Primer set and method for highly sensitive detection and quantification of diaporthe destruens

Abstract

To provide highly sensitive and accurate means for detection and quantification of Diaporthe destruens.SOLUTION: Provided is a method for detecting and quantifying Diaporthe destruens by real-time PCR using a forward primer having a sequence of GGCCTGCCCCCTTAAAAA, a reverse primer having a sequence of GCAGGCCGGCCTCTCT, and a probe having a sequence of TTTGCTCAGAGATACACTA.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to primers, primer sets, primer-probe sets used for detecting or quantifying sweet potato basal rot pathogen, and a method for detecting or quantifying sweet potato basal rot pathogen using these.

Background Art

[0002] Sweet potato basal rot is a soil-borne disease caused by the filamentous fungus Diaporthe destruens (hereinafter sometimes simply referred to as "basal rot pathogen"). Infected sweet potatoes initially show dark brown lesions at the base of the stem near the ground surface. Subsequently, the symptoms progress to the above-ground and underground parts, adversely affecting growth and resulting in poor growth. In severe cases, the plant wilts and the tuberous roots cannot be harvested. This disease was first detected in Okinawa Prefecture and Kagoshima Prefecture in 2018. Currently, it has been confirmed in almost all production areas in Japan, including major production areas, and has caused serious damage in southern Kyushu and other regions. By detecting the occurrence of this disease at an early stage and taking appropriate control measures, it is possible to reduce the damage caused by this disease. For this purpose, it is effective to grasp the occurrence in the production area at an early stage by a highly sensitive and highly accurate detection and quantification technique for the basal rot pathogen, and to evaluate the degree of contamination quickly and accurately to take appropriate control measures.

[0003] As a technique for detecting and quantifying the basal rot pathogen, for example, a primer set for an intercalator method for detecting and quantifying the basal rot pathogen from an infected plant has been developed using Real-time PCR (Patent Document 1, Non-Patent Document 1). In addition, a method for detecting the basal rot pathogen from an infected plant by LAMP (Loop-mediated isothermal amplification) has also been developed (Non-Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Literature

[0005]

Non-Patent Literature 1

Non-Patent Literature 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The above-described method was mainly developed for detection and quantification from infected plant bodies and has not been optimized for detection and quantification from soil. Although it is possible to detect and quantify the basal rot pathogen from soil by the methods of Patent Document 1 and Non-Patent Literature 1, there are cases where detection and quantification become difficult when testing soils with different types, compositions, organic matter contents, etc., or when the basal rot pathogen is present at a low density in the soil.

[0007] In addition, for the detection of the basal rot pathogen by Real-time PCR using the intercalator method, when non-specific reactions (such as primer misannealing) proceed, the measured value (Ct value) includes the non-specific reaction, which hinders detection and quantification. Therefore, when using DNA extracted from environmental samples, especially soil, where there is a lot of contamination and DNA of other organisms is mixed, false detection is likely to occur, and it may be difficult to achieve high-precision and high-sensitivity detection and quantification.

[0008] The present invention has been made under the above background, and an object thereof is to provide a highly sensitive and highly accurate detection and quantification means for the basal rot pathogen.

Means for Solving the Problems

[0009] As a result of intensive studies to solve the above problems, the present inventors have found that real-time PCR using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 4, a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a probe consisting of the nucleotide sequence set forth in SEQ ID NO: 6 can detect and quantify the basal rot pathogen with high sensitivity and high precision, and thus have completed the present invention.

[0010] That is, the present invention provides the following [1] to [8]. [1] A primer used for detecting or quantifying the basal rot pathogen of sweet potato, characterized in that it consists of a continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 2.

[0011] [2] The primer according to [1], wherein the continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 2 is the nucleotide sequence set forth in SEQ ID NO: 5.

[0012] [3] A primer set used for detecting or quantifying the basal rot pathogen of sweet potato, comprising a forward primer and a reverse primer, wherein the forward primer consists of a continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 1, and the reverse primer consists of a continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 2.

[0013] [4] The primer set according to [3], wherein the continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 1 is the nucleotide sequence set forth in SEQ ID NO: 4, and the continuous nucleotide sequence of 10 or more nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 2 is the nucleotide sequence set forth in SEQ ID NO: 5.

[0014] 〔5〕A primer-probe set used for detecting or quantifying sweet potato basal rot pathogen, comprising a forward primer, a reverse primer, and a probe, wherein the forward primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1, the reverse primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2, and the probe consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 3.

[0015] 〔6〕The primer-probe set according to 〔5〕, wherein the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1 is the base sequence set forth in SEQ ID NO: 4, the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2 is the base sequence set forth in SEQ ID NO: 5, and the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 3 is the base sequence set forth in SEQ ID NO: 6.

[0016] 〔7〕A method for detecting or quantifying sweet potato basal rot pathogen in a sample, characterized by comprising the following steps (1) and (2): (1) A step of performing PCR using the nucleic acid in the sample as a template and the primer described in 〔1〕 or 〔2〕, or the primer set described in 〔3〕 or 〔4〕. (2) A step of detecting or quantifying the sweet potato basal rot pathogen in the sample by detecting or quantifying the amplification product by the PCR.

[0017] 〔8〕A method for detecting or quantifying sweet potato basal rot pathogen in a sample, characterized by comprising the following steps (1) and (2): (1) A step of performing PCR using the nucleic acid in the sample as a template and the forward primer and reverse primer in the primer-probe set described in 〔5〕 or 〔6〕. (2) Detecting or quantifying the sweet potato basal rot pathogen in a sample by detecting or quantifying the amplification product by the above PCR with the probe in the primer-probe set described in [5] or [6].

Advantages of the Invention

[0018] The present invention provides a novel primer used for detecting or quantifying the sweet potato basal rot pathogen. By PCR using this primer, the basal rot pathogen can be detected and quantified with high sensitivity and high precision.

Embodiments for Carrying Out the Invention

[0019] Hereinafter, the present invention will be described in detail. The primer of the present invention is a primer used for detecting or quantifying the sweet potato basal rot pathogen, and is characterized by consisting of a continuous base sequence of 10 bases or more included in the base sequence described in SEQ ID NO: 2 below. The primer of the present invention can be used as a reverse primer in PCR. SEQ ID NO: 2: TCGGC GCAGGCCGGCCTCTCT GCTGA

[0020] In the base sequence described in SEQ ID NO: 2, the number of consecutive bases may be 10 bases or more, preferably 12 bases or more, and more preferably 15 bases or more. Also, the number of consecutive bases may be 26 bases or less, preferably 25 bases or less, and more preferably 20 bases or less.

[0021] As the continuous base sequence of 10 bases or more included in the base sequence described in SEQ ID NO: 2, the base sequence described in SEQ ID NO: 5 below (the underlined base sequence in SEQ ID NO: 2 above) is preferable. SEQ ID NO: 5: GGCCTGCCCCCTTAAAAA

[0022] The primer of the present invention is used for the detection or quantification of sweet potato basal rot pathogen. Here, "detection of sweet potato basal rot pathogen" means determining whether the sweet potato basal rot pathogen is present in a sample, and "quantification of sweet potato basal rot pathogen" means measuring the amount of the sweet potato basal rot pathogen present in a sample.

[0023] The forward primer used together with the primer of the present invention described above is not particularly limited, but a primer consisting of a continuous base sequence of 10 or more bases included in the base sequence described in SEQ ID NO: 1 below is preferred. SEQ ID NO: 1: TTCAG GGCCTGCCCCCTTAAAAA AGGCA

[0024] In the base sequence described in SEQ ID NO: 1, the number of continuous bases may be 10 or more, preferably 12 or more, and more preferably 15 or more. Also, the number of continuous bases may be 28 or less, preferably 25 or less, and more preferably 20 or less.

[0025] As the continuous base sequence of 10 or more bases included in the base sequence described in SEQ ID NO: 1, the base sequence described in SEQ ID NO: 4 below (the underlined base sequence in SEQ ID NO: 1 above) is preferred. SEQ ID NO: 4: GGCCTGCCCCCTTAAAAA

[0026] The above primer set (forward primer and reverse primer) may be used for any PCR, but is preferably used for real-time PCR. For real-time PCR, a method using an intercalator (intercalator method) and a method using a probe (probe method) are known, but the above primer set is preferably used in the probe method.

[0027] The probe used in the probe method is not particularly limited, but a probe consisting of a continuous base sequence of 10 or more bases included in the base sequence described in SEQ ID NO: 3 below is preferred. Sequence number 3: TTTTT TTTGCTCAGAGATACACTA TAAAA

[0028] In the nucleotide sequence set forth in SEQ ID NO: 3, the number of consecutive bases may be 10 bases or more, preferably 12 bases or more, and more preferably 15 bases or more. Also, the number of consecutive bases may be 29 bases or less, preferably 25 bases or less, and more preferably 20 bases or less.

[0029] As the nucleotide sequence of 10 or more consecutive bases included in the nucleotide sequence set forth in SEQ ID NO: 3, the nucleotide sequence set forth in SEQ ID NO: 6 below (the underlined nucleotide sequence in SEQ ID NO: 3 above) is preferred. Sequence number 6: TTTGCTCAGAGATACACTA

[0030] The probe is preferably modified with a fluorescent substance and a quencher, similar to the probes used in general probe methods. The fluorescent substance may be one commonly used in probe methods, and for example, FAM (fluorescein amidite) etc. can be used. The quencher may also be one commonly used in probe methods, and for example, TAMRA (carboxytetramethylrhodamine) etc. can be used.

[0031] Using the above probe, probe set, or primer-probe set (forward primer, reverse primer, and probe), the sweet potato black rot pathogen in a sample can be detected or quantified by a method including the following steps (1) and (2).

[0032] In step (1), PCR is performed using the above primer or primer set with the nucleic acid in the sample as a template.

[0033] The sample is not particularly limited, and examples include plant tissues, soil, and microbial cells. As the sample, for example, tissues of sweet potato suspected of being infected with basal rot pathogen, soil suspected of being contaminated with basal rot pathogen, and microbial cells that may contain basal rot pathogen can be used. As the template for PCR, DNA extracted from cultured microbial cells and conidia can be used. Alternatively, cultured microbial cells may be directly used as the template (direct PCR). Also, as the template for PCR, DNA extracted from plant tissues such as sweet potato can be used. The tissue from which DNA is extracted is not particularly limited, and examples include leaves, stems, fruits, runners, roots, tubers, and crowns. Furthermore, as the template for PCR, DNA extracted from soil can also be used. As the method for extracting DNA, known extraction methods such as the phenol-chloroform method and the CTAB method can be used. Also, DNA may be extracted using commercially available DNA extraction kits such as the DNeasy plant mini kit (Qiagen) and the DNeasy plant max kit (Qiagen).

[0034] As described above, for PCR, real-time PCR is preferred, and real-time PCR using the probe method is particularly preferred.

[0035] The Taq DNA polymerase, buffer, etc. used in PCR may be those used in general PCR.

[0036] The conditions for PCR, such as denaturation conditions, annealing-extension conditions, and the number of cycles, can be the same as those in general real-time PCR. For example, the denaturation temperature can be 90 - 100°C, the denaturation time can be 1 - 5 seconds, the annealing-extension temperature can be 50 - 75°C, the annealing-extension time can be 0.5 - 2 minutes, and the number of cycles can be 30 - 45 cycles.

[0037] In step (2), the basal rot pathogen of sweet potato in the sample is detected or quantified by detecting or quantifying the amplification product by the above-mentioned PCR.

[0038] The method for detecting or quantifying the amplification product is not particularly limited. However, when the above primer-probe set is used, the amplification product is detected or quantified by the fluorescent substance released from the probe.

Examples

[0039] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0040] 〔Experimental method〕 Design of primers and probes From the NCBI database, the nucleotide sequences of the Internal Transcribed Spacer (hereinafter referred to as ITS) 1 and ITS2 regions containing the 5.8S of the rRNA gene of sweet potato basal rot pathogen Diaporthe destruens (JN848791, MH465671, MH465672, MH465673) and sweet potato dry rot pathogen D. batatas (KU577616, MG827239, NR_152456) were obtained. The obtained nucleotide sequences were compared, and sequences specific to the basal rot pathogen were extracted and selected to determine primer and probe sequences.

[0041] DNA extraction from soil A total of 9 soils of black peat soil collected from 6 locations were used for the test. A spore suspension of the basal rot pathogen strain MAFF246953 cultured and collected on a sweet potato leaf medium (Koji Nomiyama, Hiroyuki Sekiguchi, Shigenobu Yoshida, "Mass sporulation of Diaporthe destruens, the causal agent of sweet potato basal rot, using the agar leaf method", Abstracts of the Annual Meeting of the Phytopathological Society of Japan, 2023) was adjusted to 10 1 ~10 5Spores were inoculated into each soil so that the amount of spores was 0.4 g of raw soil. The soil without inoculation of the spore suspension was set to 0 spores / 0.4 g of raw soil. Soils with each spore density were prepared in triplicate, and DNA was extracted using the Fast DNA SPIN Kit for Soil (manufactured by MP Biomedicals, CA, USA) according to Morimoto & Hoshino (2008). When adding the sodium phosphate buffer attached to the kit, 80 μl of a 20% (w / v) skim milk solution was added, and then the sample was disrupted at a rotation speed of 5.5 for 30 seconds using a disruption device (FP series of Q-BioGene). After disruption, DNA extraction was carried out according to the product manual. The extracted DNA was purified using DNA Clean & Concentrator-25 (Zymo Research, CA, USA) and eluted with 50 μl.

[0042] DNA extraction from plants Six sweet potato stems with symptoms of basal rot disease confirmed at the ground level of the plant were obtained from a sweet potato cultivation field in Kagoshima City, Kagoshima Prefecture. Furthermore, in an indoor sweet potato cultivation test, soil inoculated and mixed with a spore suspension of the basal rot pathogen strain F3-MZ so that there were 10 5 spores per 1 g of culture soil, and culture soil without inoculation of the basal rot pathogen were each filled into 9-cm pots, and a single-node seedling of sweet potato (cultivar Beniazuma) was transplanted and cultivated in a greenhouse (27 - 29°C) for 25 days. One stem showing symptoms of basal rot disease at the ground level of the seedlings cultivated in the soil inoculated with the basal rot pathogen was obtained. Also, as a control, one healthy sweet potato stem of a seedling cultivated in soil without inoculation of the basal rot pathogen was obtained. Approximately 100 mg of tissue by fresh weight was collected in triplicate from the symptomatic part of each collected sweet potato stem (any part for the control stem), and DNA was extracted and purified from each tissue using the DNeasy® Plant Mini Kit (250) (manufactured by QIAGEN, Netherlands).

[0043] DNA extraction from bacterial cells As a positive control for real-time PCR analysis, DNA of the Fusarium solani strain MAFF246953 was used. Approximately 0.1 g of the cells obtained by culturing this strain on sweet potato leaf medium was used to extract and purify DNA using the Fast DNA SPIN Kit for Soil (manufactured by MP Biomedicals, CA, USA) in the same manner as the DNA extraction method from the above soil.

[0044] Detection and quantification of basal rot pathogen by real-time PCR Regarding the novel primer-probe set and the primer set reported previously (Fujiwara et al., 2021), the detection and quantification of the Fusarium solani were compared using Step One Plus (ABI).

[0045] Using the novel primer-probe set (Forward Primer: 5’-GGCCTGCCCCCTTAAAAA-3’ (SEQ ID NO: 4), Reverse Primer: 5’-GCAGGCCGGCCTCTCT-3‘ (SEQ ID NO: 5), Probe (FAM-MGB): 5’-TTTGCTCAGAGATACACTA-3’ (SEQ ID NO: 6)), 0.9 μM of each primer, 0.2 μM of the probe, and TaqMan TM Fast Advanced Master Mix (Applied Biosystems TM ) 5 μl was added, and 1 μl of the template DNA was used as a sample to perform a PCR reaction in a 10 μl reaction system. The reaction conditions were 95°C for 2 minutes, [95°C for 3 seconds, 60°C for 1 minute] × 45 cycles.

[0046] The previously reported primer set (Dd ITS-F: 5’-GTTTTTATAGTGTATCTCTGAGC-3’ (SEQ ID NO: 7), Dd ITS-R: 5’-GGCCTGCCCCCTTAAAAA-3‘ (SEQ ID NO: 8); Fujiwara et al., 2021) was used. For each primer at a final concentration of 0.4 μM, 5 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2× conc.) and 0.2 μl of ROX Reference Dye (50× conc.) from Takara were added, and 1 μl of template DNA was used as a sample to prepare a reaction solution with a total volume of 10 μl. The PCR reaction was then carried out. The reaction conditions were as follows: 94°C for 1 minute, [96°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds] × 40 cycles, and a melting curve of [96°C for 15 seconds, 55°C for 1 minute, 55 - 96°C (15 seconds / °C)].

[0047] In addition, the amount of DNA of the basal rot pathogen in the plant was estimated using a calibration curve prepared by diluting the DNA of the basal rot pathogen strain MAFF246953, which was extracted and purified as a positive control, to 0.5 - 0.00005 ng / μl.

[0048] 〔Experimental Results〕 Detection of basal rot pathogen from soil using real-time PCR The detection and quantification sensitivities of the novel primer-probe set and the previously reported primer set were compared. The results are shown in Table 1.

Table 1

[0049] The numbers in the table indicate the number of samples detected in the three replicates. The detection limit (horizontal line in the table) was set based on the presence or absence of detection in all three replicates. The quantification was confirmed in the gray-highlighted part.

[0050] As shown in Table 1, the novel primer-probe set improved the detection sensitivity by 10 - 100 times in 7 soils and the quantification sensitivity by 10 times in 8 soils.

[0051] Detection of basal rot pathogen from plants using real-time PCR The detection sensitivity was compared between the novel primer-probe set and the previously reported primer set. The results are shown in Table 2.

Table 2

[0052] For both the novel primer-probe set and the previously reported primer set, the DNA of the basal rot pathogen was not detected from samples without symptoms. In all 21 symptomatic samples, the amount of detected DNA was larger and the detection sensitivity was higher (2 to 700 times) with the novel primer-probe set than with the previously reported primer set. In particular, in 15 samples (P1, P3, CR1-1, CR1-3, CR2-2, CR2-3, CR3-2, CC1-1, CC1-2, CC1-3, CC2-1, CC2-3, CC3-1, CC3-2, CC3-3), the DNA of the basal rot pathogen could not be detected with the previously reported primer set, but could be quantitatively detected with the novel primer-probe set.

[0053] Analysis time by real-time PCR instrument When using Step One Plus (ABI), the analysis time by the Real-time PCR instrument was about 60 minutes. Since the analysis time by the Real-time PCR instrument in the previous report (Fujiwara et al., 2021) was 127 minutes, the analysis time could be shortened by about 50%.

Industrial Applicability

[0054] The present invention can be used in the agricultural field and the like.

Claims

1. A primer used for detecting or quantifying sweet potato basal rot pathogen, characterized by consisting of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO:

2.

2. The primer according to claim 1, wherein the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2 is the base sequence set forth in SEQ ID NO:

5.

3. A primer set used for detecting or quantifying sweet potato basal rot pathogen, comprising a forward primer and a reverse primer, wherein the forward primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1, and the reverse primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO:

2.

4. The primer set according to claim 3, wherein the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1 is the base sequence set forth in SEQ ID NO: 4, and the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2 is the base sequence set forth in SEQ ID NO:

5.

5. A primer-probe set used for detecting or quantifying sweet potato basal rot pathogen, comprising a forward primer, a reverse primer and a probe, wherein the forward primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1, the reverse primer consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2, and the probe consists of a continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO:

3.

6. The primer-probe set according to claim 5, wherein the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 1 is the base sequence set forth in SEQ ID NO: 4, the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 2 is the base sequence set forth in SEQ ID NO: 5, and the continuous base sequence of 10 or more bases contained in the base sequence set forth in SEQ ID NO: 3 is the base sequence set forth in SEQ ID NO:

6.

7. A method for detecting or quantifying sweet potato basal rot pathogen in a sample, characterized by comprising the following steps (1) and (2). (1) A step of performing PCR using the nucleic acid in the sample as a template and the primer according to claim 1 or 2, or the primer set according to claim 3 or 4; (2) A step of detecting or quantifying the sweet potato black rot pathogen in the sample by detecting or quantifying the amplification product by the PCR.

8. A method for detecting or quantifying the sweet potato black rot pathogen in a sample, comprising the following steps (1) and (2): (1) A step of performing PCR using the nucleic acid in the sample as a template and the forward primer and reverse primer in the primer-probe set according to claim 5 or 6; (2) A step of detecting or quantifying the sweet potato black rot pathogen in the sample by detecting or quantifying the amplification product by the PCR with the probe in the primer-probe set according to claim 5 or 6.

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