Ibaraki CS, 5: Ibaraki H, 6: Fig FMV, 7: Shiso PMoV. [Figure 10-1] The partial sequence alignment of the Japanese (Ibarak-2018, Aichi-2020) and Chinese (FJ, HB, CG1) isolates of PCLSaV in the amplified region of each RT-LAMP primer and the annealing position of the RT-LAMP primer are shown. The arrow indicates the extension direction. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-2. [Figure 10-4] This is a continuation of Figure 10-3. [Figure 10-5] This is a continuation of Figure 10-4. [Figure 10-6] Continuation of Figure 10-5. [Figure 10-7] Continuation of Figure 10-6. [Figure 10-8] This is a continuation of Figure 10-7. [Figure 10-9] Continuation of Figure 10-8. [Figure 10-10] Continuation of Figure 10-9. [Figure 10-11] Continuation of Figure 10-10. [Figure 10-12] Continuation of Figure 10-11. [Figure 10-13] Continuation of Figure 10-12. [Figure 11] 1 shows the results of RT-LAMP reaction of PCLSaV in Aedes ricinus in Example 3. The individual "CS" represents an individual collected from a leaf with chlorotic spots, and "H" represents an individual collected from a healthy pear leaf. [Figure 12] The LAMP primer (EC5) for detecting Acanthurus nigricans is shown. [Figure 13] The position of the EC5 primer on the mitochondrial COI gene of A. cernua is shown. [Figure 14] 1 shows the results of LAMP detection of Aegilops fuscata using the EC5 primer in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in detail below. The primer set according to the present invention is a primer set for amplifying a base sequence specific to JPCSaV by RT-LAMP, which includes a primer for RT-LAMP that selectively hybridizes with a base sequence specific to JPCSaV.

[0011] JPCSaV is a negative single-stranded RNA virus belonging to the Emaravirus genus of the Fimoviridae family, whose genome consists of a single-stranded negative RNA consisting of five segments (RNA1 to 5).

[0012] RT-LAMP using a primer set for RT-LAMP prepared based on the nucleotide sequences of RNA1 to 5 of JPCSaV sequenced by the present inventors can detect JPCSaV in a sample derived from a pear with high sensitivity and speed. In addition, RT-LAMP using the primer set according to the present invention can detect PCLSaV detected in leaves exhibiting mosaic disease in Chinese pears.

[0013] The primer set according to the present invention is a primer set for amplifying a nucleotide sequence specific to JPCSaV by RT-LAMP, which includes a primer for RT-LAMP for any one of JPCSaV RNAs 1 to 5, each of which has a nucleotide sequence as set forth in SEQ ID NOs: 1 to 5, as shown in Figures 1 and 2. In the present invention, the nucleotide sequence of the JPCSaV RNA segment is represented by DNA (i.e., the nucleotide sequence of the JPCSaV genomic RNA is represented by DNA by converting U to T).

[0014] In the present invention, the term "RT-LAMP primer for the RNA segment of JPCSaV" refers to a primer used in RT-LAMP, which is designed based on the base sequence of the RNA segment of JPCSaV. The primer set according to the present invention can be obtained by designing RT-LAMP primers based on the sequence information of the RNA segment of JPCSaV using the primer design support software PrimerExplorer V5 (http: / / primerexplorer.jp / ).

[0015] The nucleic acid amplification method of the present invention, LAMP (Loop-Mediated Isothermal Amplification), is a loop-mediated isothermal amplification method developed by Notomi et al. that does not require temperature control, which is essential in the PCR method (International Publication No. 00 / 28082 pamphlet; Eiken Chemical Co., Ltd. website (http: / / loopamp.eiken.co.jp / lamp / )). This method is a nucleic acid amplification method that enables isothermal complementary chain synthesis reaction by annealing its own 3' end to a template nucleotide to serve as the origin of complementary chain synthesis, and combining a primer that anneals to the loop formed at this time. In addition, in LAMP, the 3' end of the primer always anneals to a region derived from the sample, so that the check mechanism by complementary binding of the base sequence functions repeatedly, which results in a highly sensitive and highly specific nucleic acid amplification reaction.

[0016] RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) is a nucleic acid amplification method in which LAMP is performed while synthesizing cDNA from the target gene, RNA.

[0017] The primers used in LAMP are at least four types of primers that recognize the base sequences of a total of six regions of the base sequence of the template nucleic acid, namely, the regions F3c, F2c, and F1c from the 3'-end side, and the regions B3, B2, and B1 from the 5'-end side, and are called inner primers F (FIP) and B (BIP), and outer primers F (F3) and B (B3), respectively. The complementary sequences of F1c, F2c, and F3c are called F1, F2, and F3, respectively, and the complementary sequences of B1, B2, and B3 are called B1c, B2c, and B3c. The inner primer is an oligonucleotide that recognizes a "certain specific nucleotide sequence region" on the target base sequence, has a base sequence at the 3'-end that provides a synthesis origin, and at the same time has a base sequence at the 5'-end that is complementary to any region of the nucleic acid synthesis reaction product that starts from this primer. Here, a primer containing the "F2 region" and the "F1c region" is called the inner primer F (FIP), and a primer containing the "B2 region" and the "B1c region" is called the inner primer B (BIP). On the other hand, an outer primer is an oligonucleotide having a base sequence that recognizes "a certain nucleotide sequence region present on the 3'-end side of a 'certain nucleotide sequence region'" on a target base sequence and provides a synthesis origin. Here, a primer containing the "F3 region" is called the outer primer F (F3), and a primer containing the "B3 region" is called the outer primer B (B3).

[0018] In RT-LAMP, the FIP primer is first annealed to the RNA segment of JPCSaV, and complementary cDNA is synthesized from it by reverse transcriptase. The FIP primer has an "F2 region" at the 3' end that is complementary to the "F2c region" on the RNA segment of JPCSaV, and a region identical to the "F1c region" on the RNA segment of JPCSaV at the 5' end.

[0019] Next, the F3 primer located outside the FIP anneals to the complementary sequence "F3c region" on the RNA segment of JPCSaV, and the action of reverse transcriptase peels off the single-stranded DNA extended from the already synthesized FIP while extending from the F3 primer. The F3 primer has an "F3 region" complementary to the "F3c region" on the RNA segment of JPCSaV. The single-stranded DNA from the peeled FIP forms a loop by self-annealing because the "F1c region" on the 5' end of FIP is complementary to the "F1 region" of the extended portion.

[0020] The other inner primer, BIP primer, anneals to the single-stranded DNA that has formed the loop, and is extended from there by the action of strand-displacement DNA polymerase. The BIP primer has a region identical to the "B2 region" on the RNA segment of JPCSaV at the 3' end, and a "B1c region" complementary to the "B1 region" on the RNA segment of JPCSaV at the 5' end. In addition, strand-displacement DNA polymerase means a DNA polymerase that can synthesize a complementary strand while dissociating the double strand when a double-stranded region exists in the extension direction.

[0021] Furthermore, the B3 primer located outside the BIP primer anneals to the single-stranded DNA that has formed the loop, and the single-stranded DNA is stripped from the already synthesized BIP primer by the action of strand-displacement DNA polymerase, while being extended from the B3 primer. The B3 primer has the same region as the "B3 region" on the RNA segment of JPCSaV. The single-stranded DNA extended from the stripped BIP has complementary sequences at both ends, so each of them self-anneals to form a loop. In other words, the single-stranded DNA extended from the stripped BIP forms a dumbbell-shaped structure by forming loops at both ends. This dumbbell-shaped structure becomes the starting structure for the amplification cycle in LAMP, and a series of amplification cycles are carried out.

[0022] In LAMP, in addition to the inner primer and the outer primer, another primer, i.e., a loop primer, can be used. The loop primers Floop (LF) and / or Bloop (LB) are primers having a base sequence complementary to the base sequence of the single-stranded portion of the loop structure on the 5'-end side of the dumbbell-shaped structure. The use of these primers increases the number of origins of nucleic acid synthesis, shortening the reaction time and increasing the detection sensitivity (International Publication No. 02 / 24902). The base sequence of the loop primer may be selected from the base sequence of the target gene or its complementary strand, or may be another base sequence, so long as it is complementary to the base sequence of the single-stranded portion of the loop structure on the 5'-end side of the dumbbell-shaped structure described above. The loop primer may be of one type or two types.

[0023] The length of each primer in the primer set described above is 10 bases or more, preferably 15 bases or more, and may be either chemically synthesized or natural, and each primer may be a single oligonucleotide or a mixture of multiple oligonucleotides.

[0024] As specific primer sets according to the present invention, as shown in Figures 3 to 5, we conducted extensive research into the base sequences and combinations of RT-LAMP primers that can rapidly amplify base sequences specific to JPCSaV, and as a result, we selected the following eight primer sets using JPCSaV RNAs 1 to 5 as target sequences.

[0025] (1) Primer set R1LP1: For JPCSaV RNA1, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:6; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:7; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:8; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:9; Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:10; Bloop primer consisting of the nucleotide sequence set forth in SEQ ID NO:11.

[0026] (2) Primer set R1LP2: For JPCSaV RNA1, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:12; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:13; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 14; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15; Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:16; Bloop primer consisting of the nucleotide sequence set forth in SEQ ID NO:17.

[0027] (3) Primer set R2LP3: For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:18; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:19; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 20; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:21; A Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:22.

[0028] (4) Primer set R2LP4: For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:23; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:24; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 25; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:26; Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:27; Bloop primer consisting of the nucleotide sequence set forth in SEQ ID NO:28.

[0029] (5) Primer set R3LP5: For JPCSaV RNA3, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:29; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO: 30; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:31; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:32; A Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:33.

[0030] (6) Primer set R4LP6: For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO: 34; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO: 35; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 36; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:37; A Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:38.

[0031] (7) Primer set R4LP7: For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:39; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:40; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:41; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:42; Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:43; Bloop primer consisting of the nucleotide sequence set forth in SEQ ID NO:44.

[0032] (8) Primer set R5LP8: For JPCSaV RNA5, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:45; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:46; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:47; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:48; A Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:49.

[0033] Alternatively, each primer may alternatively include a primer having a base sequence in which one or several (e.g., 1 to 10) bases are deleted, substituted or added in the base sequence shown in the sequence number of each primer, and having the respective primer function in LAMP.

[0034] Furthermore, the primer set according to the present invention can be provided as a kit for detecting JPCSaV or diagnosing JPCSaV infection by RT-LAMP, either alone or together with reagents necessary for RT-LAMP, such as strand-displacing DNA polymerase, reverse transcriptase, dNTP, buffer, sterilized water, and reagents necessary for detecting the reaction product.

[0035] The strand-displacing DNA polymerase is not particularly limited as long as it is a template-dependent nucleic acid polymerase having strand-displacing activity. Examples of such enzymes include Bst DNA polymerase (Nippon Gene Co., Ltd.), Bca (exo-) DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase I, Csa DNA polymerase (Nippon Gene Co., Ltd.), and 96-7 DNA polymerase (Nippon Gene Co., Ltd.).

[0036] The reverse transcriptase is not particularly limited as long as it has the activity of synthesizing DNA using RNA as a template, and examples of such enzymes include AMV reverse transcriptase (Promega Corporation), M-MLV reverse transcriptase (Promega Corporation), Recombinant HIV reverse transcriptase, SuperscriptII / III / IV, ReverTraAce, Thermoscript, Ominiscript, and Sensiscript.

[0037] Furthermore, when an enzyme having both reverse transcriptase activity and DNA polymerase activity, such as Bca DNA polymerase, is used, RT-LAMP can be performed with a single enzyme.

[0038] Furthermore, the present invention relates to a method for detecting JPCSaV or diagnosing JPCSaV infection (hereinafter referred to as "the method"), which uses the above-described primer set or kit according to the present invention to carry out an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP to detect or quantify JPCSaV. In other words, the presence or absence of JPCSaV infection can be examined or evaluated by the detection or quantification.

[0039] In this method, RNA is first extracted and purified from a sample derived from a pear to be detected for the presence or absence of JPCSaV infection. Examples of samples derived from pear include the whole plant, plant organs (e.g., leaves, petals, petals, stems, roots, seeds, fruits, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, etc.), and plant cultured cells. These samples may show mosaic or necrotic symptoms or may be asymptomatic. The most suitable sample for detection is a leaf, and the detection rate can be increased by using a mosaic site. In addition, the sample may be the false pear rust mite itself, which is presumed to be a vector of JPCSaV and collected from a pear. Examples of RNA extraction methods include a method using a commercially available RNA detection reagent, and a method in which the sample is crushed and diluted with Tris buffer or sterilized water to obtain crude purified RNA. Furthermore, as a simple method, the sample may be poked with the tip of a sharp instrument such as a toothpick or a syringe needle about 10 times and the sap attached to the tip may be used as a template as it is.

[0040] The resulting template RNA is then subjected to RT-LAMP. The RT-LAMP reaction solution contains, for example, 1.0 to 50 pmol of each primer contained in the primer set according to the present invention, 0.02 fg to 4 μg of template RNA, 0.5 to 15 U (units) of strand-displacing DNA polymerase, 1.0 to 10 U of reverse transcriptase, and dNTPs at a final concentration of 1.0 to 10 mM per 25 μl of reaction solution. The amplification reaction conditions for RT-LAMP include, for example, a temperature of 60° C. to 70° C. (preferably 63° C. to 65° C.) for 15 to 90 minutes (preferably 20 to 40 minutes).

[0041] Known techniques can be applied to detect the nucleic acid amplification products after the RT-LAMP reaction. For example, the turbidity generated by magnesium pyrophosphate, a by-product of nucleic acid synthesis, can be visually confirmed, or optically confirmed using a real-time turbidity measuring device (e.g., LoopampEXIA manufactured by Eiken Chemical Co., Ltd.). In addition, by adding malachite green to the reaction solution in advance and performing RT-LAMP, visual determination can be facilitated based on the color of the reaction solution (light blue: amplification, colorless: no amplification) (Nzelu et al. 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Tropica 132:1-6.).

[0042] The present invention also relates to a combination of the primer set for amplifying the nucleotide sequence specific to JPCSaV according to the present invention described above and a primer set for amplifying a nucleotide sequence specific to Aesthetica fasciata in the mitochondrial COI gene of Aesthetica fasciata by LAMP. With this combination, the nucleotide sequence specific to JPCSaV is amplified by RT-LAMP, while the nucleotide sequence specific to Aesthetica fasciata is amplified by LAMP, making it possible to confirm the occurrence, infestation, or absence of Aesthetica fasciata, a virulent insect, in pears.

[0043] The following EC5 primer set can be used as a primer set for amplifying a nucleotide sequence specific to Aedes ricinus by LAMP: EC5 primer set: F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:51; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:52; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:53; A BIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:54; Floop primer consisting of the nucleotide sequence set forth in SEQ ID NO:55; Bloop primer consisting of the nucleotide sequence set forth in SEQ ID NO:56.

[0044] Alternatively, each primer may alternatively include a primer having a base sequence in which one or several (e.g., 1 to 10) bases are deleted, substituted or added in the base sequence shown in the sequence number of each primer, and having the respective primer function in LAMP.

[0045] Furthermore, the present invention relates to a kit for detecting JPCSaV or diagnosing JPCSaV infection by RT-LAMP and detecting Aedes ricinus by LAMP, comprising the combination of the primer set. The kit can include the reagents and the like described above in the kit for detecting JPCSaV or diagnosing JPCSaV infection.

[0046] The present invention also relates to a method for detecting JPCSaV or diagnosing JPCSaV infection and detecting Aedes difficile, comprising a step of carrying out an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP and a step of carrying out an amplification reaction of a target nucleic acid region of Aedes difficile by LAMP using the combination of the primer set or a kit containing the combination. The amplification reaction of the target nucleic acid region of Aedes difficile can be carried out by LAMP in accordance with the RT-LAMP explained in the present method above. EXAMPLES

[0047] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0048] [Example 1] Detection of JPCSaV by RT-LAMP method using the primer set according to the present invention 1. Deciphering the full-length nucleotide sequence of the JPCSaV genome Since viruses of the Emaravirus genus (hereafter referred to as "Emaravirus") are known to have 5 to 9 RNA segments, JPCSaV should also have segments other than RNA 1. Taking advantage of the fact that within a single Emaravirus species, approximately 12 base sequences at the 5' and 3' ends of each RNA segment are common between segments, all RNA segments were amplified from mosaic-affected leaves by RT-PCR using primers targeting the terminal sequence of RNA 1, and the full-length sequence was determined by cloning into Escherichia coli and sequencing.

[0049] A total of five segmented RNAs were found, which were named RNA1 to RNA5 (Figure 1). In addition, comprehensive analysis was performed using next-generation sequencing technology on RNA extracted from mosaic-infected pear leaves to search for RNA with emaravirus-like sequences. Sequences similar to the above RNA1 to RNA5 were obtained, but no other sequences that could be considered emaraviruses were found.

[0050] 2. Design of primers for RT-LAMP Primers suitable for RT-LAMP amplification were designed based on the full-length sequences of RNA1 to RNA5 (Figure 2) using PrimerExplorer V5 (http: / / primerexplorer.jp / ). Two sets of primers were designed from RNA1, RNA2, and RNA4, and one set each from RNA3 and RNA5 (Figure 3). The positions of the primers on the JPCSaV genome are shown in Figures 4 and 5.

[0051] 3. Implementation of RT-LAMP Pear leaves showing chlorotic spots, a symptom of mosaic disease, and non-shown pear leaves were used, as well as two types of emaraviruses occurring in Japan, fig mosaic virus (FMV) and perilla mosaic virus (PMoV).

[0052] Pear leaves showing and not showing chlorotic spot symptoms were sampled from the "Kosui" variety of pear at the National Agriculture and Food Research Organization's Fruit Tree and Tea Research Division in Tsukuba, Ibaraki Prefecture, and at a pear field in Toyokawa, Aichi Prefecture, in 2019. Fig and perilla leaves were sampled from leaves that had been confirmed in advance to be infected with FMV and PMoV, respectively.

[0053] RNA extraction from pear leaves was performed according to Gambino et al. (2008) (Gambino et al. (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem. Anal. 19: 520-525.), and RNA extraction from fig and perilla leaves was performed using Trizol Reagent (ThermoScientific).

[0054] The RT-LAMP reaction solution was made up of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 0.8 M betaine (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mM MgSO4, 1.4 mM dNTPs, 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, 0.8 μM FLoop and BLoop primers, 8 units Bst DNA polymerase (Nippon Gene), and 1 unit AMV reverse transcriptase (Promega) to which 1 μl of template RNA was added to make up a total volume of 20 μl.

[0055] Using a real-time turbidity meter LA200 (Teramecs), the RT-LAMP reaction was performed at 63°C or 65°C for 60 minutes, and the generation of turbidity due to magnesium pyrophosphate, a by-product of nucleic acid amplification, was recorded and the time for the turbidity to reach 0.1 (LAMP reaction time) was measured (Figure 6).

[0056] 4. Simple RT-LAMP method using a syringe needle The enzyme used in RT-LAMP is said to be less susceptible to DNA amplification inhibition, and one of the advantages of RT-LAMP is that it can be used with easily prepared samples. Therefore, we investigated whether the syringe needle method, which is the simplest template preparation method, can be applied.

[0057] As above, a 23G injection needle (Terumo) was pierced about 10 times into leaves showing chlorotic spot symptoms and non-symptomatic leaves taken from the "Kosui" variety of pears collected at the Fruit Tree and Tea Research Institute of the National Agriculture and Food Research Organization in Tsukuba, Ibaraki Prefecture, and at a pear field in Toyokawa, Aichi Prefecture (Figure 7). The tip of the needle with the sap on it was immersed in the reaction solution prepared as above, and the RT-LAMP reaction was carried out at 63°C for 60 minutes (Figure 8). The primer set used was R2LP4. As controls, fig leaves infected with FMV and perilla leaves infected with PMoV were treated in the same way.

[0058] 5. Visually check the RT-LAMP reaction In addition to using a turbidity meter as described above, the positive or negative result of the RT-LAMP reaction can also be confirmed by visually observing whether the reaction solution is turbid or not. However, since turbidity of the reaction solution is generally difficult to see, adding HNB reagent or malachite green can make visual judgment easier.

[0059] Therefore, we used total RNA from leaves showing chlorotic spot symptoms and non-showing leaves collected from the "Kosui" variety of pear at the Fruit Tree and Tea Research Division of the National Agriculture and Food Research Organization in Tsukuba, Ibaraki Prefecture, and the pear fields in Toyokawa, Aichi Prefecture, as templates, added malachite green, and performed a RT-LAMP reaction to investigate whether visual diagnosis was possible.

[0060] Malachite green was added to the reaction solution to a final concentration of 0.008%, and the other components were prepared in the same manner as above. The RT-LAMP reaction was carried out at 63°C in an incubator. The primer set used was R2LP4. After 60 minutes, the tube was removed from the incubator, and if the reaction solution was light blue, it was judged to be positive, and if it was colorless, it was judged to be negative (Figure 9).

[0061] 6. Discussion In the graph shown in Figure 6, all eight primer sets tested gave positive reactions in pear leaves with chlorotic spot disease in Aichi and Ibaraki. In addition, except for R2LP3, no turbidity was detected in healthy leaves, FMV-infected fig leaves, or PMoV-infected perilla leaves. R2LP3 gave turbidity at approximately 57 minutes in healthy leaves in Aichi, but this was clearly distinguishable from a typical positive reaction that occurs within 30 minutes, and was therefore not considered to be a problem in practical use. Of the eight primer sets, R2LP4 and R4LP6, which have relatively fast reaction times, were considered to be the most suitable for specific detection.

[0062] Figure 8 shows the results of a simple RT-LAMP using the needle method with the R2LP4 primer set. A positive reaction was confirmed in about 20 minutes from all leaves showing chlorotic spot symptoms, while no reaction occurred from healthy leaves, FMV-infected fig leaves, or PMoV-infected perilla leaves. This suggests that the needle method can be used to detect JPCSaV.

[0063] Figure 9 shows an image of the reaction solution after the LAMP reaction using the same sample as in Figure 6. In leaves in Aichi and Ibaraki that showed chlorotic spot symptoms, the reaction solution turned cloudy or light blue, making it possible to judge whether the symptom was positive or negative. In particular, when malachite green was added, visibility was clearly improved, making it easier to judge whether the symptom was positive or negative.

[0064] From the above, it was determined that RT-LAMP using the primer set according to the present invention is practical as a method for detecting and diagnosing JPCSaV.

[0065] [Example 2] Possibility of LAMP detection of PCLSaV Japanese and Chinese isolates using RT-LAMP primers Figure 10 shows the alignment of partial sequences of Japanese (Ibarak-2018, Aichi-2020) and Chinese (FJ, HB, CG1) isolates of PCLSaV in the amplified region of each RT-LAMP primer, as well as the annealing positions of the RT-LAMP primers.

[0066] In the LAMP (RT) reaction, the F2 region of FIP or the B2 region of BIP anneals to the target sequence to start the amplification reaction. Therefore, if there is a mutation at the 3' end of the F2 or B2 region, the 5' end of the F1c or B1c region, or the 3' end of the F3 or B3 region, the amplification reaction may be inhibited. When examining the annealing sites of each RT-LAMP primer up to the 5th base of these sequences, R1LP2, R3LP5, and R4LP7 did not show any mutations that would affect amplification, and it was thought that both Japanese and Chinese isolates could be detected. In addition, R1LP1 showed a mutation at the 4th base from the 3' end of the B2 region in the Chinese isolate HB, and R2LP4 showed a mutation at the 5th base from the 5' end of the B1c region in the Chinese isolate FJ and CG1, respectively, but the mutation site was far from the very end and each mutation was only one base, so it was thought that Chinese isolates could also be detected. On the other hand, R2LP3 showed mutations at the second base from the 3' end of the F2 and B2 regions, and at the first base from the 3' end of the B3 region in the Chinese isolate CG1. R4LP6 showed mutations at the third base from the 3' end of the F3 region and at the second base from the 3' end of the B3 region in the Chinese isolate CG1. R5LP8 showed mutations at the second base from the 3' end of the B2 region in the Chinese isolate FJ, and at the third base from the 3' end of the B2 region and at the fourth base from the 3' end of the B3 region in the Chinese isolate CG1. These results suggest that the reaction of the three primers R2LP3, R4LP6, and R5LP8 may be delayed or amplification may be inhibited in some Chinese isolates.

[0067] [Example 3] Detection of PCLSaV from Pear Rust Mites Since the false pear rust mite is extremely small, with a body length of less than 0.2 mm, it was expected that various careless mistakes would occur during the process of extracting the virus. Therefore, it was thought that the method using glue would be effective as a simple and reliable way to extract and detect the virus from a single rust mite.

[0068] A small amount of glue stick was applied to the back of the cap of an 8-strip PCR tube with a toothpick. A flat cap was stable and easy to use. Next, while observing under a stereomicroscope, a single false pear rust mite was fished out with a hypodermic needle and attached to the glue on the back of the cap. The rust mite could be fished out by simply touching the needle lightly to the mite. While observing under the stereomicroscope, the rust mite was crushed with the same hypodermic needle. Next, to detect PCLSaV, a LAMP reaction solution containing R2LP4 primer was prepared and dispensed into an 8-strip PCR tube, the cap with the rust mite attached was gently placed on top, and the tube was gently inverted several times to bring the reaction solution into contact with the crushed rust mite. The tube was then incubated at 63°C for 1 hour to perform the RT-LAMP reaction and test for the presence or absence of the virus.

[0069] Figure 11 shows the results of the RT-LAMP reaction of PCLSaV in P. cernua. The individual "CS" represents an individual collected from a leaf with chlorotic spots, and "H" represents an individual collected from a healthy pear leaf. PCLSaV was detected in all P. cernua. individuals collected from pear leaves with chlorotic spots. However, PCLSaV was not detected in P. cernua. collected from healthy leaves. Based on this, it was thought that PCLSaV in P. cernua could be detected by attaching crushed P. cernua to the inside of the lid of an 8-strip PCR tube and contacting it with the LAMP reaction solution.

[0070] [Example 4] Detection of Rust Mites by LAMP Method The method for detecting PCLSaV accumulated in the body of A. cernua, which is thought to transmit PCLSaV, by the RT-LAMP method is as shown in Example 1. On the other hand, in Japan, other rust mites (eriophyid mites) that parasitize pears have been reported, including A. cernua and A. cernua (Fujio Kamitou (1995) Taxonomic study of woody plant parasitic eriophyid mites in Japan and ecological study of A. cernua parasitic on pears, Special Report of Chiba Agricultural Experiment Station 30 1-87), and even if most of the rust mites commonly found in pear cultivation fields are A. cernua, it is first necessary to identify the species by genetic diagnosis and then investigate the status of PCLSaV infection.

[0071] Therefore, the method for detecting A. japonica using the LAMP method is described below. Based on the registered sequence of the mitochondrial COI gene region of A. japonica (GenBank Accession No. LC325506) (SEQ ID NO: 50), a LAMP primer (EC5) for detecting A. japonica was designed using the primer design support software PrimerExplorer V5 (http: / / primerexplorer.jp / ) (Figure 12). The position of the EC5 primer on the mitochondrial COI gene is shown in Figure 13.

[0072] Pear rust mites were collected from the leaves of the "Kosui" variety in a pear field in Nagakute City, Aichi Prefecture in June 2020. DNA from the false pear rust mites was extracted from one mite each according to a previously reported method for extracting DNA from perilla rust mites (National Agriculture and Food Research Organization, National Agricultural Research Center (ed.) (2020) Manual for Detection of Perilla Rust Mites and Perilla Mosaic Virus in Large-leaved Plants, 3rd Edition, p. 34). Perilla rust mites and tomato rust mites were collected in groups of 10 each from the leaves of cultivated tomatoes or large-leaved plants in Nagakute City, Aichi Prefecture, and DNA was extracted. The LAMP reaction solution was prepared by mixing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 0.8 M betaine (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mM MgSO4, 1.4 mM dNTPs, 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, 0.8 μM FLoop and BLoop primers, and 8 units Bst DNA polymerase (Nippon Gene Co., Ltd.), and adding 1 μl of template RNA to make a total volume of 20 μl. The LAMP reaction was carried out at 65°C for 60 minutes using a real-time turbidity meter LA200 (Teramecs), and the time until the turbidity reached 0.1 was measured while recording the increase in turbidity (LAMP reaction time).

[0073] Figure 14 shows the results of LAMP detection of A. cernua using EC5 primers. A positive reaction was confirmed in about 20 minutes for all of the A. cernua tested, A to F, but no reaction was observed for A. perilla and A. tomato. This suggests that A. cernua can be specifically detected by the LAMP method using EC5 primers. [Industrial Applicability]

[0074] The present invention is not only useful for identifying, removing, and controlling JPCSaV-infected trees in pear production fields, but can also be used as an inspection method for pear seedling production and for ensuring the soundness of exported cuttings. Pear fruits and cuttings are exported in large quantities, and it is necessary to have an established inspection method in case of an inspection request from the export destination country, so it is expected that the method will be used by exporters, plant protection stations, etc. In addition, if JPCSaV occurs overseas, it is expected that the method will also be used overseas.

Claims

1. A primer set for amplifying a base sequence specific to Japanese pear chlorotic spot associated virus (JPCSaV) by RT-LAMP, comprising F3 primer, B3 primer, FIP primer and BIP primer for any one of RNA2 to RNA5 of Japanese pear chlorotic spot associated virus (JPCSaV), each of which has the base sequence set forth in SEQ ID NO: 2 to 5, or the above four primers and a Floop primer and / or a Bloop primer.

2. For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:18; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:19; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 20; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:21; The primer set according to claim 1 .

3. The primer set according to claim 2, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:

22.

4. For JPCSaV RNA2, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:23; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:24; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 25; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:26; The primer set according to claim 1 .

5. The primer set according to claim 4, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:27 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO:

28.

6. For JPCSaV RNA3, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:29; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:30; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO:31; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:32; The primer set according to claim 1 .

7. The primer set according to claim 6, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:

33.

8. For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:34; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:35; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 36; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:37; The primer set according to claim 1 .

9. The primer set according to claim 8, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:

38.

10. For JPCSaV RNA4, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:39; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:40; An FIP primer consisting of the base sequence set forth in SEQ ID NO: 41; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:42; The primer set according to claim 1 .

11. The primer set according to claim 10, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO: 43 and / or a Bloop primer consisting of the base sequence set forth in SEQ ID NO:

44.

12. For JPCSaV RNA5, F3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:45; B3 primer consisting of the nucleotide sequence set forth in SEQ ID NO:46; An FIP primer consisting of the nucleotide sequence set forth in SEQ ID NO: 47; and A BIP primer consisting of the base sequence set forth in SEQ ID NO:48; The primer set according to claim 1 .

13. The primer set according to claim 12, further comprising a Floop primer consisting of the base sequence set forth in SEQ ID NO:

49.

14. A kit for detecting JPCSaV or diagnosing JPCSaV infection by RT-LAMP, comprising the primer set described in any one of claims 1 to 13.

15. A method for detecting JPCSaV or diagnosing JPCSaV infection, comprising a step of performing an amplification reaction of a target nucleic acid region of JPCSaV by RT-LAMP using a primer set described in any one of claims 1 to 13 or a kit described in claim 14.

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