KR20160136029A - Primer Sets for Detecting Pseudomonas syringae pv. persicae, a Causal Agent of Bacterial Dieback in Stone Fruits and Methods for Detecting Pseudomonas syringae pv. persicae Using the Primer Sets - Google Patents

Primer Sets for Detecting Pseudomonas syringae pv. persicae, a Causal Agent of Bacterial Dieback in Stone Fruits and Methods for Detecting Pseudomonas syringae pv. persicae Using the Primer Sets Download PDF

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KR20160136029A
KR20160136029A KR1020150069682A KR20150069682A KR20160136029A KR 20160136029 A KR20160136029 A KR 20160136029A KR 1020150069682 A KR1020150069682 A KR 1020150069682A KR 20150069682 A KR20150069682 A KR 20150069682A KR 20160136029 A KR20160136029 A KR 20160136029A
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persicae
pseudomonas syringae
pseudomonas
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노영희
차재순
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충북대학교 산학협력단
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Abstract

The present invention relates to a primer set used to detect Pseudomonas syringae pv. persicae causing bacterial dieback in stone fruits; to a probe used to detect Pseudomonas syringae pv. persicae; to a composition including the primer set; to a kit including the composition; and to a detection method of Pseudomonas syringae pv. persicae using the primer set. According to the present invention, in the case of using the primer set or the probe, Pseudomonas syringae pv. persicae is differentiated from different strains which belong to Pseudomonas sp., and can be effectively detected. The primer set used to detect Pseudomonas syringae pv. persicae is composed of a nucleotide sequence represented by a first sequence and a second sequence on a sequence listing; or a nucleotide sequence represented by a third sequence and a fourth sequence on a sequence listing.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for detecting pseudomonas silifen pulmonary vesicle caused by bacterial leaf blight in a nuclear plant, and a method for detecting the same in a pseudomonas silifen pulmonary vesicle using the primer set for detecting Pseudomonas syringae pv. persicae, a Causal Agent of Bacterial Dieback in Stone Fruits and Methods for Detecting Pseudomonas syringae pv. persicae Using the Primer Sets}

The present invention Pseudomonas ache dog blood V buffer in Brassica (Pseudomonas syringae pv. Persicae) and a set of primers for detecting Pseudomonas ache dog blood V in the buffer Brassica (Pseudomonas using the same. syringae pv. persicae detection method. Particularly leaf blight (bacterial dieback) a (Pseudomonas Pseudomonas ache more blood to cause V-flops Katsushika from stone fruit syringae pv. persicae detection primer set, a probe, a composition comprising the same, a kit and a Pseudomonas sp. syringae pv. persicae detection method.

Bacterial dieback, which occurs in nuclear plants such as peach, nectarine or Japanese plum, is now occurring in some parts of Europe. In non-emergent areas of this disease, including Korea, Respectively. Therefore, when importing nuclear products produced in the area where the disease occurs, it must be confirmed that the pathogens are not present in the nuclear product. In order to confirm the presence of pathogens, a fast and sensitive detection method of the pathogens is required. In particular, Pseudomonas causing bacterial dieback syringae pv. Because persicae is the most common bacterial organism of the genus Pseudomonas , it has been found that many bacterial species such as Pseudomonas syringae pv. Persicae is the only effective method to detect. However, effective detection methods have not yet been developed.

On the other hand, the PCR technique is a method for amplifying a specific nucleotide sequence of a pathogen, and therefore, it is not required to cultivate or propagate a pathogen, and thus it has been recognized as the method having the highest sensitivity and accuracy among pathogen detection methods. Pathogen-specific detection techniques should be able to effectively distinguish target pathogens from other pathogens or contaminants. In the case of PCR detection techniques using nucleotide sequences, it is most important to obtain a primer using a specific nucleotide sequence of the target pathogen Do. In particular, Pseudomonas belonging to the genus Pseudomonas , which is one of the genera most abundant in plant pathogenic bacteria syringae pv. In the case of persicae , securing the pathogen-specific base sequence is very important.

The present inventors have succeeded in obtaining a specific base sequence of only Pseudomonas syringae pv. Persicae using the NGS nucleotide sequence technology, and using a primer set for PCR detection and a Pseudomonas syringe PPV ( Pseudomonas syringae pv. Persicae ) were investigated. As a result, the present inventors designed a primer for PCR by searching for an open reading frame (ORF) specific to the target bacterium Pseudomonas syringae pv. Persicae , and by using the Pseudomonas syringae pv. Confirming that Pseudomonas syringae pv. Persicae can be detected specifically and sensitively, thereby completing the present invention.

It is an object of the present invention to provide Pseudomonas spp. syringae pv. persicae ) detection primer set.

Another object of the present invention is to provide a Pseudomonas sp. syringae pv. persicae detection probe.

A further object of the present invention is to provide a method of screening for Pseudomonas spp. syringae pv. persicae ).

It is another object of the present invention to provide a pharmaceutical composition comprising Pseudomonas sp. syringae pv. persicae detection kit.

It is still another object of the present invention to provide a method for detecting a Pseudomonas sp. syringae pv. persicae ) in a sample .

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a nucleotide sequence as set forth in SEQ ID NOS: 1 and 2; Or a set of primers for detecting Pseudomonas syringae pv. Persicae consisting of the nucleotide sequence described in the third and fourth sequences of the sequence listing.

The term "primer set" in the present invention means a short nucleic acid sequence which can form a base pair with a complementary template and functions as a starting point for template strand copying. The primer set provided in the present invention is a forward and reverse primer designed to amplify an ORF DNA fragment specific to Pseudomonas syringae pv. Persicae .

According to another aspect of the present invention, there is provided a probe for detecting Pseudomonas syringae pv. Persicae comprising the nucleotide sequence set forth in SEQ ID No. 5.

In the present invention, the probe may be a TaqMan probe, and the end of the probe may be labeled with a fluorescent substance. That is, a reporter dye can be bound to the 5 'end of a TaqMan probe, and a quencher can be bound to the 3' end. The TaqMan probe method attaches a reporter fluorescent substance to the 5'-end of the probe and a quencher to the 3'-end of the probe, and intrinsic fluorescence emitted from the 5'-end of the reporter fluorescence In a quantitative manner, the sequence of this probe is a unique nucleotide sequence of the target, attached only to a specific product, and designed to suppress fluorescence by a quencher at the 3 'end in the absence of PCR reaction. When the number of PCR reactions is large, the amount of fluorescence increases proportionally, which can be an indirect measure for quantification.

As a fluorescent marker capable of binding to the 5 'end of the TaqMan probe of the present invention, any kind of fluorescent substance developed to date can be used. Specifically, 6-carboxyfluorescein Carboxyfluorescein, 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, 5-carboxy fluorescein, HEX 2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein and Cy5 (cyanine-5).

The quencher, which can be bound to the 3 'end of the TaqMan probe of the present invention, is 6-carboxytetramethylrhodamine, TAMRA (5-Carboxytetramethylrhodamine), BHQ 1, 2 and 3 (blackhole quencher 1, 2, 3). In particular, MGB (minor groove binder) attached with NFQ (non-fluorescent quencher) may be preferred.

According to another aspect of the present invention, there is provided a Pseudomonas sp. syringae pv. persicae ).

In addition, the detection composition may further comprise a probe having a nucleotide sequence described in SEQ ID NO: 5.

According to another embodiment of the present invention there is provided a pharmaceutical composition comprising Pseudomonas sp. syringae pv. persicae detection kit.

The detection kit according to the present invention can be used not only in the above primer set or probe but also in a sample such as Pseudomonas spp. syringae pv. persicae strains, polymerases, dNTPs, stabilizers or other biological reagents and chemical reagents, and the like. The configuration of such a kit can be selected by a person skilled in the art, and is not limited to a specific configuration.

In accordance with another aspect of the invention, the nucleic acid sample obtained from the subject to the using the primer set according to claim 1 comprising the step of performing PCR, Pseudomonas ache dog blood V buffer Brassica (Pseudomonas syringae pv. persicae < / RTI >

The subject of the present invention is a Pseudomonas spp. syringae pv. As a plant which is suspected of infection persicae), specifically a stone fruit.

The PCR of the present invention comprises a nucleotide sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2; Or a nucleotide sequence as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, or a Pseudomonas sp. syringae pv. The target sequence can be amplified by carrying out an amplification reaction using a primer set for detection of Persicae .

The target sequence amplified by the PCR may be labeled using a detectable labeling substance. The labeling substance may be a substance emitting fluorescence, phosphorescence, or radiation, but is not limited thereto. Specifically, the labeling substance may be Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling Cy-5 or Cy-3 at the 5 'end of the primer and the target sequence may be labeled with a detectable fluorescent labeling substance. When a radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution, the amplification product is synthesized and the radiation is introduced into the amplification product and the amplification product can be labeled with radiation .

Detection of the amplified product can be performed by DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a primer set and a probe for detecting Pseudomonas syringae pv. persicae .

(b) Also provided is a composition or kit for detecting Pseudomonas syringae pv. persicae comprising the above primer set or probe.

(c) Further, PCR is performed using the primer set or probe to detect Pseudomonas syringae pv. persicae .

(d) When using the primer set or probe of the present invention, Pseudomonas syringae pv. persicae can be effectively detected by distinguishing it from other strains belonging to the genus Pseudomonas .

Figure 1 shows the results ofPseudomonas syringae pv.persicae Gel electrophoresis of the PCR product of 14 strains. Lanes 1-14:Pseudomonas syringae pv.persicae (Negative control) CFBP 1569, CFBP 1572, NCPPB 2076, NCPPB 3686, ICMP 2126, ICMP 3708, ICMP 3980, ICMP 5786, ICMP 7090, ICMP 7092, ICMP 7096, ICMP 8783, ICMP 12391, ICMP 12392, .
Figure 2 shows the results ofPseudomonasThe results of the gel electrophoresis of the PCR product of 34 strains belonging to the genus. Lane 1:Pseudomonas syringae pv.persicae CFBP 1569 (positive control), Lane 2:P. coronafaciens ICMP 3113, Lanes 3-5:P. savastanoi pvs . glycinea LMG 5144,phaseolicola KACC 10575,savastanoi NCPPB 639, Lanes 6-35:P. syringae pvs . actinidiae KACC 10582,antirrhini ICMP 4303,aptitude DSM 50252,atrofaciens LMG 5095,berberidis NCPPB 2724,ciccaronei NCPPB 2355,delphinii ICMP 529,dysoxyli ICMP 545,eriobotryae NCPPB 2331,garcae ICMP 4323,helianthi NCPPB 1229,japonica ICMP 6305,lachrymans ATCC 11965,lapsa ATCC 10859,maculicola ICMP 3935,mellea ICMP 5711,mori ICMP 4331,morsprunorum ICMP 5795,myricae ICMP 7118,panic NCPPB 1498,papulans NCPPB 2847,passiflorae NCPPB 1386,kitty ICMP 4433,ribicola NCPPB 963,sesami NCPPB 1016,syringae NCPPB 388,tabaci ICMP 2835,tagetis ICMP 4091,tomato NCPPB 2683,ulmi NCPPB 632, Lane 36: water (negative control).
Figure 3 shows the results ofPseudomonas syringae pv.persicae Gel electrophoresis of the PCR product of 14 strains. Lanes 1-14:Pseudomonas syringae pv.persicae (Negative control) CFBP 1569, CFBP 1572, NCPPB 2076, NCPPB 3686, ICMP 2126, ICMP 3708, ICMP 3980, ICMP 5786, ICMP 7090, ICMP 7092, ICMP 7096, ICMP 8783, ICMP 12391, ICMP 12392, .
Figure 4 shows the results ofPseudomonasThe results of the gel electrophoresis of the PCR product of 34 strains belonging to the genus. Lane 1:Pseudomonas syringae pv.persicae CFBP 1569 (positive control), Lane 2:P. coronafaciens ICMP 3113, Lanes 3-5:P. savastanoi pvs . glycinea LMG 5144,phaseolicola KACC 10575,savastanoi NCPPB 639, Lanes 6-35:P. syringae pvs . actinidiae KACC 10582,antirrhini ICMP 4303,aptitude DSM 50252,atrofaciens LMG 5095,berberidis NCPPB 2724,ciccaronei NCPPB 2355,delphinii ICMP 529,dysoxyli ICMP 545,eriobotryae NCPPB 2331,garcae ICMP 4323,helianthi NCPPB 1229,japonica ICMP 6305,lachrymans ATCC 11965,lapsa ATCC 10859,maculicola ICMP 3935,mellea ICMP 5711,mori ICMP 4331,morsprunorum ICMP 5795,myricae ICMP 7118,panic NCPPB 1498,papulans NCPPB 2847,passiflorae NCPPB 1386,kitty ICMP 4433,ribicola NCPPB 963,sesami NCPPB 1016,syringae NCPPB 388,tabaci ICMP 2835,tagetis ICMP 4091,tomato NCPPB 2683,ulmi NCPPB 632, Lane 36: water (negative control).
FIG. 5 is a table showing Ct values and regression curves for respective DNA concentrations according to the results of TaqMan PCR using primer set 2 and a TaqMan probe.Pseudomonas syringae pv.persicae The DNA of CFBP 1569 strain was diluted 10-fold from 10 ng.
FIG. 6 is a table showing Ct values and regression curves for respective DNA concentrations according to the results of TaqMan PCR using primer set 2 and TaqMan probe.Pseudomonas syringae pv.persicae The DNA of CFBP 1569 strain was diluted 10-fold to 10-fold and used as a standard value.Pseudomonas syringae pv.persicae Thirteen strains of DNA were used at 10 ng. The strains used were: CFBP 1569, CFBP 1572, NCPPB 2076, NCPPB 3686, ICMP 2126, ICMP 3708, ICMP 3980, ICMP 5786, ICMP 7090, ICMP 7092, ICMP 7096, ICMP 8783, ICMP 12391, ICMP 12392.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Pseudomonas syring ae pv. persicae Primers for the specific detection of

One of the most abundant plant pathogenic bacteria is Pseudomonas spp. Therefore, it is very important to obtain a specific nucleotide sequence in order to detect pathogens belonging to the genus Pseudomonas . Pseudomonas syringae belonging to the genus Pseudomonas pv. To obtain a specific nucleotide sequence of persicae, the necDNA sequencing (NGS) technique was used to obtain Pseudomonas syringae pv. Genome sequencing analysis of persicae CFBP 1569 strain was performed to obtain a draft genome sequence and the ORF nucleotide sequence was predicted from a draft genome sequence through various programs. The ORF nucleotide sequence obtained therefrom was compared with the nucleotide sequence of a known gene in NCBI GenBank to determine the target bacterium Pseudomonas syringae pv. The ORF specific to persicae was found. Based on this, a PCR primer capable of specifically detecting Pseudomonas syringae pv. persicae was designed (Table 1).

designation Nucleotide sequence Sequence List Primer set 1 Forward 5'-CCCTTCAGCGCTTCTATCAG-3 ' One Reverse 5'-TCACACTCAGCCGATTTCTG-3 ' 2 Primer set 2 Forward 5'-GTTTGAGCCCGTTTGATGAG-3 ' 3 Reverse 5'-TCACACTCAGCCGATTTCTG-3 ' 4 TaqMan probe 5'-ACGGGGCATGGAAAATTCGA-3 ' 5

Example  2: primer  Using Set 1 PCR  Perform

Pseudomonas syringae pvt. 1 of the primer set 1 prepared in Example 1 above was used. PCR was carried out using primer set 1 in order to confirm the specificity for persicae .

Collected Pseudomonas syringae pv. Persicae DNA was extracted from 14 strains and PCR was carried out and analyzed by electrophoresis. Pseudomonas syringae pv. Primer set 1, Pseudomonas syringae pv. persicae ) was used as a control group to identify 34 specific strains belonging to the genus Pseudomonas .

Using 10 ng of genomic DNA as a template, 10 pmol of the primer set 1 was added, and PCR was performed using the Takara Ex Taq PCR Kit (Takara Co., Japan). The PCR conditions were initial denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 35 times The reaction was carried out at 72 ° C for 7 minutes in a final extension process. PCR was performed using T Gradient Thermal Cycler (Biometra Co., Germany), and the PCR amplification product was confirmed by electrophoresis at 90 V for 90 minutes on 2.0% agarose gel using 3 μl.

As a result, Pseudomonas syringae pv. The target size of 504 bp DNA was amplified from 14 strains of persicae (Fig. 1). When PCR was performed with 34 different Pseudomonas species DNAs by the same primer and PCR method, the target size DNA was not amplified (Fig. 2).

Therefore, the primer set 1 of the present invention is a Pseudomonas sp. syringae pv. persicae ) can be detected specifically.

Example  3: primer  Using Set 2 PCR  Perform

Pseudomonas syringae pvt. 1 of the primer set 2 prepared in Example 1 above was used. PCR was carried out using primer set 2 in order to confirm the specificity for persicae .

Collected Pseudomonas syringae pv. Persicae DNA was extracted from 14 strains and PCR was carried out and analyzed by electrophoresis. Pseudomonas syringae pv. Primer set 2, Pseudomonas syringae pv. persicae ) was used as a control group to identify 34 specific strains belonging to the genus Pseudomonas .

The PCR conditions were the same as in Example 2, and 3 μl of the amplified product was confirmed by electrophoresis at 90 V for 90 minutes on 2.0% agarose gel.

As a result, Pseudomonas syringae pv. The target size of 150 bp DNA was amplified from 14 strains of persicae (Fig. 3). When PCR was performed with 34 different Pseudomonas genus DNAs by the same primer and PCR method, the target size DNA was not amplified (Figure 4).

Thus, the primer set 2 of the present invention is Pseudomonas spp. syringae pv. persicae ) can be detected specifically.

Example  4: primer  Set 2 and The probe  Used Takman ( TaqMan ) PCR  Perform

Using the primer set 2 and the TaqMan probe of Example 1, Pseudomonas syringae pv. The DNA of persicae CFBP 1569 strain was diluted 10-fold to 10-fold, and TaqMan PCR was performed using the template as a template.

The TaqMan PCR conditions were initial denaturation at 95 캜 for 30 seconds, denaturation at 95 캜 for 5 seconds, annealing / extension at 60 캜 for 20 seconds, Respectively.

As a result, the Ct value regression curves r 2 Value of 0.997, indicating that the Ct value of TaqMan PCR was increased in a concentration-dependent manner (FIG. 5). Using the same primer and TaqMan probe Pseudomonas syringae pv. As a result of performing TaqMan PCR with 10 ng of DNA from 14 strains of persicae , the Ct value of all the strains used was below the limit of positive detection of 33 (Fig. 6). On the other hand, when 34 DNAs of Pseudomonas sp . Strain were used, no Ct value was shown.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Primer Sets for Detecting Pseudomonas syringae pv. persicae, a          Causal Agent of Bacterial Dieback in Stone Fruits and Methods for          Detecting Pseudomonas syringae pv. persicae Using the Primer Sets <130> PN150190 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pspp_primer1F <400> 1 cccttcagcg cttctatcag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pspp_primer1R <400> 2 tcacactcag ccgatttctg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pspp_primer2F <400> 3 gtttgagccc gtttgatgag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pspp_primer2R <400> 4 tcacactcag ccgatttctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pspp_TaqMan_probe <400> 5 acggggcatg gaaaattcga 20

Claims (9)

A nucleotide sequence set forth in Sequence Listing first and second sequences; Or a set of primers for detecting Pseudomonas syringae pv. Persicae consisting of the nucleotide sequence described in SEQ ID NO: 3 and SEQ ID NO: 4.
A Pseudomonas sp. Strain consisting of the nucleotide sequence described in SEQ ID NO: 5, syringae pv. persicae detection probe.
[3] The probe according to claim 2, wherein the probe is a TaqMan probe, wherein a fluorescent marker is bound to the 5 'end and a quencher is bound to the 3' end.
4. The method of claim 3, wherein the fluorescent reporter is selected from the group consisting of 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, Tetrachloro-6-carboxyfluorescein, 5-carboxy fluorescein, HEX (2 ', 4', 5 ', 7'- tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5 ). &Lt; / RTI &gt;
4. The method of claim 3, wherein the quencher is selected from the group consisting of 6-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine, BHQ 1,2, and 3 (black hole quencher 1, 2, 3 ). &Lt; / RTI &gt;
A composition for detecting Pseudomonas syringae pv. Persicae comprising the primer set of claim 1.
7. The composition of claim 6, wherein the composition further comprises the probe of claim 2.

A kit for detecting Pseudomonas syringae pv. Persicae comprising the composition of claim 6.
A method for detecting the presence or absence of Pseudomonas syringae pv. Persicae , comprising the step of performing PCR using a primer set according to claim 1 for a nucleic acid sample obtained from an individual.
KR1020150069682A 2015-05-19 2015-05-19 Primer Sets for Detecting Pseudomonas syringae pv. persicae, a Causal Agent of Bacterial Dieback in Stone Fruits and Methods for Detecting Pseudomonas syringae pv. persicae Using the Primer Sets KR101759334B1 (en)

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CN107828905A (en) * 2017-12-18 2018-03-23 福建省农业科学院植物保护研究所 Tobacco smoke pollution LAMP detection primer and detection method

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828905A (en) * 2017-12-18 2018-03-23 福建省农业科学院植物保护研究所 Tobacco smoke pollution LAMP detection primer and detection method

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