Development of a Diagnostic Method for Sweet Potato Pathogenic Virus by RT-LAMP Method

Abstract

To provide methods for simple and quick detection of SPFMV and SPLCV being important sweet potato pathogen viruses.SOLUTION: The disclosure provides a primer set for detecting sweet potato pathogen viruses using a LAMP method, and a diagnostic method of sweet potato pathogen viruses characterized in that diagnosis is carried out by detecting the presence or absence of sweet potato pathogen viruses by a LAMP method using the primer set. The invention enables quick and highly-sensitive detection of sweet potato pathogen viruses SPFMV and SPLCV, so that easy virus testing of sweet potato in its slip stage is enabled in expectation of expanding use of virus-free slips.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The present invention relates to a method for diagnosing sweet potato pathogenic virus using the RT-LAMP method, as well as primers and the like used therefor.

Background Art

[0002] There are various plant viruses in nature. Plant viruses sometimes cause damage to crops, and also cause great damage to sweet potatoes, which are one of the major crops in Japan.

[0003] The number of plant virus species that infect sweet potatoes is more than 20 as far as is known. In order to rapidly detect these plant viruses, a technique for detecting genes is used. Generally, the PCR method is used, and various techniques related to the PCR method for detecting viruses that infect sweet potatoes have been disclosed (Non-Patent Documents 1 to 4).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Disclosure of the Invention

Problems to be Solved by the Invention

[0005] The inventors considered that the current situation of detecting sweet potato pathogenic viruses was not sufficient. That is, from various analyses by the inventors, the inventors considered that the main pathogenic viruses among sweet potato pathogenic viruses were two types, SPFMV (sweet potato feathery mottle virus) and SPLCV (sweet potato leaf curl virus). However, at present, there is no method for quickly and highly sensitively detecting these SPFMV and SPLCV. Based on this, the inventors have developed a technology related to PCR primers for detecting sweet potato pathogenic viruses and filed a patent application (Japanese Patent Application No. 2019 - 227418).

[0006] Furthermore, the inventors considered that a simpler and quicker test method than the PCR method was needed. That is, the sweet potato pathogenic virus becomes a problem in the agricultural field. However, in the actual agricultural field, those with advanced inspection techniques are not necessarily many. In addition, at present, the detection of the sweet potato pathogenic virus usually takes place after an event where infection is suspected occurs, making it easy to be too late in taking countermeasures. Therefore, if routine inspections can be carried out on sweet potatoes at the seedling stage, it is possible to take preemptive measures, and by expanding the use of virus-free sweet potato seedlings, it becomes possible to prevent the infection and spread of the virus. From these points, the inventors considered that a method that can detect the sweet potato pathogenic virus more simply and quickly is needed.

[0007] Against the background of the above circumstances, in the present invention, the problem is to develop a method that can simply and quickly detect SPFMV and SPLCV, which are important pathogenic viruses of the sweet potato pathogenic virus.

Means for Solving the Problems

[0008] As a result of intensive research, the inventors analyzed comprehensive sequence information including subtypes of SPFMV and SPLCV, and developed primers that can be simply and quickly detected using the LAMP method, thus completing the invention.

[0009] The present invention consists of the following configurations. [1] A primer set for detecting a sweet potato pathogenic virus using the LAMP method, When the sweet potato pathogenic virus is SPFMV, (a) FIP 5’-(base sequence of SEQ ID NO: 11)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 12)-3’ (b) BIP 5’-(base sequence of SEQ ID NO: 13)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 14)-3’ (c) F3 SEQ ID NO: 5 (d) B3 SEQ ID NO: 6 When the sweet potato pathogenic virus is SPLCV, (g) FIP 5'-(base sequence of SEQ ID NO: 21)-(any base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 22)-3' (h) BIP 5'-(base sequence of SEQ ID NO: 23)-(any base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 24)-3' (i) F3 SEQ ID NO: 15 (j) B3 SEQ ID NO: 16 A primer set characterized by being composed of primers represented by the following.

[0010] [2] The primer set according to [1], wherein (a) is the primer represented by SEQ ID NO: 7 and (b) is the primer represented by SEQ ID NO: 8. [3] The primer set according to [1] or [2], wherein (g) is the primer represented by SEQ ID NO: 17 and (h) is the primer represented by SEQ ID NO: 18. [4] Further, in the case of SPFMV, the primer set according to any one of [1] to [3], including loop primers represented by the following sequences. (e) LF SEQ ID NO: 9 (f) LB SEQ ID NO: 10 [5] Further, in the case of SPLCV, the primer set according to any one of [1] to [3], including loop primers represented by the following sequences. (k) LF SEQ ID NO: 19 (l) LB SEQ ID NO: 20

[0011] [6] A method for diagnosing sweet potato pathogenic virus, characterized by detecting the presence or absence of sweet potato pathogenic virus by the LAMP method using the primer set according to any one of [1] to [5] and performing a diagnosis. [7] The method for diagnosing sweet potato pathogenic virus according to [6], wherein the diagnosis is performed using the sap collected from any one or a plurality of leaves and stems of sweet potato as a detection sample. [8] Primers represented by the sequences of (a) to (l) above. [Advantages of the Invention]

[0012] According to the present invention, it has become possible to provide a method for simply and rapidly detecting SPFMV and SPLCV, which are important pathogenic viruses of sweet potato pathogenic viruses.

Brief Description of Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Modes for Carrying Out the Invention

[0014] The primer set and the like of the present invention have been completed based on the following findings or experimental facts obtained by the inventors. (1) As important pathogenic viruses of sweet potato pathogenic viruses, SPFMV and SPLCV were selected. (2) In SPFMV, focusing on the coat protein (CP) gene, comprehensive analysis of multiple types of CP genes such as AB465608, AB509453, D86371, AB439206, MH763678, KY296450, MG656415, KY296451, etc. was carried out, and primers were designed. (3) In SPLCV, focusing on the movement protein (MP) gene, a total of 38 types of MP genes such as AB433786, AB433787, AB433788, KT992057, KT992055, KT992054, KT992052, KT992051, KT992048, etc. were analyzed, and primers were designed. (4) In the detection of viruses by the LAMP method using the designed primer sets, there was no problem with specificity, and the detection sensitivity was 100 to 1000 times higher than that of PCR. (5) The detection of sweet potato pathogenic viruses by the PCR method took about 1.5 to 2.0 hours, while the LAMP method took about 50 minutes, enabling rapid detection. (6) By using loop primers together in the LAMP method, the detection time could be further shortened to about 10 to 15 minutes, enabling even faster detection. (7) Viruses could be detected by using the sap obtained from sweet potato stems or leaves as the detection target. In this regard, simpler and more rapid detection could be achieved compared to the PCR method that requires sample purification.

[0015] When the sweet potato pathogenic virus is SPFMV, the primer set of the present invention is shown as the following primer set. Hereinafter, a set containing a series of primers corresponding to the following (a) to (d) is abbreviated as primer set 2. (a) FIP 5’-(base sequence of SEQ ID NO: 11. F1c region)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 12. F2 region)-3’ (b) BIP 5'-(Base sequence of SEQ ID NO: 13. B1c region)-(Any base sequence with 0 to 50 bases)-(Base sequence of SEQ ID NO: 14. B2 region)-3' (c) F3 SEQ ID NO: 5 (d) B3 SEQ ID NO: 6 Note that FIP is shown as inner primer F, BIP is shown as inner primer B, F3 is shown as outer primer F, and B3 is shown as outer primer B. Also, as a preferred embodiment in this case, the following loop primers can be included. Thereby, when performing the LAMP method using the primer set of the present invention, the detection time can be shortened, and it has the effect of improving the usefulness of the present invention. (e) LF SEQ ID NO: 9 (f) LB SEQ ID NO: 10 Note that LF is shown as loop primer F and LB is shown as loop primer B.

[0016] In the primer set for detecting sweet potato pathogenic virus using the LAMP method of the present invention, when the sweet potato pathogenic virus is SPLCV, it is shown as the following primer set. Hereinafter, a set containing a series of primers corresponding to the following (g) to (j) is abbreviated as primer set 3. (g) FIP 5'-(Base sequence of SEQ ID NO: 21. F1c region)-(Any base sequence with 0 to 50 bases)-(Base sequence of SEQ ID NO: 22. F2 region)-3' (h) BIP 5'-(Base sequence of SEQ ID NO: 23. B1c region)-(Any base sequence with 0 to 50 bases)-(Base sequence of SEQ ID NO: 24. B2 region)-3' (i) F3 SEQ ID NO: 15 (j) B3 SEQ ID NO: 16 Also, as a preferred embodiment in this case, the following loop primers can be included. Thereby, when performing the LAMP method using the primer set of the present invention, the detection time can be shortened, and it has the effect of improving the usefulness of the present invention. (k) LF SEQ ID NO: 19 (l) LB SEQ ID NO: 20

[0017] It is known as common general technical knowledge that in the inner primer of each primer set, there may be a non-specific base sequence region (linker) that does not participate in amplification between the F2 region and the F1c region, or between the B2 region and the B1c region. Regarding the length of the sequence of the non-specific region, it may be a sequence length that does not affect amplification, typically 0 to 50 sequences, more preferably 0 to 40 sequences, and particularly preferably 0 to 30 sequences. As such an inner primer, in primer set 2, the primer represented by (a) can be the primer of SEQ ID NO: 7, and (b) can be the primer of SEQ ID NO: 8. In primer set 3, the primer represented by (g) can be the primer of SEQ ID NO: 17, and (h) can be the primer of SEQ ID NO: 18.

[0018] As another aspect of the present invention, it can be configured as a sweet potato pathogenic virus diagnosis method. That is, the sweet potato pathogenic virus diagnosis method of the present invention is characterized in that the presence or absence of the sweet potato pathogenic virus is detected and diagnosed by the LAMP method using the above primer set.

[0019] A specimen sample is required for the detection of sweet potato pathogenic virus by the LAMP method. As this specimen sample, any commonly used specimen sample can be used. Typically, a specimen sample can be prepared from the leaves, stems, and tuberous roots of sweet potato, or any one or more of these parts.

[0020] When the sweet potato sample is a tuberous root, a sample for nucleic acid extraction may be prepared using the one with the epidermis cut off. When the sweet potato sample is a leaf, a sample for nucleic acid extraction may be prepared using the one cut into multiple pieces or the one punched out. Also, the sap prepared by pricking with a needle from the leaf vein part may be used. When the sweet potato sample is a stem, a sample for nucleic acid extraction may be prepared using the one cut into multiple pieces. Also, the sap prepared by pricking the stem with a needle may be used. Among these, it is preferable to use the sap collected from any one or a plurality of sweet potato leaves and stems. This enables a non-invasive, simple, and rapid virus test for sweet potatoes at the seedling stage, and it is expected to expand the use of virus-free sweet potato seedlings. In the present invention, the sap can be prepared by various commonly known methods. As an example, it is prepared by pricking a needle into sweet potato leaves, stems, etc., and suspending the tip portion in sterilized water.

[0021] For the specimen sample for detecting sweet potato pathogenic virus, pretreatment for the commonly used nucleic acid extraction operation can be performed, and it can be used as a sample for the LAMP method. For example, after crushing the plant tissue, nucleic acid extraction and purification are performed using phenol and chloroform, or nucleic acid obtained using a commercially available extraction kit (for example, ISOSPIN Plant RNA of Nippon Gene) is extracted and used as a specimen.

[0022] Once the specimen sample is prepared, an operation for nucleic acid amplification is performed using the aforementioned primer set. As the operation during this nucleic acid amplification, for example, a commercially available kit (for example, Loopamp) containing at least four types of primers (inner primer F and B, outer primer F and B), a template-dependent nucleic acid synthase, deoxynucleotide triphosphate, etc. in the reaction solution TM can be used for incubation at 60 to 65°C for about 15 minutes to 1 hour. Also, as described above, a loop primer can be further used.

[0023] The enzyme used for nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Examples of such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Csa DNA polymerase, Gsp DNA polymerase (large fragment), GspSSD DNA polymerase (large fragment), Tin DNA polymerase (large fragment), and the Klenow fragment of Escherichia coli DNA polymerase I, etc.

[0024] After the nucleic acid amplification reaction operation is completed, the nucleic acid amplification product is detected. For the detection of the nucleic acid amplification product, any commonly used detection method can be used. For example, detection using a labeled oligonucleotide that specifically recognizes the amplified base sequence, the fluorescent intercalator method, detection by agarose gel electrophoresis, etc. In agarose gel electrophoresis, the LAMP amplification product is detected as a ladder-like pattern of multiple bands with different base lengths. In addition, in the LAMP method, visual detection may be possible, which is preferable when rapid detection of sweet potato pathogenic virus is required. That is, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and the by-product pyrophosphate reacts with coexisting magnesium to form magnesium pyrophosphate, causing the reaction solution to become turbid enough to be visually confirmed, so that the sweet potato pathogenic virus can be detected. In this case, it is also possible to confirm the detection of the sweet potato pathogenic virus using a measuring instrument such as a spectrophotometer that can optically observe the increase in turbidity during the reaction and the turbidity after the reaction is completed.

[0025] When performing the detection of nucleic acid amplification using the primers of the present invention, various reagents required can be pre-packaged and made into a kit. Specifically, various oligonucleotides required as the primers or loop primers of the present invention, four types of dNTPs that serve as substrates for nucleic acid synthesis, a template-dependent nucleic acid synthase having strand displacement activity, buffers and salts that provide suitable conditions for the enzyme reaction, a protective agent that stabilizes the enzyme and the template, and further reagents required for the detection of the reaction product if necessary are provided as a kit.

Example

[0026] The present invention will be described in detail based on experimental examples.

[0027] <<I. Primer Design>> Analysis of the gene information of SPFMV and SPLCV was performed, and primers considered to be optimal were designed.

[0028] 1. In the genomic map of SPFMV, attention was paid to the coat protein (CP) gene region for design. (1) As genes known as CP, comprehensive analysis of multiple types such as AB465608, AB509453, D86371, AB439206, MH763678, KY296450, MG656415, KY296451, etc. was performed. As a result, primers were designed as shown in Table 1. Hereinafter, the primers shown in Table 1 are referred to as primer set 1. (2) The positional relationship between each primer in primer set 1 and the nucleic acid sequences of D86371, AB509453, and AB465608 is shown in FIG. 1. (3) Furthermore, primers were designed by improving primer set 1 (Table 2. Hereinafter, "primer set 2"). The positional relationship between each primer in primer set 2 and the nucleic acid sequences of various genes is shown in FIG. 2.

[0029]

Table 1

Table 2

[0030] 2. In the genomic map of SPLCV, attention was paid to the movement protein (MP) gene region for design. (1) As genes known as MPs, a total of 38 types of gene information were analyzed to cover multiple types such as AB433786, AB433787, AB433788, KT992057, KT992055, KT992054, KT992052, KT992051, KT992048. As a result, primer design was carried out as shown in Table 3 (hereinafter, "primer set 3"). (2) The positional relationship between each primer in primer set 3 and the nucleic acid sequences of AB433787, AB433786, and AB433788 is shown in Figure 3.

[0031] [Table 3]

[0032] [[II. Evaluation of Primers]] Experiments were conducted for the purpose of detecting by the LAMP method using each primer set and evaluating its performance.

[0033] [Experimental Method] [LAMP Method] 1. Using the primers shown in Tables 1 to 3, an amplification reaction was carried out by the LAMP method. Also, Loopamp (registered trademark) was used for amplification by the LAMP method. 2. As a positive control, nucleic acid extraction was performed from the leaves of sweet potatoes infected with either or both of SPFMV and SPLCV, and the purified product was used. 3. The amplification reaction was carried out at 63°C for 10 to 60 minutes in a dedicated tube. 4. After the reaction was completed, the reaction solution was subjected to 2.0% agarose gel electrophoresis and Gelred staining. 5. In addition, when real-time detection was carried out, the fluorescence intensity was measured using a fluorescence measurement device (Genelyzer FII).

[0034] [PCR Method] 1. Using the primers described in the patent application (Japanese Patent Application No. 2019-227418) developed by the inventors, PCR was performed. Table 4 shows the results for each primer. 2. To amplify by PCR, PrimeScript High Fidelity One Step RT-PCR Kit manufactured by Takara Bio Inc. was used, and the reaction reagents were prepared so that the concentration of each reagent in the final reaction solution of 25 μL was as follows. 3. To 20 μL of the above reaction reagent, 5.0 μL of the sample solution at each concentration containing template DNA or RNA was added to make the final reaction solution 25.0 μL, and each PCR reaction was performed twice. 4. The temperature cycle conditions for the PCR reaction were as follows: for reverse transcription, the reaction was carried out at 45 °C for 10 minutes and 94 °C for 2 minutes, and then thermal denaturation at 94 °C for 10 seconds, annealing at 56 °C for 15 seconds, and polymerase extension reaction at 72 °C for 10 seconds. A total of 40 cycles of these series of reactions were performed as one cycle. 5. After the reaction was completed, the reaction solution was subjected to 2.0% agarose gel electrophoresis and Gelred staining was performed.

[0035] [Table 4]

[0036] <Experimental Results, Performance Evaluation of Primer Set 1> 1. The results of the amplification experiment by the LAMP method using primer set 1 are shown in Figure 4. (1) In the negative control (Lane 1), faint staining was observed, and although non-specific amplification was slightly observed. (2) In the samples containing the positive control (Lanes 2 to 4), ladder-like bands were detected in all samples, and the bands became darker as the reaction time increased. However, even after 40 minutes of the reaction, the bands were not sufficiently dark, and fluorescence in the reaction solution could not be confirmed. 2. From these results, although primer set 1 was capable of nucleic acid amplification by the LAMP method, its amplification rate was not sufficient, and it was considered necessary to improve. 3. As an improved method, the preparation of loop primers was considered, but due to the nature of the sequence, it was not suitable for the design of loop primers.

[0037] <Confirmation of Specificity of Primer Set 2 and Primer Set 3, Experimental Results> 1. The results of the amplification experiment by the LAMP method using each primer set are shown in Fig. 5. Note that loop primers were not used in this experiment. (1) In RT-PCR as a comparison target, the detection of bands of SPFMV in Lane 3, SPLCV in Lane 4, SPFMV and SPLCV in Lane 5 was confirmed. From these results, it was confirmed that the nucleic acid gene to be detected was included in each positive control.

[0038] (2) Ladder-like bands were confirmed in Lane 8. That is, it was confirmed that the nucleic acid gene of SPFMV could be detected by Primer Set 2. (3) Ladder-like bands were confirmed in Lane 11. That is, it was confirmed that the nucleic acid gene of SPLCV could be detected by Primer Set 3. (4) On the other hand, detection could not be confirmed in Lane 9. That is, it was confirmed that Primer Set 2 did not detect SPLCV which was not the detection target. Similarly, it was confirmed that Primer Set 3 did not detect SPFMV which was not the detection target (Lane 10).

[0039] (5) Furthermore, in samples infected with both SPFMV and SPLCV, ladder-like bands were confirmed in both Primer Set 2 and Primer Set 3 (Lanes 12, 13). From this, it was found that Primer Set 2 to Primer Set 3 could detect even samples with complex infections.

[0040] 2. From these results, the following was found. (1) Primer set 2 enabled specific amplification of SPFMV by the LAMP method. Moreover, no non-specific amplification was observed. (2) Primer set 3 enabled specific amplification of SPLCV by the LAMP method. Moreover, no non-specific amplification was observed.

[0041] <Experimental results, confirmation of detection sensitivity> 1. The results of primer set 2 are shown in the upper part of Fig. 6. (1) In PCR as a comparison target, the band became thinner as the nucleic acid amount of the sample decreased, and it reached a level where it could not be detected at 100 fg / tube. (2) On the other hand, in the LAMP method using primer set 2, although the band also became thinner as the nucleic acid amount of the sample decreased, ladder-like bands could be confirmed even at 1 fg / tube, and it was at a level where detection was sufficiently possible. (3) Note that the reaction time was about 1.5 to 2 hours for PCR and about 50 minutes for the LAMP method.

[0042] 2. The results of primer set 3 are shown in the lower part of Fig. 6. (1) In PCR as a comparison target, the band became thinner as the nucleic acid amount of the sample decreased, and it reached a level where it could not be detected at 100 pg / tube. (2) On the other hand, in the LAMP method using primer set 3, although the band also became thinner as the nucleic acid amount of the sample decreased, ladder-like bands could be confirmed even at 10 pg / tube, and it was at a level where detection was sufficiently possible. (3) Note that the reaction time was about 1.5 to 2 hours for PCR and about 50 minutes for the LAMP method.

[0043] 3. From these results, the following was found. (1) The detection by the LAMP method using primer set 2 had a detection sensitivity 1000 times higher than that by the PCR method. (2) The detection by the LAMP method using the primer set 3 had a detection sensitivity 100 times higher than that by the PCR method. (3) In either case, the detection by the LAMP method took less time.

[0044] <Experimental results, detection using loop primers> 1. Fig. 7 shows the results of comparing the presence or absence of loop primers. Regarding the notation of each line, the primer set number used is shown, and in parentheses, the presence or absence of loop primers and the presence or absence of the virus to be detected are shown in this order. (1) In samples positive (+) for SPFMV or SPLCV, an increase in fluorescence intensity was observed, while no fluorescence was observed in all negative (-) samples. (2) In the detection of SPFMV, when there was no loop primer, an increase in fluorescence intensity was observed from about 25 minutes after the start of the reaction, and it reached a plateau at about 35 minutes. On the other hand, when there was a loop primer, an increase in fluorescence intensity was observed from about 13 minutes, and it reached a plateau at about 20 minutes. (3) In the detection of SPLCV, when there was no loop primer, an increase in fluorescence intensity was observed from about 25 minutes after the start of the reaction, and it reached a plateau at about 35 minutes. On the other hand, when there was a loop primer, an increase in fluorescence intensity was observed from about 13 minutes, and it reached a plateau at about 33 minutes.

[0045] 2. From these results, the following was found. (1) The presence of loop primers does not inhibit the detection specificity. (2) In any reaction, the presence of loop primers enables faster detection.

[0046] <Experimental results, detection using sweet potato-infected samples> 1. The results of detecting SPFMV-infected samples are shown in Fig. 8. For each of the samples from A to H, in the LAMP method, for samples A and B, sap was collected from the main stem, and for the others, sap was collected from the main stem and leaves for the amplification reaction. On the other hand, in the PCR method, nucleic acids were extracted from the mesophyll of all samples for use as samples. (1) In the LAMP method, detection of C, E, G, and H was possible, but detection was not achieved for the others. (2) On the other hand, in the PCR method, thick bands were confirmed for E, G, and H, and thin bands were also confirmed for A, B, and C.

[0047] 2. The results of detecting SPLCV-infected samples are shown in Fig. 9. (1) In the LAMP method, detection of 2, 3, and 4 was possible, but detection of 1 was not achieved. (2) On the other hand, in the PCR method, thick bands were confirmed for 3 and 4, and a thin band was also confirmed for 2.

[0048] 3. For both SPFMV and SPLCV, the results of the LAMP method and the PCR method did not completely match, but it is not appropriate to make a general comparison. (1) In the LAMP method, the collected sap was directly used for the amplification reaction. In this regard, the inspection can be carried out simply and quickly. (2) On the other hand, in the PCR method, the collected sap was purified and used for the amplification reaction. In this regard, it is presumed that the virus concentration of the sample to be detected has increased compared to the sap sample used in the LAMP method, making it an easy-to-detect condition. Also, in the PCR method, since such a purification operation is required, there is a drawback of lacking rapidity. (3) Generally, the localization of plant viruses may vary depending on the growth stage. From this, by collecting sap from multiple locations or increasing the number of examples in the future, and clarifying the appropriate sap collection site according to the time, it is expected that the usefulness of the LAMP method diagnostic method according to the present invention will be further enhanced.

Industrial Applicability

[0049] Although SPFMV is an RNA virus and SPLCV is a DNA virus, in this LAMP method, the sap is used as a direct test sample and visual determination is possible, so it can be used by agricultural corporations and farmers. As a result, the introduction of technology into a large-scale stable supply system of virus-free seedlings for general farmers can be expected.

Claims

Claim 1 A primer set for detecting sweet potato pathogenic virus using the LAMP method, wherein when the sweet potato pathogenic virus is SPFMV, it consists of the following (a), (b), (c) and (d), (a) FIP SEQ ID NO: 7 (b) BIP SEQ ID NO: 8 (c) F3 SEQ ID NO: 5 (d) B3 SEQ ID NO: 6 wherein when the sweet potato pathogenic virus is SPLCV, it consists of the following (g), (h), (i) and (j), (g) FIP SEQ ID NO: 17 (h) BIP SEQ ID NO: 18 (i) F3 SEQ ID NO: 15 (j) B3 SEQ ID NO: 16 and is characterized by being composed of primers shown by the above. Claim 2 Furthermore, in the case of SPFMV, in addition to (a), (b), (c) and (d), the primer set according to claim 1, which includes loop primers shown by the following sequences. (e) LF SEQ ID NO: 9 (f) LB SEQ ID NO: 10 Claim 3 Furthermore, in the case of SPLCV, in addition to (g), (h), (i) and (j), the primer set according to claim 1, which includes loop primers shown by the following sequences. (k) LF SEQ ID NO: 19 (l) LB SEQ ID NO: 20 Claim 4 A method for diagnosing sweet potato pathogenic virus, characterized by detecting the presence or absence of sweet potato pathogenic virus by the LAMP method using the primer set according to any one of claims 1 to 3 and performing a diagnosis. Claim 5 The method for diagnosing sweet potato pathogenic virus according to claim 4, wherein the diagnosis is performed using a sap collected from any one or a plurality of leaves and stems of sweet potato as a detection sample.

Images