KR102737155B1 - Xanthomonas hotorum pv. carotae-specific primer set and method for detecting Xanthomonas hotorum pv. carotae using the same - Google Patents

Abstract

The present invention relates to a primer set for detecting Xanthomonas hotorum pv. carotae , comprising an oligonucleotide primer of sequence number 1 and an oligonucleotide primer of sequence number 2; a composition comprising the same; a kit comprising the same; and a method for detecting Xanthomonas hotorum pv. carotae using the same.

Description

{Xanthomonas hotorum pv. carotae-specific primer set and method for detecting Xanthomonas hotorum pv. carotae using the same}

The present invention relates to a primer set for detecting Xanthomonas hotorum pv. carotae , comprising an oligonucleotide primer of sequence number 1 and an oligonucleotide primer of sequence number 2; a composition comprising the same; a kit comprising the same; and a method for detecting Xanthomonas hotorum pv. carotae using the same.

Xanthomonas is a genus of bacteria involved in denitrification of soil nitrogen. Some of these are pathogenic bacteria that cause peach bacterial hole, radish black rot, citrus canker, rice white leaf blight, and soybean fire blight. Xanthomonas hortorum causes bacterial spot disease. At first, it forms water-soaked spots mainly on old leaves, which gradually enlarge and form brown to black spots with yellow borders. If the disease is severe, the entire leaf turns black and dries up, and the lesions do not form black granules, so it has different characteristics from spot diseases caused by fungal diseases.

Dot-blot, polymerase chain reaction (PCR), etc. are used as genetic identification methods for plant pathogenic bacteria. In particular, PCR is a method that has been widely used recently because it is more economical than the Dot-blot method in terms of time, effort, and manpower. The PCR (polymerase chain reaction) identification method contacts (anneals) a specific DNA fragment (primer) consisting of 10 to 20 bp to the genomic DNA of the bacteria, then adds heat-stable DNA polymerase (Taq polymerase) and performs the synthesis reaction repeatedly, rapidly amplifies the region bound by the primer, and identifies DNA polymorphisms of bacterial species, such as the presence or absence or difference in shape of DNA bands that appear after electrophoresing the PCR amplification product on an agarose gel or an acrylamide gel and staining the DNA with ethidium bromide and silver stain. Currently, this PCR-based bacterial identification method is mainly used to identify bacterial diseases in humans or animals, but recently, PCR identification has been developed to identify plant pathogens, and is used for disease identification and quarantine of imported and exported agricultural products. In addition, the PCR identification method is economical because it takes less time than other methods, the cost of identification is low, and many samples can be tested simultaneously. Therefore, among the recent Xanthomonas identification methods, the PCR identification method that amplifies nucleic acids, which are genetic material, is the most preferred.

However, existing PCR identification methods have difficulty in differentiating between species due to genetic similarity, and a solution to overcome this is needed.

Among the existing Xanthomonas genus microorganisms, studies have been conducted on individual PCR primers used for the identification of various microorganisms, including the rice wilt pathogen Xanthomonas oryzae pv. oryzae, the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria, the cruciferous black rot pathogen Xanthomonas campestris pv. campestris, the citrus canker pathogen Xanthomonas axonopodis pv. citri, and the soybean wilt pathogen Xanthomonas axonopodis pv. glycines. However, there has been no study yet on a method for identifying Xanthomonas hotorum pv. carotae from the genus Xanthomonas. Therefore, the development of a method for identifying Xanthomonas hotorum pv. carotae is required.

Korean Publication Patent No. 10-2010-0048028

Accordingly, the present invention has as its technical task the provision of a primer set capable of detecting Xanthomonas hotorum pv. carotae , the causative bacterium of carrot bacterial blight, with high specificity and high sensitivity, and suitable for real-time PCR, thereby enabling confirmation of infection and direct quantification (density measurement) without extracting DNA from a specimen.

The present inventors confirmed the specificity of the hypothetical protein gene of Xanthomonas hotorum pv. carotae (GenBank accession NZ_CM002307.1, protein ID WP_220655539.1, location= 2448580..2449011) registered in Genbank (www.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) by producing a primer set to specifically detect Xanthomonas hotorum pv. carotae using the blastn and blastx programs.

Accordingly, a novel primer set was created based on the above-described hypothetical protein gene to amplify a 300-bp fragment in Xanthomonas hotorum pv. carotae , and PCR and real-time PCR amplification tests were performed on several microbial strains using the same. As a result, it was confirmed that the primer set specifically generates an amplification product only in Xanthomonas hotorum pv. carotae , thereby completing the present invention.

Based on the above, the present invention provides a primer set for detecting Xanthomonas hotorum pv. carotae, comprising an oligonucleotide primer of SEQ ID NO: 1 and an oligonucleotide primer of SEQ ID NO: 2.

The present invention also provides a composition for detecting Xanthomonas hotorum pv. carotae , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides a kit for detecting Xanthomonas hotorum pv. carotae , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention provides a method for detecting Xanthomonas hotorum pv. carotae , comprising the steps of (i) performing a DNA amplification reaction on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set represented by the base sequences of SEQ ID NO. 1 and SEQ ID NO. 2; and (ii ) detecting a DNA amplification product.

In addition, the present invention provides a method for diagnosing Xanthomonas hotorum pv. carotae infection, comprising the steps of performing real-time PCR on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and analyzing the gene amplification result by the real-time PCR.

Using the primer set according to the present invention, Xanthomonas hotorum pv. carotae can be detected specifically and efficiently within a short period of time.

In particular, the primer set according to the present invention has a high detection sensitivity for Xanthomonas hotorum pv. carotae , so that it can be detected with excellent efficiency even when the amount of sample is small.

In addition, real-time PCR using the primer set according to the present invention can directly confirm the presence or absence of infection with Xanthomonas hotorum pv. carotae without a DNA extraction process from a specimen, and can quantitatively analyze the pathogenic organisms to confirm the density of the pathogenic organisms in the specimen.

Figure 1 shows the results of a blastn search using the hypothetical protein gene of Xanthomonas hotorum .
Figure 2 is a diagram showing the results of PCR amplification of a hypothetical protein using primers.
Figure 3 is a diagram showing the results of real-time PCR amplification of a hypothetical protein of Xanthomonas hotorum pv. carotae .
Figure 4 is a diagram showing the results of analyzing the sensitivity of real-time PCR in a standard dilution series of genomic DNA.
Figure 5 is a diagram showing the results of analyzing the sensitivity of real-time PCR in a standard dilution series of cloned DNA.
Figure 6 is a diagram showing the results of analyzing the sensitivity of real-time PCR in a standard dilution series of bacterial cells.

The present inventors confirmed the specificity of the hypothetical protein gene of Xanthomonas hotorum pv. carotae (GenBank accession NZ_CM002307.1, protein ID WP_220655539.1, location= 2448580..2449011) registered in Genbank (www.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) by producing a primer set to specifically detect Xanthomonas hotorum pv. carotae using the blastn and blastx programs.

Accordingly, a novel primer set was created to amplify a 300-bp fragment from Xanthomonas hotorum pv. carotae based on the hypothetical protein gene.

The present inventors performed PCR and real-time PCR amplification tests on several microbial strains including Xanthomonas hotorum pv. carotae using the above primer set, and confirmed that the primer set specifically generates an amplification product only in Xanthomonas hotorum pv. carotae .

Accordingly, in one aspect, the present invention relates to a primer set for detecting Xanthomonas hotorum pv. carotae , comprising an oligonucleotide primer of SEQ ID NO: 1 and an oligonucleotide primer of SEQ ID NO: 2.

The RS22715_300F primer represented by the base sequence of the above sequence number 1 is a forward primer, and the RS22715_300R primer represented by the base sequence of the above sequence number 2 is a reverse primer.

The term "primer" as used herein refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be replicated, and can act as a starting point for the synthesis of a primer extension product. When designing a primer, there are various restrictions, such as the content ratio of A, G, C, and T of the primer, prevention of primer dimer formation, prohibition of repeating the same base sequence more than three times, and in addition, for a single PCR reaction condition, the amount of template DNA, concentration of primer, concentration of dNTP, concentration of Mg2 ⁺ , reaction temperature, and reaction time must be appropriate.

The primer set according to the present invention may also include a base sequence having one or more bases deleted, substituted or added to the base sequences represented by SEQ ID NOS: 1 and 2. In addition, the primer set according to the present invention may include additional features that do not change the basic properties. That is, the base sequences may be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologues of the nucleotides, and modification of the nucleotides to uncharged linkers such as phosphonates, phosphotriesters, phosphoramidates or carbamates, or to charged linkers such as phosphorothioates or phosphorodithioates. In addition, the nucleic acids may have one or more additional covalently bonded moieties, such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, intercalators such as acridine or psoralens, chelating agents such as metals, radioactive metals, iron-oxidizing metals, and alkylating agents.

Additionally, the base sequence of the primer set according to the present invention can be modified using a label capable of directly or indirectly providing a detectable signal. The primer set can include a label capable of being detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include ³² P, fluorescent dyes, electron dense reagents, enzymes (typically those used in ELISA), biotin or haptens and proteins for which antisera or monoclonal antibodies are available.

The primer set according to the present invention can be chemically synthesized using any other well-known method including cloning and restriction enzyme digestion of appropriate sequences and direct chemical synthesis methods such as the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68:90-99), the diethylphosphoramidite method of Beaucage et al. (1981, Tetrahedron Lett. 22: 1859-1862), and the solid support method of U.S. Pat. No. 4,458,066.

Methods for amplifying the target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification, amplification via replicase, or any other suitable method known in the art for amplifying a nucleic acid molecule.

Among these, "polymerase chain reaction (PCR)" is a method of amplifying a target nucleic acid from a set of primers that specifically bind to the target nucleic acid using a polymerase. The PCR of the present invention encompasses general (non-quantitative) PCR and quantitative PCR, and includes, for example, general PCR and Real-time PCR. PCR is the most well-known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, in order to enhance the specificity or sensitivity of PCR, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying the traditional PCR procedure. Additionally, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For a detailed discussion of PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000).

Real-time PCR is a technology that monitors and analyzes the increase in PCR amplification products in real time. The PCR reaction can be monitored by recording the amount of fluorescence emission at each cycle during the exponential phase, when the increase in PCR products is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the Ct value (threshold cycle). When the amount of PCR amplification product reaches a detectable amount by fluorescence, the amplification curve begins to occur, and the signal increases exponentially and then reaches a plateau. The larger the initial amount of DNA, the fewer the number of cycles for the amplification product to reach a detectable amount, so the amplification curve appears faster. Therefore, when real-time PCR is performed using a stepwise diluted standard sample, an amplification curve is obtained that is lined up at equal intervals in order of the initial amount of DNA. Here, if the threshold is set at an appropriate point, the Ct value at which the threshold and the amplification curve intersect is calculated.

In one embodiment, the primer set according to the present invention may be used in PCR.

In another embodiment, the primer set according to the present invention may be used in Real-time PCR.

In the present invention, the amplified target sequence can be labeled with a detectable labeling substance. In one embodiment, the labeling substance can be a substance that emits fluorescent, phosphorescent or radioactivity, but is not limited thereto. When the 5'-end of the primer is labeled with Cy-5 or Cy-3 during amplification of the target sequence and the amplification reaction is performed, the target sequence can be labeled with a detectable fluorescent labeling substance. In addition, when labeling using a radioactive substance, when a radioactive isotope such as ³² P or ³⁵ S is added to the amplification reaction solution during the amplification reaction, the radioactivity is incorporated into the amplification product as the amplification product is synthesized, so that the amplification product can be radioactively labeled.

In the present invention, the detection of the amplification product can be performed through, but is not limited to, fluorescence measurement, DNA chip, gel electrophoresis, radioactivity measurement, or phosphorescence measurement. As one of the methods for detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product.

Another aspect of the present invention relates to a composition for detecting Xanthomonas hotorum pv. carotae , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Another aspect of the present invention relates to a kit for detecting Xanthomonas hotorum pv. carotae , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In one embodiment, the kit may be a PCR kit.

In another embodiment, the kit may be a kit for Real-time PCR.

In one embodiment, the kit may further include, in addition to the primer set, reagents for nucleic acid extraction, amplification and product detection, and instructions therefor. For example, the kit may include reagents for nucleic acid extraction, deoxynucleotides, reagents necessary for performing a nucleic acid amplification reaction, a heat-stable nucleic acid polymerase, a buffer, and dNTPs. The primer set and the reagents for nucleic acid extraction, amplification and product detection may be provided in a lyophilized state in a plastic tube, and the nucleic acid may preferably be DNA.

Another aspect of the present invention relates to a method for detecting Xanthomonas hotorum pv. carotae , comprising the steps of: (i) performing a DNA amplification reaction on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and (ii) detecting a DNA amplification product.

The sample in step (i) above may include anything suspected of being infected with Xanthomonas hotorum pv. carotae , for example, the pathogen itself, or leaves, branches, stems, roots, flowers, etc. of plants expected to be infected with the pathogen.

In one embodiment, the DNA amplification reaction of step (i) may be Real-time PCR. The primer set according to the present invention is suitable for Real-time PCR, so that it is possible to directly confirm the presence or absence of infection with Xanthomonas hotorum pv. carotae without a DNA extraction process from a sample, and to quantitatively analyze the pathogenic organisms to confirm the density of the pathogenic organism in the sample.

In one embodiment, the step (i) may further include a step of isolating genomic DNA from a sample suspected of being infected with Xanthomonas hotorum pv. carotae . The method for isolating genomic DNA from the sample may utilize a method known in the art, for example, the CTAB method may be utilized, or the Wizard kit (Promega) may be utilized.

In one embodiment, in the step (i), when isolating genomic DNA from a sample suspected of being infected with Xanthomonas hotorum pv. carotae , the DNA amplification reaction can be performed by PCR. The PCR reaction can be performed using a PCR reaction mixture containing various components known in the art necessary for a PCR reaction. The PCR reaction mixture contains, in addition to genomic DNA and the primer pair provided in the present invention, an appropriate amount of DNA polymerase, dNTP, PCR buffer solution, and water.

The detection of the DNA amplification product of the above step (ii) can be performed by a DNA chip, electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement, thereby confirming whether the target gene is amplified. Gel electrophoresis can be performed as one of the methods for detecting the amplification product. Depending on the size of the amplification product, gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis.

In one embodiment, the detection of the DNA amplification product of step (ii) can be performed by electrophoresis, and at this time, whether the target gene is amplified can be confirmed by the amplification of a 300 bp-sized fragment of the hypothetical protein gene of Xanthomonas hotorum pv. carotae .

Another aspect of the present invention relates to a method for diagnosing Xanthomonas hotorum pv. carotae infection, comprising the steps of performing real-time PCR on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and analyzing the result of gene amplification by the real-time PCR.

In one embodiment, the diagnostic method is characterized in that real-time PCR is performed on a sample itself without a process of isolating genomic DNA from a sample suspected of being infected with Xanthomonas hotorum pv. carotae .

The real-time PCR diagnostic method according to the present invention can directly detect the pathogen in a sample suspected of being infected with Xanthomonas hotorum pv. carotae without a DNA extraction process. In addition, the real-time PCR diagnostic method can quantitatively analyze Xanthomonas hotorum pv. carotae individuals in the sample to confirm the density of the pathogen in the sample.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

Example

Example 1: Culturing bacterial strains and extracting total RNA

(1) Bacterial strains and culture conditions

Other microorganisms, including Xanthomonas hotorum pv. carotae , used in the present invention were provided by the Korean Agricultural Culture Collection, Republic of Korea (KACC), and their sources are summarized in Table 1. Xanthomonas strains were cultured for two days at 27°C on Tryptic Soy Agar (TSA) medium.

No. Scientific name Source ^a Host ^b note 1 X. hotorum pv. carotae This study carrot JJ2001 2 X. hotorum pv. carotae This study carrot JJ2004 3 X. hotorum pv. carotae This study carrot JJ2006 4 X. axonopodis pv. KACC 10317 N.D. N.D 5 X. campestris pv. zinnia KACC 17126 N.D. Zinnia 6 X. campestris pv. glycines KACC 10491 N.D. Soybean 7 X. campestris pv. KACC 10460 N.D. N.D 8 X. campestris pv. KACC 10377 N.D. N.D 9 X. axonopodis pv. glycines KACC 11144 N.D. Soybean 10 X. campestris pv. vitians KACC 19681 N.D. lettuce 11 X. arboricola pv. pruni KACC 18153 N.D. plum 12 X. euvesicatoria KACC 18722 N.D. pepper 13 X. perforans KACC 16356 N.D. Tomato

KACC: Korean Agricultural Culture Collection, Republic of Korea

RDA: Rural Development Administration, Republic of Korea

^a Superscript "T" indicates type strain

^a ND, Not determined

(2) Extraction of total DNA

To extract total DNA from the cultured strain, a genomic DNA extraction kit (Wizard ^® Genomic DNA Purification Kit) from Promega (Madison, WI, USA) was used.

Example 2: Preparation of a specific primer set

(1) Analysis of the use of the blastn program to search for specific base sequence information

To generate a primer set to specifically detect Xanthomonas hotorum pv. carotae , the genome information of Xanthomonas hotorum pv. carotae (GenBank accession NZ_CM002307.1, protein ID WP_220655539.1, location= 2448580..2449011) registered in Genbank of the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/) was used to confirm the specificity using the blastn and blastx programs (Fig. 1).

(2) Production of special primer

The RS22715_300F/R candidate primer set was prepared from Xanthomonas hotorum pv. carotae , which was designed to amplify a 300 bp fragment, and the sequences of the primers are as follows (Table 2). The primers were used to amplify a 300 bp fragment from Xanthomonas hotorum pv. carotae registered in GenBank. It was produced after confirming the specificity of the hypothetical protein gene sequence information of the strain using the blastn program.

Primer Base sequence Size (bp) order
number RS22715_300F '5-TGGCCGGCTGCTCAAAGA-3' 300 Sequence number 1 RS22715_300R '5-CCGCATCCGAATCAGAATACAACT-3' Sequence number 2

Example 3: PCR amplification reaction

PCR amplification reactions were performed using a PTC-200 ^TM thermocycler (MJ research, Watertown, mass), and the final volume of the reaction solution for each PCR amplification was 25 μl. The composition of the reaction solution was a mixture of 1X reaction buffer, 0.2 mM dNTP, 10 pM each of Forward/Reverse primers, and 1.25 unit GoTaq ^® Flexi DNA polymerase (Promega Madison, WI, USA). The amount of bacterial genomic DNA used in the PCR reaction was adjusted to 20 ng.

The PCR reaction was denatured at 95°C for 5 minutes, and then repeated 30 times at 95°C for 60 seconds, 60°C for 30 seconds, and 72°C for 60 seconds, and then reacted at 72°C for 7 minutes. After the amplification reaction, 10 μl of the PCR product was stained with Loading Star dye (DYNEBIO, Republic of Korea) to stain the DNA, and electrophoresed on a 1.5% agarose gel, and the amplified DNA band was confirmed under a UV transilluminator.

As a result, it was confirmed that a 300 bp sized nucleic acid fragment was specifically amplified only in Xanthomonas hotorum pv. carotae (Fig. 2).

Example 4: Real-time PCR specificity analysis

Real-time PCR amplification reaction was performed using the CFX96 Real-time PCR system (Bio-Rad laboratories, Inc., USA). The reaction was performed using 5 pM of each RS22715-300F/R primer and 10 ㎕ QGreenBlue 2X Green qPCR Master Mix (CellSafe Bio, Inc., Korea) for a total of 20 ㎕ of Real-time PCR mixture.

The amount of bacterial genomic DNA used in real-time PCR was adjusted to 5 ng using Nanodrop ^TM NP80 (Implen, Germany) and used in the reaction. The reaction was denatured at 95°C for 30 sec, repeated 40 times at 95°C for 5 sec and 60°C for 30 sec, reacted at 95°C for 15 sec, and then increased by 0.5°C every 5 sec from 65°C to 95°C for a melting curve and melting peak.

As a result, a melting peak at 85℃ was confirmed only in Xanthomonas hotorum pv. carotae (Fig. 3).

Example 5: Real-time PCR sensitivity analysis

(1) Real-time PCR of genomic DNA

Real-time PCR amplification reactions were performed using a CFX96 Real-time PCR system (Bio-Rad laboratories, Inc., USA). Each reaction was performed in a total volume of 20 ㎕ with a real-time PCR mixture containing 5 pM each of QGreenBlue 2X Green qPCR Master Mix (CellSafe Bio, Inc., Korea) and RS22715-300F/R primers. Xanthomonas hotorum pv. carotae genomic DNA was diluted 10-fold from 5 ng.

As a result, standard curves were generated at the threshold cycle (Ct), also known as the intersection point of the Xanthomonas hotorum pv. carotae genomic DNA standard dilutions, and all curves showed ^R2 > 0.995, and the melting peak results confirmed that the amplified product was at a melting temperature (Tm) of approximately 85°C (Fig. 4).

(2) Real-time PCR of clone DNA

The genomic DNA of strain 1 described in Table 1 above was generated using the primers described in Table 2 above and the PCR conditions described in Example 3, and the PCR product was cloned into the pGEMT-easy ^TM vector (Promega Co., The cloned DNA was diluted 10-fold from approximately 1 ng to investigate the detection limit.

As a result, the standard curve was determined using Xanthomonas hotorum pv. carotae It was generated at the threshold cycle (Ct), also known as the intersection point of the cloned DNA standard dilution, and all curves showed ^R2 > 0.997, and the melting peak results confirmed the amplified product at a melting temperature (Tm) of approximately 85℃ (Fig. 5).

(3) Real-time PCR of bacterial cells

The bacterial cell suspension was adjusted to 8.10 X 10 ⁷ CFU/ml, diluted 10-fold, and added to the real-time PCR mixture. The real-time PCR amplification reaction was performed by denaturing at 95°C for 30 sec, repeating 40 cycles of 95°C for 5 sec and 60°C for 30 sec, reacting at 95°C for 15 sec, and then increasing the temperature by 0.5°C every 5 sec from 65°C to 95°C for a melting curve and melting peak.

As a result, the standard curve was determined using Xanthomonas hotorum pv. carotae It was generated at the threshold cycle (Ct), also known as the intersection point of the bacterial cell standard dilution, and all curves showed ^R2 > 0.997, and the melting peak results confirmed that the amplified product was at a melting temperature (Tm) of approximately 85℃ (Fig. 6).

In addition, since the R ² value and E value of the standard curve are within the appropriate range, it can be determined as valid data, and it was confirmed that Xanthomonas hotorum pv. carotae can be stably quantitatively detected even at 8.10Х10 ⁵ CFU/ml in bacterial cell suspension (Table 3).

Cloned DNA Genomic DNA Cell suspension Weight/μl reaction mix Ct ± SD ^a
( n = 3) Weight/μl reaction mix Ct ± SD
( n = 3) CFU/ml reaction mix Ct ± SD
( n = 3) 5 ng 22.22±0.07 1 ng 9.31±0.10 8.10×10 ⁷ 24.28±0.11 500 pg 25.59±0.39 100 pg 13.21±0.14 8.10×10 ⁶ 28.88±0.21 50 pg 29.36±0.03 10 pg 17.41±0.19 8.10×10 ⁵ 33.31±0.18 5 pg 32.58±0.26 1 pg 20.94±0.36 8.10×10 ⁴ 38.03±0.19 500 fg 35.46±0.08 100 fg 24.73±0.43 8.10×10 ³ ND 10 fg 28.32±0.21 8.10×10 ² ND 8.10×10 ¹ ND

^a SD, Three reactions standard deviation.

^b ND, Not detected.

Therefore, through the sensitivity analysis of the above Real-time PCR, it can be determined that the R ² value and E value of the standard curve are within the appropriate range, which means that it is valid data, and it was confirmed that Xanthomonas hotorum pv. carotae can be stably quantitatively detected at 5 pg of genomic DNA, 10 fg of cloned DNA, and 8.10Х10 ⁵ CFU/ml in bacterial cell suspension.

In conclusion, the RS22785-257F/R primers, which were constructed from a specific DNA fragment of the hypothetical protein gene of Xanthomonas hotorum pv. carotae , can be used as PCR primers for the specific detection of R. fascians , and it was confirmed that the corresponding symptoms can be detected directly from the sample without a DNA extraction process.

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Xanthomonas hotorum pv. carotae-specific primer sets and methods for detecting Xanthomonas hotorum pv. carotae using the same <130> PD22-039 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RS22715_300 Forward primer <400> 1 tggccggctg ctcaaaga 18 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RS22715_300 Reverse primer <400> 2 ccgcatccga atcagaatac aact 24

Claims (15)

A primer set for detection of Xanthomonas hotorum pv. carotae , comprising an oligonucleotide primer of sequence number 1 and an oligonucleotide primer of sequence number 2.
In the first paragraph, the primer set is a primer set used in polymerase chain reaction (PCR).
In the first paragraph, the primer set is a primer set used in real-time polymerase chain reaction (real-time PCR).
A composition for detecting Xanthomonas hotorum pv. carotae , comprising a primer set according to any one of claims 1 to 3.
A kit for detecting Xanthomonas hotorum pv. carotae , comprising a primer set according to any one of claims 1 to 3.
A kit according to claim 5, wherein the kit is for PCR (polymerase chain reaction).
A kit according to claim 5, wherein the kit is for real-time PCR.
(i) a step of performing a DNA amplification reaction on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set of any one of claims 1 to 3; and
(ii) comprising a step of detecting a DNA amplification product;
Method for detecting Xanthomonas hotorum pv. carotae .
A method in claim 8, wherein the DNA amplification reaction in step (i) is real-time PCR.
A method according to claim 8, further comprising a step of isolating genomic DNA from a sample suspected of being infected with Xanthomonas hotorum pv. carotae in step (i).
A method in claim 10, wherein the DNA amplification reaction in step (i) is PCR.
A method in claim 8, wherein detection of the DNA amplification product of step (ii) is performed by electrophoresis.
A method in claim 12, wherein the amplification of a target gene is confirmed by amplifying a fragment of 300 bp in size.
A method for diagnosing Xanthomonas hotorum pv. carotae infection, comprising: performing real-time PCR on a sample suspected of being infected with Xanthomonas hotorum pv. carotae using a primer set of any one of claims 1 to 3; and analyzing the results of gene amplification by the real -time PCR.
A method in claim 14, wherein real-time PCR is performed on a sample itself without a process of isolating genomic DNA from a sample suspected of being infected with Xanthomonas hotorum pv. carotae .