JP2023084968A - Oligonucleotides for detection of potato cyst nematodes - Google Patents

Abstract

To provide a method that can efficiently detect only living potato cyst nematodes (Globodera pallida), an important potato pest.SOLUTION: Provided is a method for detecting living potato phyllocyst nematodes, comprising performing an amplification reaction of a target nucleic acid region of Potato cyst nematode by RT-PCR using a potato phyllocyst nematode-specific primer set designed on the sequence of Y45F10D.4 gene of potato phylocyst nematode.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting live potato phyllocyst nematode, which is an important pest of potatoes, for example.

Currently, Abashiri City, Shari Town, etc. are conducting emergency control of potato cyst nematode (Globodera pallida; hereinafter sometimes referred to as "Gp"). Surveys are being conducted by the Plant Protection Station to determine whether to continue or terminate the control after the fiscal year. This survey is conducted by the cup examination method, but it is a problem that it requires a long period of two months or more. On the other hand, detection methods based on molecular biological methods using DNA have been developed so far, but since DNA can be detected even from dead individuals, the pest control effect tends to be judged to be lower than it actually is. Therefore, there is a demand for the development of a method that can efficiently detect and evaluate only living individuals.

Conventional techniques for detecting only living nematode individuals in soil include the following two methods.
One is the cup examination method (Non-Patent Documents 1 and 2), which is currently being implemented for emergency control of Gp as described above. In this method, transparent plastic cups are filled with field soil, seed potatoes of potato cyst nematodes (Globodera rostochiensis; hereinafter sometimes referred to as "Gr") resistant potato varieties are planted, and cultured for about 70 days. The presence or absence of cysts formed later on the surface of the root is investigated from the outside of the cup. However, the cup screening method requires a long period of time (about 70 days) to obtain results, and the cysts formed inside the cup cannot be seen, so the sensitivity is somewhat low.

The other is a method in which mRNA is targeted for detection. This method utilizes the fact that gene expression (mRNA synthesis) is a phenomenon specific to living cells and that RNA is easily degradable, and is applied to the detection and evaluation of living individuals. A technique for detecting viable Gp by reverse transcription real-time PCR using Gp mRNA as a template has been reported so far (Non-Patent Documents 3 and 4). However, the reverse transcription real-time PCR method developed so far has the problem of low specificity, as it detects other closely related species such as Gr and Globodera ellingtonae (Non-Patent Document 3), and the use of fluorescent dye probes. Therefore, there is a problem that the running cost increases (Non-Patent Documents 3 and 4).

Simple detection and density estimation method of potato cyst nematodes using plastic cups, Hokkaido Agricultural Research Results Information, 2007 (http://www.naro.affrc.go.jp/org/harc/seika/h19/304. html) Imajo, T. (2017). Emergency control of potato cyst nematodes. Plant Protection 71(7), 484-486. Mimee, B., Soufiane, B., Dauphinais, N., & Belair, G. (2017). A qRT-PCR method to evaluate viability of potato cyst nematode (Globodera spp.). Canadian Journal of Plant Pathology, 39( 4), 503-513. Beniers, J. E., Been, T. H., Mendes, O., van Gent-Pelzer, M. P., & van der Lee, T. A. (2014). Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA- specific Real-Time PCR. Nematology, 16(10), 1219-1232.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method capable of efficiently detecting only viable potato phyllocyst nematode (Gp).

As a result of intensive research to solve the above problems, a live nematode detection method using RNA was adopted, and various gene regions were examined to select sequences that can ensure high species specificity. To prevent DNA-derived amplification, the primers were designed such that an intron of about 500 bp was sandwiched between the forward and reverse primers. This made it possible to omit the step of degrading genomic DNA during RNA extraction, further improving efficiency and reducing costs. As a result of the examination, we selected Gp-specific primers designed on the sequence of the Y45F10D.4 gene of potato phyllocyst nematode (Gp), and performed RT-PCR using the designed primers. As a result, the present inventors have found that only living Gp can be efficiently detected, leading to the completion of the present invention.

That is, the present invention includes the following.
[1] A primer set for specifically detecting viable Gp by amplifying a Gp-specific nucleotide sequence by RT-PCR, including the following primers (1) and (2).
(1) a primer consisting of the nucleotide sequence set forth in SEQ ID NO: 1; and
(2) a primer consisting of the base sequence of SEQ ID NO:2;
[2] A kit for detecting viable Gp by RT-PCR, comprising the primer set described in [1].
[3] A method for detecting viable Gp, comprising the step of performing an amplification reaction of the target nucleic acid region of Gp by RT-PCR using the primer set of [1] or the kit of [2].
[4] The method of [3], wherein the RT-PCR is reverse transcription real-time PCR (RT-qPCR) by the intercalator method.

INDUSTRIAL APPLICABILITY According to the present invention, living potato phyllocyst nematodes (Gp), which are important pests of potatoes, can be easily and specifically detected.

4 shows the alignment of the Y45F10D.4 gene of potato phyllostomy nematode (Gp) and its related species and the annealing positions of the primers according to the present invention. From the top, the genomic DNA sequence of the Y45F10D.4 gene of G. pallida, the cDNA sequence of the same gene of G. pallida, the cDNA sequence of the same gene of G. rostochiensis, and the Globodera ellingtonae (G. ellingtonae) and that of the tobacco cyst nematode (Globodera tabacum; G. tabacum). The solid line indicates the annealing position of primer Y45_0803_F1 (46th to 65th bases in the figure), and the dashed line indicates the annealing position of primer Y45_0715_R4 (952nd to 970th bases). The 4th base from the 3' end of primer Y45_0715_R4 is originally A, but it is intentionally replaced with T in order to reduce primer dimers and improve specificity. This is a continuation of Figure 1-1.

The present invention will be described in detail below.

The primer set according to the present invention includes a pair of primers that selectively hybridize with a nucleotide sequence specific to potato cyst nematode (Globodera pallida; Gp). Primer set for amplification by reaction (RT-PCR) and specific detection of viable Gp.

FIG. 1 shows the alignment of the Y45F10D.4 genes of Gp and its related species and the annealing positions of the primers according to the present invention. The Y45F10D.4 gene is a gene encoding an enzyme involved in iron-sulfur cluster formation in vivo. In the present invention, Gp-specific primers were placed on the Y45F10D.4 gene sequence of Gp by 500 between the forward and reverse primers to prevent amplification from genomic DNA that is also detected from dead Gp. It was designed to sandwich an intron of about bp. By performing RT-PCR using the designed primers, it is possible to specifically detect Gp RNA (mRNA), and specifically detect living Gp from detection of RNA derived from living Gp. can do.

A primer set according to the invention comprises the following pair of Gp-specific primers:
(1) a primer (forward primer Y45_0803_F1) comprising or consisting of the nucleotide sequence (CAAAATGACCCATCGGTTG) set forth in SEQ ID NO: 1; and
(2) A primer (reverse primer Y45_0715_R4) containing or consisting of the nucleotide sequence (GCAATGAATGCAACGTTCG) of SEQ ID NO:2.

Alternatively, the primer set according to the present invention has 1 or several (eg, 1 to 10, 1 to 5, 1 to 3, preferably 1 or 2) in the nucleotide sequence indicated by the SEQ ID NO of each primer. ) has a nucleotide sequence in which the nucleotides of ) are deleted, substituted, inserted or added, and primers having the respective primer functions (under stringent conditions for cDNA reverse transcribed from mRNA derived from Y45F10D.4 gene of Gp A primer that hybridizes (preferably under highly stringent conditions) can alternatively be included. Here, "stringent conditions" refer to, for example, conditions for hybridization such as "5×SSPE, 5×Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg/mL salmon sperm DNA, at 42° C. "Night", the conditions for washing are "0.5×SSC, 0.1% SDS, 42° C.". "Highly stringent conditions" are, for example, conditions for hybridization such as "5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL salmon sperm DNA, overnight at 42 ° C. ” and “0.2×SSC, 0.1% SDS, 65° C.” as conditions for washing.

The present invention also relates to a kit for detecting viable Gp by RT-PCR, containing the primer set of the present invention. The kit includes, for example, reverse transcriptase and DNA polymerase used for RT-PCR; substrates for nucleic acid synthesis (dNTPs) used for RT-PCR, buffers, salts, containers, etc.; reagents necessary for detecting RT-PCR amplification products (eg, agarose gel, ethidium bromide, nucleic acid-detectable staining reagents (intercalators), etc.); instructions for use;

Furthermore, the present invention provides a method for detecting viable Gp ( hereinafter referred to as "this method").

In this method, first, for example, an isolate containing cysts and/or eggs separated from a field soil sample containing or suspected of containing Gp (for example, a field soil sample with a history of potato cultivation), or the field soil sample Extract and purify RNA from cysts and/or eggs isolated from Examples of the RNA extraction method include a method using a commercially available RNA extraction reagent. In addition, the primer set according to the present invention has 500 primers between the forward primer and the reverse primer in order to prevent amplification derived from genomic DNA that is also detected from dead Gp on the sequence of the Y45F10D.4 gene of Gp. It is designed to sandwich an intron of about bp, and since it is not amplified from genomic DNA containing the intron by PCR due to its long base sequence, the step of degrading the genomic DNA can be omitted during RNA extraction.

Then, when using the one-step RT-PCR method, the purified RNA is used as a template for PCR using the primer set according to the present invention. The PCR reaction solution contains, for example, 300 to 400 nM (preferably 400 nM) final concentration of each primer contained in the primer set according to the present invention, 1 to 2 µL (preferably 1 µL) of template RNA, and the manufacturer's manual per 10 µL of the reaction solution. Prepare to contain each volume of DNA polymerase, reverse transcriptase, and dNTP according to . Thermal cycling conditions for PCR include, for example, reverse transcription reaction: 42°C for 5 minutes → initial denaturation: 95°C for 10 seconds → PCR reaction: denaturation: 95°C for 5 seconds, and annealing and extension at 62 to 64°C (preferably 63°C). ° C.) 33-35 seconds (preferably 35 seconds) for 30-40 cycles (preferably 40 cycles).

Next, after the PCR reaction, the reaction solution is subjected to agarose gel electrophoresis and stained with ethidium bromide or the like to confirm the presence or absence of an amplification product of the expected size (367 bp amplification product). The presence of viable Gp can be detected by the presence or absence of amplification products.

In this method, when quantitative RT-PCR (RT-qPCR) such as reverse transcription (RT-) real-time PCR is used, for example, in addition to the reagents used in RT-PCR, staining reagents capable of detecting nucleic acids ( intercalator; a method using an intercalator is referred to as an "intercalator method"), or a fluorochrome probe that binds complementary within the amplified region (e.g., a specific PCR is performed using a fluorescent dye probe having an annealing base sequence, a reporter labeled at the 5' end and a quencher labeled at the 3' end, etc.), and a staining reagent or fluorescent dye that can detect the nucleic acid Amplification products can be quantified using the fluorescence signal from the probe as an indicator.

In particular, in this method, it is preferable to perform reverse transcription real-time PCR (RT-qPCR) by the intercalator method as RT-PCR. By using the intercalator method, the running cost can be kept low compared to the case of using a fluorescent dye probe.

In this method, when the two-step RT-PCR method is used, a reverse transcription reaction is performed using the purified RNA as a template to prepare cDNA, and then a PCR reaction is performed in another tube. Specifically, using as a template an isolate containing cysts and/or eggs isolated from a field soil sample, or RNA from cysts and/or eggs isolated from a field soil sample, reverse transcriptase, random hexamer, etc. cDNA is prepared using the primers of Thereafter, PCR is performed using the prepared cDNA as a template and the primer set according to the present invention.

EXAMPLES 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.

[Example 1] Method for detecting viable potato phyllocyst nematode (Gp) and primer set used in the method In this example, a method for detecting viable nematodes using RNA was adopted to keep running costs low. , aimed at detection by the intercalator method without using a probe. In addition, while examining various gene regions and selecting a sequence that can ensure high species specificity, in order to prevent amplification from the genomic DNA of the target gene, an intron of about 500 bp between the forward primer and the reverse primer The primers were designed to sandwich the This made it possible to omit the step of degrading genomic DNA during RNA extraction, further improving efficiency and reducing costs.

1. Primer Design As a result of examination, Gp-specific primers designed on the sequence of the Y45F10D.4 gene of Gp were selected (Table 1 and Figure 1; product size: 367 bp).

2. Intercalator-based reverse transcription real-time PCR (RT-qPCR) using Gp and its relatives
Using the designed primers shown in Table 1, reverse transcription real-time PCR (RT-qPCR) by the intercalator method was performed using Gp and its related species. Table 2 shows the nematode species from which the template nucleic acid used for RT-qPCR was derived, the number of cysts or eggs, and the type of template nucleic acid.
(1) RNA extraction:
RNA was extracted from each nematode sample using Takara's NucleoSpin RNA.
(2) Preparation of PCR reaction solution:
Reverse transcription quantitative PCR was performed using Takara's One Step TB Green (registered trademark) PrimeScript ^™ PLUS RT-PCR kit. The composition of the PCR reaction mixture is 2×One Step TB Green RT-PCR Buffer 5 μl, Takara Ex Taq HS Mix 0.6 μl, PrimeScript PLUS RTase Mix 0.2 μl, Forward Primer Y45_0803_F1 (10 μM) 0.4 μl, Reverse Primer Y45_0715_R4 (10 μM) μM) 0.4 μl, RNase free water 2.4 μl, template RNA 1 μl (10 μl in total).
(3) PCR temperature conditions:
The temperature conditions for PCR were as follows: reverse transcription reaction: 42°C 5 minutes → initial denaturation: 95°C 10 seconds → PCR reaction: denaturation 95°C 5 seconds → annealing/extension 63°C 35 seconds (40 cycles). → Melting curve analysis: 95°C 15 seconds → 60°C 1 minute → 0.3°C/second from 60°C to 95°C.
(4) Results of RT-qPCR by Intercalator Method Table 2 shows the results of RT-qPCR by the intercalator method.

As shown in Table 2, "Detectability", Gp RNA could be specifically detected. In addition, this method was able to detect even very low-density conditions, such as only 10 viable Gp eggs.

3. Reverse transcription real-time PCR (RT-qPCR) by intercalator method using Gp cysts in soil treated with nematicidal 1,3-dichloropropene
Using Gp cysts in soil treated with nematicides with 1,3-dichloropropene (active ingredient of nematicide "DD", hereinafter referred to as "1,3-D"), we conducted an inoculation test on potatoes. , RNA was extracted and RT-qPCR was performed. RT-qPCR was performed in the same manner as RT-qPCR for Gp and its relatives in Section 2 above.
The results are shown in Table 3 below.

As shown in Table 3, Gp survival in the inoculation test correlated well with nucleic acid amplification in RT-qPCR, and nucleic acid amplification was not observed in the sample considered to be dead.

In addition, when PCR was performed on DNA, nucleic acid amplification was observed in all samples. At this time, the composition of the PCR reaction mixture was 5.0 μl of Dream Taq (TM) Hot Start Green Master Mix, 0.3 μl each of primers (ITS5 and PITSp4, both at a concentration of 10 μM), 1 μl of template DNA, and 3.4 μl of RNase free water. (10 μl synthetic). The sequence information of ITS5 is from White et al. (1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: Innis, M.A., Gelfand, D.H., Snisky, J.J. and White, T.J., Eds., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 315-322 (DOI:10.1016/B978-0-12-372180-8.50042-1), the sequence information of PITSp4 is from Bulman and Marshall (1997) Differentiation of Australasian potato cyst nematode ( PCN) populations using the polymerase chain reaction (PCR), New Zealand Journal of Crop and Horticultural Science, Volume 25, Issue 2, pp. 123-129 (DOI: 10.1080/01140671.1997.9513998). These primers are primers targeting the ITS (Internal Transcribed Spacer) of rDNA. The temperature conditions for PCR were as follows: initial denaturation: 2 minutes at 94°C → PCR reaction: denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds (35 cycles). The PCR product was subjected to agarose gel electrophoresis to confirm the presence or absence of a band (265 bp).

4. Summary From the above, according to the RT-qPCR by the intercalator method using the Gp-specific primers designed in this example, it can be performed more quickly than the cup screening method (more than 2 months) without using expensive fluorescent dye probes. Within a few days (within a few days) viable Gp can be specifically detected. As a result, RT-qPCR using the Gp-specific primers designed in this example enables the specific detection and evaluation of viable Gp at a low cost and quickly, requiring more than 2 months. It can be expected to significantly improve the efficiency and speed of the current evaluation of control effects.

Claims (4)

Amplify a nucleotide sequence specific to potato cyst nematode (Globodera pallida), including the following primers (1) and (2), by RT-PCR, and specifically detect surviving potato cyst nematodes primer set for
(1) a primer consisting of the nucleotide sequence set forth in SEQ ID NO: 1; and
(2) a primer consisting of the base sequence of SEQ ID NO:2; A kit for detecting viable potato phyllocyst nematodes by RT-PCR, comprising the primer set according to claim 1. A method for detecting viable potato psyllocyst nematode, comprising the step of performing an RT-PCR amplification reaction of a target nucleic acid region of potato psyllocyst nematode using the primer set of claim 1 or the kit of claim 2. . The method according to claim 3, wherein the RT-PCR is reverse transcription real-time PCR (RT-qPCR) by the intercalator method.

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