KR20170034648A - PNA probe for detecting Yersinia pestis having resistance against ciprofloxacin antibiotic and the uses thereof - Google Patents

Abstract

The present invention relates to a PNA probe for the detection of pest bacteria resistant to ciprofloxacin antibiotics and a use thereof, wherein the PNA-mediated PCR clamping method is applied through the present invention to produce an antibiotic with a gene similar to a single base mutation It is possible to provide a diagnostic system capable of detecting rapidly and highly sensitively the mutant strains of P. pastoris showing tolerance, and it is anticipated that it will be possible to promptly respond to bioterrorism using drug resistant strains to prevent or minimize the occurrence of large scale damage.

Description

PNA probes for the detection of pest bacteria resistant to ciprofloxacin antibiotics and their uses {PNA probe for detecting Yersinia pestis having resistance against ciprofloxacin antibiotic and the uses thereof}

The present invention relates to a PNA probe and a clamping primer set for detecting pest bacteria resistant to ciprofloxacin antibiotics.

Antibiotic resistant bacteria are increasing worldwide with threat of new infectious diseases and the rapid spread of antibiotic resistant bacteria is emerging as a big problem in many countries of the world. According to the data of Seoul National University Hospital, which is the world's first antibiotic resistance rate due to abuse of antibiotics prior to the division of labor in 2000, Korea has suffered from a great difficulty in treating various bacterial infectious diseases, 'The possibility of appearing is higher than any other country. In 2002, antibiotic resistance of resistant bacteria in Korea was investigated. Enterococcus faecium has a resistance rate of 97% in ampicillin, vancomycin has a resistance rate of 33% and staphylococcus aureus is known to show 75% resistance to methacillin. If the antibiotic resistance rate is high, treatment of infectious diseases in the clinical field becomes ineffective, and it is impossible to cope with the problem of bacterial food poisoning with antibiotic resistance in the food field.

Since the September 2001 terrorist attacks on the World Trade Center in the United States and bioterrorism using anthrax bacteria in mailings, fear and concern about terrorism have spread worldwide and the world is struggling to prepare countermeasures for the protection of its citizens from all over the world. Biological weapons are economically cheaper than conventional weapons, they can be easily concealed and sprayed, and it is difficult to detect in the early days due to the time difference in patient outbreak after spraying, and the lethal dose is extremely small. Once contaminated, · Has the characteristic of spreading. In addition, biological weapons are not easy to identify contaminated areas because they have a latent period of exposure for a certain period of time and do not immediately show signs of infection. When the infection is confirmed, it is likely to have already spread to a large area and many people. The possibility of bioterrorism and concerns are increasing due to the characteristics of these biological weapons, the international situation surrounding North Korea and Iraq, and the large-scale events such as APEC, G20 and G50. Therefore, Korea now has an anthrax, Botulinum toxinosis, pest, and viral hemorrhagic fever have been designated as legal infectious diseases.

From the viewpoint that individuals, groups, or large groups that are doing bad things can weaponize sufficiently pathogenic pathogens, there is a growing likelihood of research into antibiotic-resistant bacteria, viruses that fade away the vaccine, have. In addition, reports on drug resistance due to gene mutation of high-risk pathogens have been steadily developed. Therefore, we have developed a rapid detection technique for drug resistance gene mutation to promptly respond to bioterrorism using drug-resistant strains, By providing information, it is possible to prevent or minimize the occurrence of large-scale damage caused by impeachment. Currently, the use of antibiotics such as ciprofloxacin, doxycycline, and penicillin is based on the use of antibiotics, but basic diagnosis of disease is essential and resistance to the drug may be a problem. Although it has not yet been found that wild-type pests present in the natural state are resistant to ciprofloxacin and doxycycline, it has been reported that resistant bacteria can be induced in vitro, and antibiotic- And the like.

Recently, PNA-mediated PCR clamping (PNA-mediated PCR clamping), a method using peptide nucleic acids (PNA), has been developed. PNA has resistance to exonuclease (5 → 3) of Taq DNA polymerase and can bind strongly to the complementary chain of DNA. If one base does not coincide, Tm is greatly reduced, so a clamp primer ).

In recent years, a PNA clamping technique has been developed that selectively detects mutations by a method of inhibiting amplification of a wild-type wild-type using a PNA (peptide nucleic acid) probe that specifically binds to a wild-type. PNA was first reported in 1991 as a pseudo-DNA in which the nucleic acid base is linked by a peptide bond rather than a phosphate bond (Nielsen et al., Science, 254: 1497-1500, 1991). PNA forms a double strand by causing a hybridization reaction with a natural nucleic acid of a complementary base sequence. When the number of nucleic acid bases is the same, the PNA / DNA double strand is more stable than the DNA / DNA double strand, and the PNA / RNA double strand is more stable than the DNA / RNA double strand. The basic backbone of peptide nucleic acids is most often used in the case of N- (2-aminoethyl) glycine repeatedly linked by amide bonds as a basic skeleton of PNA. Unlike the basic structure of a negatively charged natural nucleic acid It is electrically neutral.

The four nucleobases present in the PNA occupy a space similar to that of the DNA and the distance between the nucleotides is almost the same as that of the native nucleic acid. PNA is chemically more stable than natural nucleic acid, and biologically stable because it is not degraded by nuclease or protease. Since PNA is also electrically neutral, the stability of PNA / DNA, PNA / RNA double strands is not affected by salt concentration. Because of this property, PNAs are better able to recognize complementary nucleic acid sequences than natural nucleic acids and are therefore applicable for diagnostic or other biological and medical purposes.

The PNA clamping technology utilizes the advantages of the PNA described above, so that when the PNA probe is perfectly coupled, the enzyme does not recognize the amplification reaction, and when the point mutation is present, the PNA probe does not bind perfectly, It is a method that uses the principle that occurs, and it is widely used because it can detect a mutation which exists in a very small amount compared to a wild type, quickly and accurately.

Korean Patent Laid-Open Publication No. 2008-0011257 discloses a kit for detecting microorganisms in a water-borne pathogen. However, PNA probes for detecting pest bacteria resistant to ciprofloxacin antibiotics and their uses have been disclosed in the present invention There is no bar.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for rapidly and rapidly obtaining a high-risk pathogen pest mutant strain which shows antibiotic resistance above a gene such as a single base mutation by applying a PNA- And to develop a diagnostic system with high sensitivity. For this purpose, a specific mutation of the gyrA gene from the P. pastoris mutant strain was first reported to show resistance to ciprofloxacin antibiotics, and PNA probes were specifically prepared for these mutant sites. As a result of analyzing the PNA-mediated clamping real-time PCR using the PNA probe and the optimal primer selected by the present invention, it was found that in the wild-type and mutant genes, the PNA- Although the synthesis of the gene did not occur, it was confirmed that the gene was amplified by the dissociation phenomenon of PNA in the mutated gene. As a result, the gyrA Specific PNA probes and primers can be used to accurately diagnose antibiotic-resistant Pseudomonas sp. Strains. Thus, the present invention has been completed.

In order to solve the above problems, the present invention provides a DNA gyrase A gene comprising the guanine (G) in the 241st nucleotide sequence of GyrA (DNA gyrase A) comprising the nucleotide sequence of SEQ ID NO: 11 or the guanine Or a plasmid that is resistant to ciprofloxacin antibiotics comprising a nucleotide sequence comprising a guanine (G) in the 248th nucleotide sequence or a cytosine (C) in the 249th nucleotide sequence or a complementary base sequence thereof ( Yersinia (PNA) probe for detection of pestis .

The invention also plague bacteria (Yersinia with ciprofloxacin (ciprofloxacin) antibiotic resistance, including the above-GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) probes and the GyrA gene-specific primer sets clamping PNA-mediated clamping real-time PCR for pestis detection.

In addition,

Real-time PCR was performed on the GyrA gene of Yersinia pestis using a GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) probe and a GyrA gene-specific clamping primer set ; And

(b) analyzing the result of gene amplification by the real-time PCR to determine the presence or absence of mutation of the GyrA gene of a P. pastoris microbes, which is resistant to ciprofloxacin antibiotics.

By applying the PNA-mediated PCR clamping method according to the present invention, it is possible to provide a diagnostic system capable of detecting rapidly and highly sensitively a mutant strain of Pest fungus showing resistance to antibiotics above a gene such as a single base mutation. It is anticipated that rapid response to bioterrorism using drug-resistant strains will prevent or minimize the occurrence of large-scale damage.

Figure 1 is a Yersinia pestis group (wild type DNA) and a PNA2, screening in the present invention to target the control group (mutant type DNA) samples primer (F / R) / probe (P) for mutation detection of Yersinia pestis gyrA gene The results are shown in Fig. (A) is selected after F2 / R1 / P3, (B) F2 / R2 / P3, (C) F2 / R3 / P3 and (D)
Fig. 2 shows a standard curve confirmed by PNA-mediated clamping real-time PCR of pest bacteria against the gyrA gene target of P. aeruginosa.

In order to accomplish the object of the present invention, the present invention provides a GyrA (DNA gyrase A) gene comprising the nucleotide sequence of SEQ ID NO: 11, which contains guanine (G) in the 241st nucleotide sequence or guanine (G) Or a ciprofloxacin antibiotic consisting of a nucleotide sequence comprising a guanine (G) in the 248th nucleotide sequence or a cytosine (C) in the 249th nucleotide sequence, or a complementary base sequence thereof. (Peptide Nucleic Acid) probe for detection of Yersinia pestis .

In the PNA probe according to an embodiment of the present invention, the GyrA gene-specific PNA probe comprises guanine (G) in the 241th base sequence of the GyrA (DNA gyrase A) gene consisting of the nucleotide sequence of SEQ ID NO: (G) in the second base sequence or guanine (G) in the 248th base sequence, or 15 to 25 base sequences including the cytosine (C) in the 249th base sequence, and preferably But is not limited to, the nucleotide sequence of SEQ ID NO: 10 or a complementary base sequence thereof.

The present invention also provides a method for detecting Yersinia pestis having antibiotic resistance to ciprofloxacin comprising the GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) probe and the GyrA gene-specific clamping primer set A kit for PNA-mediated clamping real-time PCR is provided.

In the kit for PNA-mediated clamping real-time PCR according to an embodiment of the present invention, the GyrA gene-specific clamping primer set may be, but not limited to, a primer set consisting of the nucleotide sequences of SEQ ID NOS: 2 and 3. The GyrA The set of gene-specific clamping primers is not limited to the above-described primer set, only the best combination is selected by checking the real time PCR graph, the fluorescence color size and the size of the PCR product.

In addition,

(a) Real-time PCR (real-time PCR) of the GyrA gene of Yersinia pestis using a GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) probe and a GyrA gene-specific clamping primer set ); And

(b) analyzing the result of gene amplification by the real-time PCR to determine the presence or absence of mutation of the GyrA gene of a P. pastoris microbes, which is resistant to ciprofloxacin antibiotics.

The method of the present invention can detect pest bacteria resistant to antibiotics only by amplification cycle difference with wild type using PNA (clamping) probe through PNA-mediated clamping real-time PCR, and can detect a large amount of wild-type amplification , Thereby improving the detection sensitivity of the mutant, thereby enabling rapid and accurate detection of a mutated mutant having a very small amount with high sensitivity.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1. Screening of target genes in Pest bacteria

Yersinia pestis ) genome project in the nucleotide sequence of the NCBI database. The nucleotide sequences of ciprofloxacin antibiotics target bacteria type Ⅱ enzyme (DNA gyrase (GyrA and GyrB), topoisomerase IV (ParC and ParE)), which is an antibiotic target gene in P. aeruginosa, were analyzed. The ciprofloxacin resistance was determined by the change of the amino acid sequence around the active site of the enzyme, confirming the nucleotide sequence of the quinolone resistance-determining region (QRDR) gene and confirming the nucleotide sequence variation of the gene having general and antibiotic resistance.

The gene sequence and amino acid sequence of the QRDR of P. pasty were confirmed and compared with the nucleotide sequence of the antibiotic resistance mutant of the reported papers, it was confirmed that there was a nucleotide sequence variation in the gyrA gene. The 248th nucleotide sequence was changed from G to A, the 241st nucleotide sequence was changed from G to T, the 248th nucleotide sequence was changed from G to T, and the 249th nucleotide sequence was changed from C to A / G in the mutant nucleotide sequence. Among them, the 249th nucleotide sequence was changed from C to G, and the same amino acid sequence as that from C to A was confirmed. For the genes identified above, the nucleotide sequence and amino acid sequence of the GyrA gene of P. aeruginosa were confirmed using the NCBI gene database, and the nucleotide sequence of each type of gene was confirmed by alignment analysis. Protein sequences were compared and analyzed.

Variations in the nucleotide sequence of gyrA in pest-resistant drug resistant mutants gyrA Nucleotide Amino acid One G242 → A G81 → D 2 G241 → T G81 → C 3 G248 → T S83 → I 4 C249 → A S83 → R 5 C249 → G S83 → R

The selection gene by a control oligonucleotide having a group with a modified nucleotide sequence of the nucleic acid synthesis were cloned into E. coli (E. coil) strain was used as a template for PCR. In addition, the nucleotide sequence of the experimental group gene was designed to include all nucleotide sequence variations of each gene, and the mutated and synthesized genes were cloned into a plasmid and constructed as shown in Table 2 below.

A plasmid having a modified base sequence of the gyrA gene of the present invention and a plasmid having a wild type sequence LMO Name Formation plasmid name Insertion gene
(Size bp) Mutation location E-YP-gyrAW pGEM-YP-gyrAW gyrAW
(nt1-660)
(660 bp) wild type E-YP-gyrAM pGEM-YP-gyrAM gyrAM
(nt1-660)
(660 bp) G241T
G242A
G248T
C249A

Example  2. Real-time PCR ( Real - time PCR)  Condition optimization

Three sets of specific primers and probes were selected for each gene of each bacterium, and real - time PCR conditions were optimized by checking the reaction concentration, reaction temperature, reaction time, and synthesis curve for each set. We designed a primer containing a site for diagnosis in the target gene and tried to synthesize and confirm the gene through conventional PCR. The nucleotide sequences of primers designed for each gene are shown in Table 3. In order to amplify the size of gene synthesis to about 150-600 bp according to the combination of forward / reverse primer, we tried to select the same conditions for synthesizing these various sizes of PCR products by conventional PCR. The combination of each primer and thus the size of the PCR synthetic gene is shown in Table 3 and Table 4 below.

Primer base sequence for diagnosis of resistance mutant strains of Pest fungus division Name Sequence (SEQ ID NO) size
(mer) GC
(%) GyrA
Forward F1 cgtgtagtcggggacgttat (1) 20 50.0 F2 gcgtactgtttgcgatgaat (2) 20 50.0 GyrA
Reverse R1 cgctaacaattcgtgagcaat (3) 20 50.0 R2 ccagcatatagcgcagtgag (4) 20 50.0 R3 atcgacggaaccgaagttac (5) 20 50.0

When GyrA gene of Pest bacterium was used to determine the temperature at which the primer binds to the template, gradient PCR at 50-60 ° C was performed to confirm that the PCR reaction proceeded at the same reaction time as the temperature of 50-60 ° C, PCR products were confirmed by electrophoresis using 2% agarose gel.

As a result of the above experiment, conventional PCR showed that the PCR reaction of each gene was smooth at annealing temperature of 50-60 ℃, and PCR reaction product of control group and experimental group was performed at 55 ℃ using control and experiment group genes. In comparison, a single set of bands was identified in the control group, and a set of primers in which a single or more bands were identified in the experimental group. The primer sets in which such nonspecific PCR synthesis bands were identified were excluded from the candidates for real-time PCR. Therefore, the selected conventional PCR conditions are shown in Table 5 below.

Real-time PCR was performed using CYBR Taq-polymerase in combination with the selected conventional PCR conditions and the primer combinations shown in Table 4 above. Based on the combination of the above primers, probe positions in each gene were selected as shown in Table 6, and FAM, HEX and Cy5 were selected as fluorescence in consideration of real-time PCR instrument.

The probe nucleotide sequence used in the diagnosis of pest fungus division Name Sequence (SEQ ID NO) size
(mer) GC
(%) GyrA P-01 FAM gcgatgcgttataccgaaat BHQ1 (6) 20 50.0 P-02 HEX agcgcggtctacgacactat BHQ1 (7) 20 55.0 P-03 Cy5 cgtgtagtcggggacgttat BHQ2 (8) 20 50.0

Real-time PCR was performed at annealing temperatures of 50 ° C and 55 ° C, respectively, which were confirmed by conventional PCR using combinations of primers and probes according to position of each gene. The concentration of the template DNA in the experimental group and the control group of each gene was measured, and the Cp value of the experimental group and the control group was corrected after real-time PCR.

Real-time PCR was carried out at annealing temperatures of 50 ° C and 55 ° C, respectively. At 50 ° C, the synthesis of the gene proceeded, but the probe bound to the gene template and showed no fluorescence as indicated by hydrolysis by endo-nuclease. 55 ℃ showed gene synthesis and fluorescence value. Therefore, the primer and probe annealing temperature of real-time PCR was determined to be 55 ° C.

Example  3. PNA  medium Clamping  real time PCR To use PNA Of design

In this experiment, PNA designed to distinguish the control DNA from the genetic mutation DNA was designed using the real-time PCR conditions determined above. In the control group, since the clamping was performed due to the complete complementary binding of PNA to the gene template, In the case of PNA binding to the gene template, if the nucleotide sequence does not match, the Tm is greatly decreased and the PCR synthesis proceeds from the gene template to identify the nucleotide sequence variation of the gene.

PNA was designed with the base sequence mutation in the target gene of P. pastoris and synthesized through PANAGENE. Typical PNAs are synthesized to a length within about 18 mer, and it is recommended that the base sequence containing the mutations be placed in the center of the PNA. In addition, PNAs of the same base sequence were also synthesized in both forward and reverse directions to select more optimized PNAs for the reaction. The synthesized PNA is shown in Table 7 below.

PNA nucleotide sequence used in the diagnosis of P. pastoris gene mutation division Name Sequence (SEQ ID NO) size
(mer) GC
(%) GyrA YP-GyrA-PNA-01 gcat gg tgaca gc gcg (9) sense 16 68.8 GyrA YP-GyrA-PNA-02 cgc gc tgtca cc atgc (10) anti-sense 16 68.8

Example  4. PNA  medium Clamping  real time PCR Optimal condition of

PNA was added to the reaction solution in an amount of 50 pmole. For the Taq polymerase of this experiment, AccuPower Plus DualStar qPCR PreMix from Bioneer was used. Since the appropriate primers were not yet determined according to the reaction temperature, a sufficient time (about 20 seconds) was selected for the synthesis of a gene having a maximum of 600 bp, and the synthesis time was changed after the determination of appropriate primers and probes.

The final concentrations of primers and probes were maintained at 10 pmole, and each DNA was diluted to 10 ng / ul by measuring the concentration after plasmid isolation. The reaction temperature of PNA-added real-time PCR was adjusted to 70 ° C, the binding temperature of the gene template, and the binding temperature of the primer and the gene template also started at 55 ° C. The mixing conditions of real-time PCR mixed with PNA are shown in Table 8 below.

Mixing conditions of real-time PCR using PNA template DNA (10 ng) 1 μl Primer (10 pmole) forward 1 μl reverse 1 μl Probe (10 pmole) 1 μl PNA (10 pmole) 5/10 or 0 ul 2 x DualStar qPCR PreMix 10 μl DW ul total volume 20 μl

Comparing the reaction with and without PNA added to the control gene, it was confirmed that the reaction value (Cp value) that performs the role of clamping by adding PNA was higher than the reaction value without addition, And shifted to the right. Also, when the PNA binding temperature was changed to 68 ° C, it was confirmed that the difference in reaction value was not greater than 70 ° C, and it was confirmed that the initial reaction value (Cp) was delayed by about 3 to 5 cycles. PCR was carried out by changing the temperature to 60 ° C according to the advice that the temperature to bind to the primer's gene template was 55 ° C. However, it was confirmed that the value of the reaction was lowered, and it was confirmed that the original binding temperature was the optimal condition. Therefore, all PNA-mediated clamping real-time PCR in the following cases was carried out at a binding temperature of 70 ° C and a binding temperature of the primer with the gene template of 55 ° C under the basic conditions as shown in Table 9 below.

Generalized PNA-Mediated Clamping Real-time PCR was performed using the combination of directive / competitive PNAs, primers, and probes for screening gene mutations using real-time PCR conditions as shown in Table 10 below.

Example  5. Pest fungus QRDR  For related gene segmentation PNA  medium Clamping  real time PCR Optimal for use in primer , Probe  And PNA Selection of

Thirteen combinations of primers and probes of the gyrA gene of P. aeruginosa resulted in the number of 26 cases including PNA. Among the 26 combinations, the results for PNA were analyzed. When the PNA1 (forward) and PNA2 (reverse) were compared, it was found that the reaction value of the mutant gene in PNA1 and PNA2 was lower than that of the normal gene. In the case of PNA2, PNA2 was selected because it was confirmed that PNA2 plays a more stable clamp than PNA1 because the difference in the reaction value of the common nucleotide sequence is higher. Also, the combination of the primer and the probe was confirmed by distinguishing the difference of the reaction values in the order of F2 / R1 / P3> F2 / R2 / P3> F2 / R3 / P3> F2 / R1 / P2 (FIG.

Therefore, the combination of primers, probes, and PNAs for identifying QRDR-related genes in P. aeruginosa and real-time PCR conditions for final PNA-mediated clamping are summarized in Table 11 below.

The primer / probe / PNA base sequence for each gene selected for the resistance mutant strain of P. pasty division Name Sequence (SEQ ID NO) size
(mer) GC
(%) YP-
GyrA F2 gcgtactgtttgcgatgaat (2) 20 50.0 R1 cgctaacaattcgtgagcaat (3) 20 50.0 P-03 Cy5 cgtgtagtcggggacgttat BHQ2 (8) 20 50.0 PNA-02 cgc gc tgtca cc atgc (anti-s) (10) 16 68.8

Mixture composition of PCR for diagnosis of resistant mutant strains of P. aeruginosa template DNA (10 ng) 1 μl Primer (10 pmole) forward 1 μl reverse 1 μl Probe (10 pmole) 1 μl PNA (10 pmole) 1 or 0 ul 2 x DualStar qPCR PreMix 10 μl DW ul total volume 20 μl

Example  6. In the present invention, PNA  medium Clamping  Real-time PCR ( PNA - mediated  clamping real - time PCR Selection of drug-resistant high-risk pathogen bacterium using optimal conditions

PNA-mediated Clamping To construct a drug resistance gene mutation detection system of Pest bacteria using real-time PCR method, PNA-mediated clamping real-time PCR with primers, probes and PNAs of antibiotic-associated genes selected in the present invention was used to detect drug resistance However, the absence of mutant pest bacteria in Korea and the generation of drug-resistant mutants of high-risk pathogens using transfection have not been achieved due to international conventions and restrictions.

Therefore, the wild-type DNA and the control (mutant type DNA) DNA of the yeast strain of the present invention can be used as a template using the plasmid having the modified base sequence shown in Table 2 and the plasmid having the wild type sequence as the template Standard curve and copy number of each gene were calculated to confirm detection limit and sensitivity of PNA-mediated clamping real-time PCR method. The concentration of the test DNA in the experimental group and the control DNA solution was adjusted to 10 ng and the detectable concentration was determined by stepwise dilution (stepwise dilution was 1:10).

After diluting the gene and performing PNA-mediated clamping real-time PCR for 45 cycles, the reaction value of the mutation-containing gene progressed and the mutant-free gene was amplified about 10 times The reaction value is displayed (FIG. 2). Therefore, when the concentration of the gene is high, the reaction value appears in a low cycle, so that the response value of the wild type gene that does not include the mutation also appears rapidly. Pest The minimum concentration of the mutation detected by the real-time PCR method of PNA-mediated clamping of the wild type and mutation of the gyrA gene is about 0.000001 ng. At this concentration, the reaction occurs in the mutant gene but the reaction does not occur in the control group. However, as the total number of reaction cycles decreases, the differential limit is reached and should be considered.

<110> The Armed Forces Medical Command <120> PNA probe for detecting Yersinia pestis having resistance against          ciprofloxacin antibiotic and the uses thereof <130> PN15276 <160> 11 <170> KoPatentin <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cgtgtagtcg gggacgttat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcgtactgtt tgcgatgaat 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgctaacaat tcgtgagcaa t 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccagcatata gcgcagtgag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atcgacggaa ccgaagttac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 gcgatgcgtt ataccgaaat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 agcgcggtct acgacactat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 8 cgtgtagtcg gggacgttat 20 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 9 gcatggtgac agcgcg 16 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <400> 10 cgcgctgtca ccatgc 16 <210> 11 <211> 2676 <212> DNA <213> Yersinia pestis <400> 11 atgagcgacc ttgccagaga aataacaccg gtcaacatcg aggaagagct gaaaagctcc 60 tatctggatt atgcgatgtc cgttattgtc ggacgtgcgt taccagatgt ccgtgatgga 120 ctgaaaccgg tgcaccgtcg cgtactgttt gcgatgaatg tactgggtaa tgactggaat 180 aaaccataca aaaaatcggc ccgtgtagtc ggggacgtta tcggtaaata ccacccgcat 240 ggtgacagcg cggtctacga cactatcgtg cgtatggccc agccgttctc actgcgctat 300 atggggtgg atgggcaggg taacttcggt tccgtcgatg gtgactccgc cgcggcgatg 360 cgttataccg aaatccgtat gtctaaaatt gctcacgaat tgttagcgga tttagaaaaa 420 gataccgttg acttcgtgcc taactatgat ggtacggaac aaattccggc tgttatgccg 480 accagaatcc ctaacctgct ggtaaacggt tcgtcgggta ttgcggtagg gatggcaacc 540 aatattccgc cacataatct ttctgaggtt attgatggct gtctggctta tatagaagat 600 gaaaacatca gcattgaagg gctgatggag tatatccctg gcccagactt cccaacagca 660 gccattatta atggccgccg tggtattgaa gaggcttatc gtactggtcg cggtaaggtc 720 tatatccgcg cacgtgctga agtcgaagcg gatgccaaaa cgggccgtga aaccattatt 780 gttcatgaga tcccgtatca ggtgaacaaa gcacggttga ttgagaaaat cgccgagctg 840 gtaaaagaaa aacgtgtcga aggcatcagc gcgttacgcg atgagtctga taaagacggc 900 atgcgcatcg tgattgaaat caaacgtgat gcggtcgggg aagtggtgct gaataacctc 960 tactctctga cacaattgca ggtgactttc ggtatcaata tggtggcctt gtctcagggg 1020 caaccgaagc tgcttaacct gaaagacatt ctggttgctt ttgttcgtca ccgtcgtgaa 1080 gtggtgactc gccgtaccat ttttgaactg cgtaaagcac gtgatcgcgc ccatatcctt 1140 gaagcgttgg cgattgcact ggctaacatt gatccgatta tcgaattgat tcgccgtgca 1200 tcgacccctg cggaagcaaa agccggtttg attgccagtc cttgggagtt aggtaacgtt 1260 gcttccatgc tggaacgtgc tggtgatgac gcggcccgcc ctgagtggct ggaagcggaa 1320 tttggtatcc gtgacggtaa atattacctt accgagcaac aggctcaggc tattttggat 1380 ctgcgtttgc agaaactgac aggtctggag catgagaaac tgctggatga gtataaagag 1440 ctgctcacct taattggcga gctgatcttt attctagaaa atccggatcg cctgatggag 1500 gttatccgcg aagaattagt ggcgatcaaa gagcaatata acgatcttcg tcgcactgag 1560 atcaccgcga atacctctga tatcaatatt gaagacctga ttaatcaaga agatgtggtt 1620 gtgacgttgt cccatcaggg ctacgtcaaa taccagccgt tgagtgatta cgaagctcag 1680 cgtcgtggtg gtaaaggtaa atctgctgca cgtatcaaag aagaagactt tattgatcgc 1740 ctgctcgttg ccaataccca cgataccatc ttgtgcttct ccagccgtgg ccgtctctat 1800 tggatgaagg tctatcaact gccggaagcc agccgtgggg ctcgtggccg tcccatcgtc 1860 aacttgttgc cgcttgagcc aaatgagcgt attaccgcca ttctgcctgt gcgcgaatat 1920 gaagaaggct gtcatgtctt tatggctacc gccagcggta ctgtgaagaa aacggcattg 1980 acggagttca gtcgcccacg tagcgccggt attatcgccg ttaatttgaa cgaaggtgat 2040 gagcttatcg gtgtggatct gacggatggc agcaatgaag ttatgctgtt ctcagcattg 2100 ggtaaagtgg ttcgtttccc agaagggcag gttcgctcta tgggccgtac cgcaaccggt 2160 gtccgtggga tcaacctcaa tggcgatgat cgggttattt ctctgatcat tcctcgtggt 2220 gatggtgaaa tcctgaccgt aactgaaaac ggttatggta agcgtactgc agtggcagaa 2280 tatccaacca agtcccgtgc gactcagggg gttatctcca ttaaagtcag tgagcgtaat 2340 ggtaaggttg tcggtgctgt tcaagttgca ccaactgacc aaatcatgat gatcactgat 2400 gctggcacac tggtacgtac tcgcgtatcc gaagtgagtg tcgtagggcg taatacccaa 2460 ggtgtgaccc tgatccgtac cgcagaagat gaacatgttg ttggtttgca acgtgtcgct 2520 gaacctgaag aggatgatga tatccttgat ggcgaatcat cagaaggtga agagggcagc 2580 gaagaaaacg cggcactaaa tgcaccatca gatgatgttg ccgatgaaga cactgatgca 2640 gaagacgaca ctgatgtaga agacgacaac gcataa 2676

Claims (5)

(G) in the 241st nucleotide sequence of the GyrA (DNA gyrase A) gene consisting of the nucleotide sequence of SEQ ID NO: 11, or guanine (G) in the 242nd nucleotide sequence, or guanine ) Or a yeast infection resistant to ciprofloxacin antibiotics consisting of a nucleotide sequence comprising cytosine (C) in its 249th nucleotide sequence or a complementary base sequence thereof PES (Peptide Nucleic Acid) probe for detection of. 2. The PNA (Peptide Nucleic Acid) probe according to claim 1, wherein the GyrA gene-specific PNA probe comprises the nucleotide sequence of SEQ ID NO: 10 or a complementary base sequence thereof. Claim 1 or claim 2 GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) Yersinia pestis (Yersinia with ciprofloxacin (ciprofloxacin) antibiotic resistance comprising a specific probe and primer sets clamping GyrA gene pestis ) detection Kit for Real-time PCR. 4. The kit according to claim 3, wherein the GyrA gene-specific clamping primer set is a primer set consisting of the nucleotide sequences of SEQ ID NOS: 2 and 3. The GyrA gene of Yersinia pestis was amplified by real-time PCR using a GyrA (DNA gyrase A) gene-specific PNA (Peptide Nucleic Acid) probe and a GyrA gene-specific clamping primer set of claim 1 or 2 -time PCR); And
(b) analyzing the result of gene amplification by the real-time PCR to determine the presence or absence of mutation of the GyrA gene of a P. pastoris microbial strain, which is resistant to ciprofloxacin antibiotic.

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