KR102348237B1 - Recombinase polymerase amplification primer sets for detecting Bursaphelenchus xylophilus and using thereof - Google Patents

Abstract

The present invention relates to a primer for recombinase polymerase amplification (RPA) for detecting Bursaphelenchus xylophilus and uses thereof. The present invention is detected at a concentration of about 1000 times lower than that of the conventional PRA primer, thereby having an effect of being detected even in a low concentration sample.

Description

Primer for Recombinase Polymerase Amplification (RPA) for detecting pine wilt nematode and its use {Recombinase polymerase amplification primer sets for detecting Bursaphelenchus xylophilus and using thereof}

The present invention relates to a primer for Recombinase Polymerase Amplification (RPA) for detecting pine wilt nematodes and their use.

The pine wilt nematode ( Bursaphelenchus xylophilus ) is a pathogen of pine wilt disease that causes serious damage to forests. The pine wilt nematode is a species introduced from North America and causes serious damage to pine trees that are susceptible to pine wilt nematodes.

There are three main methods for diagnosing pine wilt disease in Korea. There are a method of determining with the naked eye, a method of isolating and identifying pine wilt nematodes from a wood sample under a microscope, and a method of amplifying and confirming a part of a pine wilt gene by polymerase chain reaction (PCR: ploymerase chain reaction).

In the first method, it is not possible to determine whether the disease is caused by drought or other diseases in the method of distinguishing symptoms with the naked eye, so the opinion of an experienced pest expert is required. make it impossible

The second method uses the funnel method to isolate pine wilt nematodes from wood chips, which takes at least 18 - 24 hours, and in the absence of expert knowledge on nematode classification, Bursaphelenchus mucronatus ) cannot be identified using the morphological classification key, and such confusion may lead to errors in the determination of pine wilt disease.

에서 증폭 반응이 진행되며, 이에 따라 생성되는 증폭 산물의 에어로졸에 의한 진단 시료 간 교차오염이 발생할 수 있어 위양성 반응 (false positive reaction)이 쉽게 일어난다는 단점이 있다.Third, the existing PCR method requires at least 30 minutes of experimental time and other experimental equipment such as a centrifuge to extract pine wilt nematode genomic DNA from wood, and isothermal amplification using the existing LAMP (loop mediated isothermal amplification) method Similarly, it takes at least 30 minutes to extract the pine wilt nematode genomic DNA, and it takes at least 1 hour to confirm the amplification. In addition, these methods are high temperature (65

The amplification reaction proceeds in the amplification reaction, and cross-contamination between diagnostic samples by aerosols of the generated amplification product may occur, so there is a disadvantage in that a false positive reaction easily occurs.

The present invention was devised to overcome the above problems, and the present inventors studied a primer set with higher sensitivity than a primer set specific to the existing pine wilt nematode. By confirming that it can be detected in a sample at a concentration about 1000 times lower than that of a primer specific for pine wilt nematode, the present invention has been completed.

An object of the present invention is to provide a primer set for detecting pine wilt nematodes, comprising a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2.

Another object of the present invention is to provide a composition for detecting pine wilt nematodes comprising the primer set.

Another object of the present invention is to provide a kit for detecting pine wilt nematodes comprising the primer set.

Another object of the present invention is to provide a method for detecting pine wilt nematodes using the primer set.

The present invention has been devised to solve the above problems, and the present invention can provide a primer set for detecting pine wilt nematodes comprising a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2 have.

The present invention may also provide a composition for detecting pine wilt nematodes comprising the primer set.

The present invention may also provide a kit for detecting pine wood nematodes comprising the primer set.

The kit may include sodium hydroxide (NaOH), polyethylene glycol (PEG), and dimethyl sulfoxide (DMSO).

According to a preferred embodiment of the present invention, the concentration of NaOH in the composition may be 1 mM to 50 mM.

According to another preferred embodiment of the present invention, the concentration of PEG in the composition may be 1% (v/v) to 10% (v/v), most preferably 5% (v/v). have.

According to another preferred embodiment of the present invention, the concentration of DMSO in the composition may be 1% (v/v) to 10% (v/v), most preferably 5% (v/v). have.

In addition, the present invention provides a method for detecting a pine wilt nematode comprising the following steps.

i) extracting the pine wilt nematode gDNA;

ii) using the extracted Pine wilt nematode gDNA as a template and amplifying a target sequence by performing a polymerase amplification reaction using the primer set; and

iii) detecting the amplification product.

내지 42

에서 수행될 수 있다. According to another preferred embodiment of the present invention, the polymerase amplification reaction is 31

to 42

can be performed in

According to another preferred embodiment of the present invention, the polymerase amplification reaction may be performed for 1 minute to 20 minutes, and more preferably for 15 minutes.

According to another preferred embodiment of the present invention, the polymerase amplification reaction may be a Recombinase Polymerase Amplification (RPA) technique.

The composition of the present invention does not separate the pine wilt nematode, which is a factor of pine wilt disease, unlike the existing domestic and foreign diagnostic methods for pine wilt disease, and collects wood from trees suspected of being infected with pine wilt disease in 10 minutes. It has the advantage of being able to determine the presence or absence of pine wilt disease within a short time using extraction and recombinase polymerase amplification (RPA). have. In addition, compared to the conventional PRA primer, it is detected at a concentration that is about 1000 times lower, so it has the effect of being detected even in a sample with a low concentration.

1 shows the sequences corresponding to the target positions of two types of primer sets specific for the Bx-ITS2 gene of the pine wilt nematode of the present invention.
2 is a schematic diagram of an RPA isothermal amplification reaction experiment process using the Bx-ITS2 marker of the present invention.
3 is a detailed view of a Portable Optical Isothermal Device (POID).
4 is a non-specific amplification reaction assay result for nematode-like pine wood nematodes.
5 is a result of testing the presence or absence of a non-specific amplification reaction caused by the RPA reagent and the pine wood sample.
6 is a result of testing the detection limit of the RPA isothermal amplification method.

Hereinafter, the present invention will be described in more detail.

Definition of Terms

In the present invention, the term “NaOH” refers to sodium hydroxide or sodium hydroxide, “PEG” refers to polyethylene glycol, and “DMSO” refers to dimethyl sulfoxide. it means. The term "extraction" refers to any method for dissolving and isolating certain components from a sample, more specifically a wood sample. "gDNA" means genomic DNA as the total DNA in a cell. A "primer" is a nucleic acid sequence with a short free 3' hydroxyl group, capable of forming a base pair with a complementary template, and serving as a starting point for template strand copying. refers to a nucleic acid sequence.

The present invention provides a primer set for detecting pine wilt nematodes comprising a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2.

Two types of primer sequences specific to the Pine wilt nematode Bx-ITS2 gene division name base sequence size SEQ ID NO: 1 Bx_ITS2_F 5'Biotin-GCACGTTGTGACAGTCGTCTCGCATTGTTC-3' 30 SEQ ID NO: 2 Bx_ITS2_R 5'FAM-TCGAGCACGAAGCCCTCTCGCCCCGCACGG-3' 34

The present inventors studied a primer set with higher sensitivity than a primer set specific to the existing pine wilt nematode, and as a result, the primer specific to the pine wilt nematode of the present invention is a sample at a concentration about 1000 times lower than that of the existing primer specific to the pine wilt nematode. By confirming that it can also be detected, the present invention has been completed. The wood fragment and its gDNA extraction solution (NaOH (Sodium Hydroxide), PEG (Polyethylene Glycol) and DMSO (dimethyl sulfoxide)) were reacted to extract gDNA, and the primer of the present invention and the gDNA of the wood fragment were reacted using a portable isothermal amplifier. and absorbance was measured to confirm positive or negative (FIG. 3). 5 pine wilt nematode- like species genetically similar to pine wilt nematode (Bt: B. thailandae , Bd: B. doui , Bh: B. hylobianum , BmA: B. mucronatus Asia type, BmE: B. mucronatus Europe type) As a result of confirming the presence or absence of non-specific amplification reaction, no amplification reaction did not occur (FIG. 4a), and major forest pests, such as Pinecium Mealyte (Mm), Northern Capricorn (Ms), Monochamus alternatus (Ma), and Alachnidaria (Ac) As a result of confirming the presence or absence of a non-specific amplification reaction targeting , no amplification reaction occurred ( FIG. 4b ). As a result of checking the presence or absence of a non-specific amplification reaction that may be induced from the RPA reagent itself and the pine wood sample, no amplification reaction occurred ( FIG. 5 ). In addition, in order to confirm the detection limit of the primer of the present invention, as a result of concentration-dependent amplification of pure Pine wilt nematode DNA, when pure Pine wilt nematode DNA was used, it was amplified at a DNA concentration of 1.6 fg (10-15) level (Fig. 6a). On the other hand, in the 'rapid gDNA extraction method and method for diagnosing pine wilt nematodes using recombinase polymerase amplification reaction' using the existing Bx_IGS_F and Bx_IGS_R primer sets, when pure pine wilt DNA was used, the pine wilt nematode DNA concentration was 1.6 pg (10-12). up to (Fig. 6b).

The primer is for a polymerase amplification reaction, and the polymerase amplification reaction may be Recombinase Polymerase Amplification (RPA), and these amplification techniques are exemplary, and the present invention is not limited thereto. Those skilled in the art to which the present invention pertains will readily understand. Specific conditions, objects, or materials necessary for the RPA reaction are well known in the prior art, and thus detailed description thereof will be omitted in the present specification.

The primer for detecting pine wilt nematodes of the present invention specifically maintains binding to the target gene of the pine wilt nematode, and 80% or more, 90% or more, 95% or more, 97% or more of the base sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 or greater or greater than 99% homology.

The present invention may also provide a composition for detecting pine wilt nematodes comprising the primer set.

The present invention may also provide a kit for detecting pine wood nematodes comprising the primer set.

The kit may include sodium hydroxide (NaOH), polyethylene glycol (PEG), and dimethyl sulfoxide (DMSO).

The concentration of NaOH may be 1 mM to 50 mM, preferably 25 mM.

The concentration of the PEG may be 1% (v/v) to 10% (v/v), preferably 5% (v/v).

The concentration of DMSO may be 1% (v/v) to 10% (v/v), preferably 5% (v/v).

The composition of the present invention may include distilled water or a buffer, and one or more additional acceptable carriers in addition to the active ingredient may be included to formulate a composition for extraction.

The type and collection method of the wood sample to be extracted according to the present invention are not particularly limited. In addition, the kind of wood from which wood pieces are collected is not specifically limited. Trees of the genus Pinus, such as Japanese black pine, Japanese red pine, Ryukyu island pine, Korean pine, Japanese white pine, Europe Scots pine, European black pine, maritime pine, mugo pine, radiata pine, Monterey pine, ponderosa pine, western white pine pine), Taiwan black pine, Chinese black pine, Himalayan white pine, slash pine, pitch pine, lodgepole pine, strobe eastern white pine, loblolly pine, longleaf pine, jack pine, table Mountain pine, lacebark pine and Taiwan red pine ) is there.

The extraction kit of the present invention may include a composition of one or more other components suitable for the extraction method, and may further include other solutions or devices. Preferably, the extraction kit may further include components necessary for performing RPA. Extraction kits include, in addition to each primer set specific for a marker gene, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. enzymes, DNase, RNase inhibitors, DEPC-water, sterile water, and the like.

The present invention provides a method for detecting pine wilt nematodes comprising the following steps.

i) extracting the pine wilt nematode gDNA;

ii) using the extracted Pine wilt nematode gDNA as a template and amplifying a target sequence by performing a polymerase amplification reaction using the primer set; and

iii) detecting the amplification product.

내지 42

에서 수행될 수 있다. The polymerase amplification reaction is 31

to 42

can be performed in

The polymerase amplification reaction may be performed for 1 to 20 minutes, preferably for 15 minutes.

The polymerase amplification reaction may be Recombinase Polymerase Amplification (RPA), and these amplification techniques are exemplary, and it is understood that the present invention is not limited thereto. You will be able to understand easily. Specific conditions, objects, or materials necessary for the RPA reaction are well known in the prior art, and thus detailed description thereof will be omitted in the present specification.

The method for detecting the amplified pine wilt nematode DNA, which is the fourth step, is not particularly limited, but at least one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement and phosphorescence measurement, etc. Methods known in the art can be used.

The gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product.

In the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the marker with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence can be measured using a fluorometer. can

Hereinafter, the present invention will be described in detail by way of Examples. However, these Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

Example 1. Pine wilt nematode gDNA extraction from wood samples

5 wood samples (8 X 4 X 1.5 mm) were collected from trees infected with pine wilt disease by combining an 8 mm drill bit with a pine sampler manufactured by the National Institute of Forest Science. Pine wilt nematode gDNA extraction containing 25 mM NaOH (Sodium Hydroxide), 5% (v/v) PEG (Polyethylene Glycol, average molecular weight 190-210) and DMSO (Dimethyl Sulfoxide) at 5% (v/v) concentration A composition was prepared. 10 wood samples (8 X 4 X 1.5 mm) infected with the pine wilt nematode were placed in a 2.0 ml eppendorf tube. After putting 1 ml of the composition for extraction into the tube, it was mixed with a wood sample, and then, by reacting at room temperature for 10 minutes, gDNA of pine nematodes was extracted from the wood sample. 3 μl of the wood extract was used as a template for a PCR reaction with a volume of 20 μl or a recombinase polymerase amplification (RPA) with a volume of 50 μl.

Example 2. Recombinase Polymerase amplification (RPA)

(1) Preparation of primer for RPA specific to the pine wilt nematode species

Among the pine wilt nematodes ( Bursaphelenchus xylophilus ) and 5 species of other pine wilt nematode genera ( Bursaphelenchus mucronatus Asia-type, Bursaphelenchus mucronatus Europe-type, Bursaphelenchus thailandae , Bursaphelenchus doui , Bursaphelenchus hylobianum ) are the closest related to the pine wilt nematode Bursaphelenchus hylobianum. mucronatus ), ITS2 sequences present between the 5.8S rDNA and 28S rDNA gene sequences were compared to prepare two types of primers specific to Pine wilt nematodes (FIG. 1 and Table 1).

(2) Recombinase polymerase amplification reaction product combination and amplification reaction process

에서 15분 반응하였다. 증폭반응 종료된 반응물 25 μl은 25 μl SYBR mixture [1 μl SYBR® Green I 시약 (이하 미국, Lonza사 제품), 24 μl 3차 증류수] 와 섞은 후, 휴대용 등온증폭기기를 사용하여 흡광도를 측정하여, 양성 또는 음성 판단을 하였다. (흡광도 수치 건전목 < 50 -≤ 감염목) (도 3 및 도 5).The reaction mixture of the recombinase polymerase amplification reaction is as follows: 29.5 μl of 1X rehydration buffer (UK, TwistDX), Twistamp nfo dried pallet 1 tube, 1.6 μl of Bx_ITS2_F1 (5 pmol/μl), 1.6 μl of Bx_ITS2_R1 (5 pmol/μl), 11.8 μl of tertiary distilled water, 2.5 μl of MgAc (280 mM), and 3 μl of an extract extracted from pine wood using DAP buffer. combine For the recombinase polymerase amplification reaction, a portable isothermal amplification device (POID: Portable Optical Isothermal Device) was used, 37

reacted for 15 min. After the amplification reaction was completed, 25 μl of the reaction was mixed with 25 μl SYBR mixture [1 μl SYBR® Green I reagent (hereinafter, Lonza, USA), 24 μl tertiary distilled water], and absorbance was measured using a portable isothermal amplifier. A positive or negative judgment was made. (Absorbance values in healthy trees < 50 -≤ infected trees) ( FIGS. 3 and 5 ).

Example 3. Specificity and non-specificity amplification reaction assay result of ITS2 marker of Pine wilt nematode

(1) Pine wilt nematode genetic sequence specific amplification test result

In addition to the pine wilt nematode, which is a major factor causing pine wilt disease, other nematodes, fungi, bacteria, and microscopic animals live in trees. In order to find out whether a false-positive reaction is shown in the amplification reaction due to the genomic DNA of these other organisms, the pine wilt nematode ( B. xylophilus ) and five species of nematodes of the genus pine wilt nematode ( B. mucronatus Asia-type, B. mucronatus Europe-type, B. thailandae , B. doui , B. hylobianum ) Using 16 ng of genomic DNA, a recombinase polymerase amplification reaction was performed to confirm whether non-specific amplification occurred ( FIG. 4a ). As a result of the test, under the optimal amplification test condition of 37°C for 15 minutes, only the genomic DNA sample of Pine wilt nematode showed specific color change and amplification product, and the color change and amplification product (false positive reaction) did not appear in 5 nematodes of the genus Pine wilt nematode. .

(2) Non-specific amplification reaction assay results of pine wilt nematodes

In order to find out whether other forest pests non-specifically amplify, the major forest pests, such as briar beetle (Mm), borealis borealis (Ms), monochamus alternatus (Ma), and alpacas (Ac) were targeted. The presence or absence of a non-specific amplification reaction was checked. Non-specific amplification by performing a recombinase polymerase amplification reaction using 16 ng of the genomic DNA of pinecula beetle (Mm), boreal capricorn (Ms), monochamus alternatus (Ma) and alfalfa (Ac) I checked to see if this is happening. As a result of the experiment, the amplification reaction did not occur in the major forest pests, such as briar beetle (Mm), borealis capricorn (Ms), monochamus alternatus (Ma), and alpacas (Ac) (FIG. 4b).

(3) Non-specific amplification reaction assay results of pine wilt nematodes

In order to confirm the presence or absence of a non-specific amplification reaction that may be induced from the RPA reagent itself and the pine wood sample, 3 ul of a healthy pine wood sample extracted using a genomic DNA extract, 3 ul of tertiary distilled water, the Bx_ITS2 Recombinase polymerase amplification of the reaction solution except for the marker was performed to check whether non-specific amplification occurred. As a result of the experiment, as a result of confirming the presence or absence of a non-specific amplification reaction that may be induced from the RPA reagent itself and a healthy pine wood sample, all absorbance values were less than 50, so that no amplification reaction occurred ( FIG. 5 ).

Example 4. Detection limit test result of RPA isothermal amplification method

The detection limit of the RPA isothermal amplification method of the Bx_ITS2_F and Bx_ITS2_R primer sets was overcome by the detection of the RPA isothermal amplification method using the Bx_IGS_F and Bx_IGS_R primer sets used in the existing 'rapid gDNA extraction method and pine wilt nematode diagnosis method using recombinase polymerase amplification reaction' In order to compare and test the limits, the RPA isothermal amplification reaction under the same conditions was performed under the same conditions as Bx_ITS2_F and Bx_ITS2_R by setting the pure pine wood nematode gDNA concentration to 16 ng, 1.6 ng, 160 pg, 16 pg, 1.6 pg, 160 fg, 16 fg, 1.6 fg, respectively. Comparisons were made according to the use of the primer set (FIG. 6A) and the Bx_IGS_F and Bx_IGS_R primer sets (FIG. 6B).

The detection limit of the RPA isothermal amplification reaction of the present invention was amplified even at a pine wilt nematode DNA concentration of 1.6 fg (10-15) when pure pine wilt DNA was used (FIG. 6a). In the 'rapid gDNA extraction method and the pine wilt nematode diagnosis method using the recombinase polymerase amplification reaction' using the existing Bx_IGS_F and Bx_IGS_R primer sets, it was possible to amplify up to a pine wilt nematode DNA concentration of 1.6 pg (10-12) (Fig. 6b). . Therefore, it was confirmed that the method using the Bx_ITS2_F and Bx_ITS2_R primer sets was improved by about 1,000 times in detecting the pine wilt nematode DNA.

The above description of the invention is illustrative of the present invention. In addition, the above description shows and describes preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes or modifications are possible within the scope of the concept of the invention disclosed herein, the scope equivalent to the written disclosure, and/or within the scope of skill or knowledge in the art. Various modifications required in the specific field of application and use of the present invention are also possible. Accordingly, the detailed description of the present invention is not intended to limit the present invention to the disclosed embodiments.

<110> KOREA FOREST RESEARCH INSTITUTE <120> Recombinase polymerase amplification primer sets for detecting Bursaphelenchus xylophilus and using thereof <130> 1067728 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Bx_ITS2_F <400> 1 gcacgttgtg acagtcgtct cgcattgttc 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Bx_ITS2_R <400> 2 tcgagcacga agccctctcg ccccgcacgg 30

Claims (5)

A primer set for detecting pine wilt nematodes, comprising a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2.
A composition for detecting pine wood nematodes, comprising the primer set of claim 1.
A kit for detecting pine wood nematodes, comprising the primer set of claim 1.
According to claim 3, wherein the kit includes NaOH (Sodium Hydroxide), PEG (Polyethylene Glycol) and DMSO (dimethyl sulfoxide), the kit for detecting pine wilt nematodes.
i) extracting the pine wilt nematode gDNA;
ii) using the extracted Pine wilt nematode gDNA as a template and amplifying a target sequence by performing a polymerase amplification reaction using the primer set of claim 1; and
iii) detecting the amplification product;
A method for detecting a pine wilt nematode comprising a.

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