KR101953823B1 - Primer set for detection of Xanthomonas fragariae and diagnostic kit using the same - Google Patents

Abstract

The present invention relates to a PCR primer set for detecting Xanthomonas fragariae , and more particularly, to a PCR primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a bacterium of strawberry bacteria including the primer set, And a method for detecting a bacterium of strawberry bacterium using the same. Since the primer set of the present invention has a gene sequence that is specific to the bacterium of Streptococcus faecalis, it can be effectively used for the diagnosis and detection of Staphylococcus aureus.

Description

[0001] The present invention relates to a PCR primer set for detecting bacterium of strawberry bacteria, and a diagnostic kit using the PCR primer set.

The present invention relates to a PCR primer set for detecting a bacterium of Staphylococcus aureus and a diagnostic kit using the same, and more particularly to a PCR primer set for detecting a bacterium of Staphylococcus aureus, A primer set, a kit for detecting a bacterium of the genus Streptococcus comprising the same, and a method for detecting a bacterium of a strawberry bacterium using the same.

Dot-blot and polymerase chain reaction (PCR) are used for the genetic diagnosis of plant pathogenic bacteria. In particular, PCR is a widely used method because it is economical in terms of time, effort, and manpower than the dot-blot method. PCR (polymerase chain reaction) is a diagnostic method in which a specific DNA fragment (primer) composed of 10-20 bp is annealed to a genomic DNA of a bacterium and then a heat-resistant DNA polymerase The PCR amplification product is electrophoresed on an agarose gel or an acrylamide gel and ethidium bromide is added to the reaction mixture, The DNA polymorphism of bacterium species such as the presence or absence of DNA bands after DNA staining with bromide and silver stain is detected and diagnosed. Currently, the PCR method is mainly used to diagnose bacterial diseases in humans and animals. Recently, PCR diagnosis method has been developed for diagnosis of plant pathogens, and is used for quarantine of agricultural products in addition to diagnosis of diseases . In addition, the PCR diagnosis method is shorter in time than other methods, is low in cost for diagnosis, and is also economical because many samples can be assayed at the same time. Recently, a PCR method for amplifying nucleic acid as a genetic material among Xanthomonas detection methods is most preferred.

Strawberry fungus is an important disease in the production of strawberries, and economic losses due to it are not well documented, but it is known to reduce production by 8% per year in Florida, 75% in Wisconsin and 10% in Europe by 20%. In addition, strawberry bacterial fungus disease is caused by infestation of calyx, and if disease occurs in petiole and buds, systemic infection and seedling die.

Of the existing janto Pseudomonas spp, rice huinip diamond pathogens (Xanthomonasoryzae pv. Oryzae), bacterial spots pathogens (Xanthomonascampestris pv. Vesicatoria), cruciferous heukbu pathogens (Xanthomonascampestris pv. Campestris), citrus gweyangbyeong pathogens (Xanthomonasaxonopodis pv. Citri) and kongbul diamond pathogen (Xanthomonas axonopodis pv. glycines), such as research on the individual PCR primers used for the detection of various microorganisms has been made, there is still a research to bar with respect to the base pattern strawberry bacterial germs (Xanthomonas fragariae).

An object of the present invention is to provide a PCR primer set for detecting Xanthomonas fragariae , which is represented by SEQ ID NO: 1 and SEQ ID NO: 2.

Another object of the present invention is to provide a kit for diagnosing a bacterium of Staphylococcus aureus comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

It is another object of the present invention to provide a method for detecting DNA comprising: (a) separating DNA from a sample; (b) amplifying the DNA extracted in the step (a) by PCR using the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2; And (c) confirming the amplified PCR product of step (b) by electrophoresis.

In order to design a PCR primer set that can be used for the detection of Staphylococcus aureus ( Xanthomonas fragariae ), the inventors of the present invention have found that when the nucleotide sequence of 23S rRNA is changed to a species We found some sequences of 23S rRNA specific to Staphylococcus aureus, and invented a primer set using it.

The present invention provides a PCR primer set for detecting a bacterium of Staphylococcus aureus, which is represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention provides a kit for diagnosing a bacterium of Staphylococcus aureus comprising the primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

(A) separating DNA from the sample; (b) amplifying the DNA extracted in the step (a) by PCR using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2; And (c) confirming the amplified PCR product of step (b) by electrophoresis.

Hereinafter, the present invention will be described in detail.

The term 'primer' in the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary sequence and serving as a starting point for complementary strand duplication. In the present specification, the terms "forward primer" and "reverse primer" refer to primers that bind to the 3 'and 5' ends of a certain region of a gene amplified by PCR, respectively.

The present invention relates to a PCR primer set for detecting a bacterium of the genus Streptococcus, as shown in SEQ ID NO: 1 and SEQ ID NO: 2.

A variety of genes such as rRNA and house keeping genes have been used in the production of polymerase chain reaction primers specific to microbial genes. Among them, 16S rRNA and 23S rRNA are stable to evolution and belong to the same species or subclass Because genetic variation is rare, it is ideal as a polymerase chain reaction primer for the diagnosis of hospital germs. In the present invention, the PCR primer set targeted a specific region of species-conserved 23S rRNA in the gene sequence of the strawberry bacterium fungus genome. Therefore, the microorganism gene other than the bacterium Staphylococcus aureus Is characterized by being able to specifically detect only a bacterium of Staphylococcus aureus without specific binding.

The PCR primer set for detecting bacterium bacterium of the present invention is characterized by having a specific sequence existing only in a bacterium of Staphylococcus aureus, as compared with other microorganisms belonging to the genus Xanthomonas. In the present invention, the 23S rRNA gene of the known Mantus monascus bacterium and the 23S rRNA gene of the Staphylococcus aureus bacterium were compared and analyzed to find that a primer set (XF1F <forward direction, SEQ ID NO: 1> & XF3R (Reverse direction, SEQ ID NO: 2)) was selected (see Example 1). In order to verify whether the selected primer set has specificity only for the bacterium of Staphylococcus aureus, 34 strains of strains belonging to 27 genus of Xanthomonas including Staphylococcus aureus were isolated, PCR reaction was performed with the primer set, and electrophoresis was performed (FIG. 1). As a result, it was confirmed that only the bacterium of Strawberry bacterium was specifically detected.

In addition, the present invention provides a PCR primer set for detecting a bacterium of Staphylococcus aureus having a sequence complementary to SEQ ID NO: 1 and SEQ ID NO: 2. The primer sets of the present invention can be modified (for example, addition, deletion, substitution) within a range that does not affect the specific detection of the bacterium Staphylococcus aureus.

The present invention provides a kit for diagnosing a bacterium of Staphylococcus aureus comprising the primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

In the kit of the present invention, various reagents (e.g., PCR) necessary for detecting a bacterium of Strawberry bacterium using the primer set of the present invention and a reagent necessary for confirming the result, for example, a PCR composition, a restriction enzyme, an agarose, A buffer solution necessary for hybridization and electrophoresis, and the like may be further included. More specifically, the PCR composition may further comprise a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermococcus litorialis) in addition to the pair of complementary DNA synthesized by the reverse transcription reaction and the PCR primer pair provided in the present invention Or thermostable DNA polymerase from Pyrococcus furiosus (Pfu)), dNTP, PCR buffer solution and water (dH ₂ O). The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. At this time, MgCl ₂ concentration greatly affects amplification specificity and yield. Generally, if an excess of the Mg ^{2 +} increase the non-specific PCR amplification products, and reduce the yield of a PCR product if the Mg ^{2 +} insufficient. The PCR buffer solution includes, but is not limited to, Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin bovine serum albumin (BSA)) may be further included.

In addition, the kit may further include a DNA polymerase and a PCR reaction buffer solution having the above composition in order to facilitate the PCR reaction, and a kit for performing electrophoresis, May be further included in the kit of the present invention.

(A) separating DNA from a sample; (b) amplifying the DNA extracted in the step (a) by PCR using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2; And (c) confirming the amplified PCR product of step (b) by electrophoresis.

Polymerase chain reaction (PCR) is a technique in which a small amount of DNA present in a living body is separated and used as a template, and a pair of primers consisting of oligonucleotides complementary to both ends of the target DNA sequence is used. . By using such PCR, microorganisms infected by plants can be detected quickly and accurately. In the present invention, a detection method including the above-mentioned PCR was used in order to detect a bacterium of strawberry bacteria.

In step (a), the DNA may be separated from the bacterium of Strawberry bacterium according to a method commonly used in the art, and may be carried out using a commercially available DNA extraction kit have.

In step (b), the 23S rRNA gene of Staphylococcus aureus can be amplified using the DNA isolated in step (a) as a template and the primer pair of the present invention. This step is performed by repeating cycles including i) denaturation step, ii) target nucleic acid annealing step of the primer, and iii) elongation step. The term " cycle " in the present invention means a process of generating one copy of a target nucleic acid. The conditions for the modification, the temperature and the time at the target nucleic acid binding step and the extension step of the primer can be selected by those skilled in the art, and are not limited to a specific range, but are preferably (i) 55 to 65 seconds (Ii) annealing at 55 to 70 ° C for 55 to 60 seconds, and (iii) elongation at 170 to 190 seconds at 65 to 75 ° C for 30 cycles And (iv) an additional elongation step at 70 to 75 &lt; 0 &gt; C for 5 to 10 minutes. The time and number of cycles in each step can be determined according to the conditions commonly practiced in the art.

The step (c) can separate the PCR products by DNA size according to methods well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or fluorescence analysis apparatus. In this case, in order to use the fluorescence analysis apparatus, the PCR should be performed in the step (b) using a pair of primers having a fluorescent dye well known in the art. The PCR amplification result can preferably be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. For a general reverse transcription reaction, PCR, and the result analysis method, a method well known in the art can be used.

The present invention relates to a PCR primer set for detecting Xanthomonas fragariae , and more particularly, to a PCR primer set for detecting Xanthomonas fragariae , comprising a PCR primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, And a method for detecting a bacterium of strawberry bacterium using the same. Since the primer set of the present invention has a gene sequence specific to the bacterium of Staphylococcus aureus, it is effective for the diagnosis and detection of the bacterium of the strawberry bacteria.

Figure 1 shows the results of Xanthomonas campestris pv. 23S-F6, X-23S-F7 and X-23S-R7 primers to the campestris 23S rRNA gene (NC010688).
FIG. 2 is a schematic diagram showing the position where the XF1F / XF3R primer binds to the Xanthomonas fragariae 23S rRNA gene. FIG.
Figure 3 shows the results of detection of 27 species of Xanthomonas using XF1F / XF3R PCR primers (1 to 8; X. fragariae , 9; X. axonopodis pv. Axonopodis , 10; X. bromi , 11; X. campestris pv. campestris, 12; X. cassavae, 13 ; X. cucurbitae, 14; X. pisi, 15;. X. oryzae pv oryzae, 16; X. alfalfae subsp alfalfae, 17;.. X. compestris pv cucurbitae, 18; X .. axonopodis pv vasculorum, 19; X. hyacinthi, 20; X. populi, 21; X. translucens pv translucens, 22;. X. vesicatoria, 23; X. cynarae, 24; X. sacchari, 25; X. melonis , 26; X. codiaei, 27; X. theicola, 28; X. gardneiri, 29; X. perforans, 30; X. euvesicatoria, 31; X. citri, 32; X. arboricola pv juglandis, 33;. X. arboricola pv. fragariae , 34; NC, M; Molecular marker)
Fig. 4 shows the result of investigation of detection limit of XF1F / XF3R PCR primer.
FIG. 5 shows the result of comparing the nucleotide sequence of Staphylococcus aureus with the nucleotide sequence of Campestris and other strains.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: Strawberry bacterium &lt; RTI ID = 0.0 &gt; Xanthomonas fragariae ) Design of primer pair for detection

<1-1> Search for information of specific base sequence

Among the rRNA and housekeeping genes that are used in PCR primer development to select nucleic acids having specific bases of the bacterium of Staphylococcus aureus Staphylococcus aureus, Were targeted to the 23S rRNA region, which is ideal for conserved, sparse PCR primers for the diagnosis of pathogenic bacteria.

In the present invention, Xanthomonas campestris pv., Which is registered in GenBank and whose whole nucleotide sequence is analyzed, The 23S rRNA gene (NC010688) of campestris was compared with the 23S rRNA gene of Staphylococcus aureus using ClustalW multiple alignment program installed in BioEdit program (Version 7.0.9.0).

The combination of the PCR primers used in the analysis of the 23S rRNA gene sequence of the bacterium of Staphylococcus aureus was as follows: Xanthomonas campestris pv. The binding site to the 23S rRNA gene of campestris is shown in FIG.

X-23S-F5: (2) TGGTCAAGCCGCACGGATCATTAGTAT (28)

X-23S-R6: (2,828) ACGTGGATAGCCTGCGAAAAGTGTC (2,805)

X-23S-F7: (643) GAGACCGCCCCAGTCAAACTAC (664)

X-23S-R7: (2,339) ACCTTTTGTATAATGGGTCAACG (2,317)

As a result of the experiment, it was confirmed that some of the nucleotide sequences of 23S rRNA of the bacterium of Staphylococcus aureus were specifically present only in the above microorganisms. The nucleotide sequence of Staphylococcus aureus was compared with that of other strains such as Campestris and the results are shown in Fig.

<1-2> Construction of primer pairs for amplifying specific sequences

Using the 23S rRNA gene of which specificity was confirmed in Example <1-1>, forward and reverse primer pairs of polymerase chain reaction specific to Staphylococcus aureus were prepared as shown in Table 1 below. The binding positions of the following pair of XF1F / XF3R primers are shown in Fig.

SEQ ID NO: primer Base sequence direction One XF1F 5'- (1302 *) cttcgtgagctgaagcttaga (1322) -3 ' Forward 2 XF3R 5'- (2569) aaggtgatagccctgtatttg (2549) -3 ' Reverse

Example 2: Investigation of the specificity of the primer pairs produced

A PCR reaction was carried out to investigate the specificity of the primer set of Example 1 for the bacterium of Staphylococcus aureus. The specificity of the XF1F / XF3R PCR primer was investigated against 27 strains of the genus Staphylococcus spp. Including Staphylococcus aureus. Total DNAs of the strains were isolated using Genomic-tip 100 / G (Qiagen, Cat. No. 10243). To the PCR buffer, 0.2 mM dNTP, 1.5 mM MgCl ₂ , 10 pmole of XF1F primer, 10 pmole of XF3R primer and 2 units of Taq DNA polymerase were added and the amount was adjusted with distilled water. The buffer solution was amplified 30 times at 95 DEG C for 1 minute, 68 DEG C for 1 minute, and 72 DEG C for 3 minutes, and then treated at 72 DEG C for 7 minutes. The PCR product was electrophoresed on agarose gel. The result is shown in FIG.

As shown in FIG. 3, bands appeared only for the bacterium Staphylococcus aureus, and no bands appeared in the remaining microorganisms. As a result, it can be confirmed that the primer set of Example 1 specifically amplified only 23S rRNA of Staphylococcus aureus.

In addition, the detection limit of the XF1F / XF3R PCR primer pair is shown in FIG. 4, and the detection limit of the pathogenic bacteria in the XF1F / XF3R PCR primer pair is 10 ² .

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Primer set for detection of Xanthomonas fragariae and diagnostic          kit using the same <130> P16-275, 2016-0451-10-A <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> XF1F <400> 1 cttcgtgagc tgaagcttag a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> XF3R <400> 2 aaggtgatag ccctgtattt g 21 <210> 3 <211> 2714 <212> DNA <213> Xanthomonas fragariae <400> 3 acacacctga cctatcaacc acgtagtcta catggttcct ttagggggct tgtgccccgg 60 gaagtctcat cttgaggcgc gcttcccgct tagatgcttt cagcggttat cgcttccgaa 120 catagctacc cggcaatgcc actggcgtga caaccggaac accagaggtt cgtccactcc 180 ggtcctctcg tactaggagc agcccctctc aaacttccaa cgcccatggc agatagggac 240 cgaactgtct cacgacgttc tgaacccagc tcgcgtacca ctttaaatgg cgaacagcca 300 tacccttggg accgactaca gccccaggat gtgatgagcc gacatcgagg tgccaaacac 360 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc 420 gttgagcgat ggcccttcca tacagaacca ccggatcact aagacctact ttcgtacctg 480 cttgatccgt cgatcttgca gtcaagcacg cttatgcctt tgcacacagt gcgcgatgtc 540 cgaccgcgct gagcgtacct tcgtgctcct ccgttactct ttaggaggag accgccccag 600 tcaaactacc caccatacac ggtccctgat ccggataacg gatctaggtt agaacgtcaa 660 gcacgacagg gtggtatttc aaggatggct ccactgcagc tagcgccaca gtttcatagc 720 ctcccaccta tcctacacag acgaactcaa cgttcagtgt aaagctatag taaaggttca 780 cggggtcttt ccgtcttgcc acgggaacgc tgcatcttca cagcgatttc aatttcactg 840 agtctcgggt ggagacagcg ccgctgtcgt tacgccattc gtgcaggtcg gaacttaccc 900 gacaaggaat ttcgctacct taggaccgtt atagttacgg ccgccgttta ctggggcttc 960 gatcaagagc ttcgccttgc ggctgacccc atcaattaac cttccagcac cgggcaggcg 1020 tcacacccta tacgtccact ttcgtgtttg cagagtgctg tgtttttgat aaacagtcgc 1080 agcggcctgg tttctgcgac cctcttcagc tatagctcgc atgagccacc aaaaagggtg 1140 caccttctcc cgaagttacg gtgccatgtt gcctagttcc ttcacccgag ttctctcaag 1200 cgcctgagaa ttctcatcct acccacctgt gtcggtttac ggtacggtct tcgtgagctg 1260 aagcttagaa gcttttcctg gaagcgtggt atcagtgact ttgccataaa ggctcgtctc 1320 ggtgctcggt cttaaagaat cccggatttg ccaaagattc aaacctaccg cctttccccg 1380 ggacaaccaa cgcccggtac acctaacctt ctccgtccct ccatcgcact cacgcgaggt 1440 gcaggaatat taacctgctt cccatcgact acggctttcg ccctcgcctt agggaccgac 1500 taaccctgcg tcgattaacg ttgcgcaagg aaaccttggg ctttcggcgt gcgggctttt 1560 cacccgcatt atcgttactc atgtcagcat tcgcacttcc gatacctcca gcagacttct 1620 caatctacct tcacaggctt acggaacgct cctctaccgc gcataaaaca agttttatgc 1680 accccaagct tcggttcact gcttagcccc gttaaatctt ccgcgcagac cgactcgacc 1740 agtgagctat tacgctttct ttaaagggtg gctgcttcta agccaacctc ctggctgtct 1800 gtgcctttcc acatcgtttt ccacttagca atgaatttgg gaccttagct gtgggtctgg 1860 gttgtttccc ttttcacgac ggacgttagc acccgccgtg tgtctcccgg atagtacgta 1920 ctggtattcg gagtttgcaa tggtttggta agtcgcgatg accccctagc cataacagtg 1980 ctctaccccc agtagtattc gtccgaggcg ctacctaaat agctttcgag gagaaccagc 2040 tatctccggg ttcgattagc ttttcactcc taatcacagc tcatccccgt cttttgcaac 2100 agacgtgggt tcgggcctcc agtacctgtt acggcacctt caccctggcc atgactagat 2160 cacccggttt cgggtctact gcccgcgact atgcgccctt atcagactcg gtttcccttc 2220 gcctccccta tacggttaag cttgccacga acagtaagtc gctgacccat tatacaaaag 2280 gtacgcagtc actcttgcga gctcctactg cttgtacgca cacggtttca ggatctattt 2340 cactcccctc tccggggttc ttttcgcctt tccctcacgg tactggttca ctatcggtcg 2400 gtcaggagta tttagccttg gaggatggtc cccccatatt cagacagggt ttcacgtgcc 2460 ccgccctact cgtcttcact ggagtggccc tttcaaatac agggctatca ccttctatgg 2520 ccaatctttc cagattgttt ttctaaaacc attccagctt aagggctgtt ccccgttcgc 2580 tcgtcactac ttagggaatc tcggttgatt tcttttcctc cggttactta gatatttcag 2640 ttcaccgggt tcgcttccag cagttatgaa ttcactgcag gatactgccg aagcagtggg 2700 tttccccatt cgga 2714

Claims (3)

A primer represented by SEQ. ID. NO: 1 for detecting the 1324th adenine of the 23S rRNA nucleotide sequence of Staphylococcus aureus ( Xanthomonas fragariae ) shown in SEQ ID NO: 3; And
And a primer represented by SEQ ID NO: 2 for detecting the 2551st cytosine of the 23S rRNA base sequence of the bacterium of Staphylococcus aureus represented by SEQ ID NO:
A primer set for detection of strawberry bacterium ( Xanthomonas fragariae ). A kit for detecting Staphylococcus aureus ( Xanthomonas fragariae ) comprising the primer set of claim 1. (a) separating DNA from a sample;
(b) amplifying the DNA extracted in the step (a) by PCR using the primer set of claim 1; And
(c) identifying the amplified PCR product of step (b) by electrophoresis. &lt; Desc / Clms Page number 19 &gt;

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