JP2003052391A - Method for performing detection of gonococcus and new quinolone medicine sensitivity evaluation of gonococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for performing the detection of gonococcus and new quinolone medicine sensitivity of the gonococcus, by which the detection and new quinolone medicine sensitivity detection of the gonococcus can rapidly be preformed. SOLUTION: This method for performing the detection of the gonococcus and new quinolone medicine sensitivity evaluation of the gonococcus includes the following steps: (1) a step for specifically amplifying a target DNA containing a region encoding a specific polymorphic site in gonococcus gyr A gene; and (2) a step for analyzing the fusion temperature of the target DNA with a nucleic acid probe which can specifically hybridize with a portion containing the polymorphic site.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting Neisseria gonorrhoeae and assessing sensitivity to new quinolone agents. The present invention is
It can be used for diagnosis and treatment of gonorrhea infection. Further, it is possible to prevent improper use of the new quinolone drug, thereby suppressing the increase of quinolone drug resistant bacteria. Therefore, it can be used for preventive medicine.

[0002]

2. Description of the Prior Art New quinolone agents have excellent antibacterial activity against Neisseria gonorrhoeae (Hooper, DC, and JS Wolfso
n. 1991. Fluoroquinolone antimicrobial agents. N.
Engl.J. Med. 324: 384-394.), Some of the new quinolones have been recommended as first-line drugs for Neisseria gonorrhoeae infections (Ce
nters for Disease Control and Prevention. 1993.Sex
ually transmitted diseases treatment guidelines. M
Orbid. Mortal. Weekly Rep. 42: 57-59.). Due to the heavy use of new quinolone drugs and the improper use of some of them, resistant gonococci against new quinolone drugs have begun to appear (Birley, H., P. McDonald, and P.
Carey. 1994. High-level ciprofloxacin resistance i
n Neisseria gonorrhoeae. Genitourin. Med. 70: 292-2
93., Centers for Disease Control and Prevention. 1
994. Decreased susceptibility of Neisseria gonorrh
oeae to fluoroquinolones-Ohio and Hawaii, 1992-199
4.Morbid. Mortal. Weekly Rep. 42: 57-59., Gransden,
WR, CA Warrenm I. Phillips, M. Hodges, and
D. Barlow. 1990. Decreased susceptibility of Neisse
ria gonorrhoeae to ciprofloxacin. Lancet 335: 51.,
Jephcott, AE, and A. Turner. 1990. Ciprofloxaci
n resistance in gonococci. Lancet 335: 165., Kam,
KM, PW Wong, MM Cheung, NKY Ho, and
KK Lo. 1996. Quinolone-resistant Neisseria gonor
rhoeae in Hong Kong. Sex. Transm. Dis. 23: 103-10
6., Tanaka, M., J. Kumazawa, T. Matsumoto, and I.
Kobayashi. 1994. High prevalence of Neisseria gon
orrhoeae strains with reduced susceptibility to fl
uoroquinolones in Japan. Genitourin. Med. 70: 90-9
3., Tapsall, JW, EA Limnios, C. Thacker, B.
Donovan, SD Lynch, LJ Kirby, KA Wise, and
CJ Carnody. 1995. High-level quinolone resistan
ce in Neisseria gonorrhoeae: a report of two case
S. Sex. Transm. Dis. 22: 310-311.). New quinolone drug ineffective cases have been reported even in actual treatment, and the usefulness of the new quinolone drug is gradually being impaired, which has become a major problem in the world including Japan (Tanaka, M., T. Matsu).
moto, M. Sakumoto, K. Takahashi, T. Saika, I. Kobay
ashi, J. Kumazawa, and The Pazufloxacin STD Group.
1998. Reduced clinical efficacy of pazufloxacin a
gainst gonorrhea due to high prevalence of quinolo
ne-resistant isolates with the GyrA mutation. Anti
microb. Agents. Chemother. 42: 579-582.).

On the other hand, the resistance mechanism of Neisseria gonorrhoeae, which is resistant to new quinolone drugs, has been investigated (Belland, R.
J., SG Morrison, C. Ison, and WM Huang. 199
4. Neisseria gonorrhoeae acquires mutations in ana
logous of gyrA and parC in fluoroquinolone-resista
nt isolates. Mol. Microbiol. 14: 371-380., Corkill,
JE, A. Percival, and M. Lind. 1991. Reduced upt
ake of ciprofloxacin in a resistant strain of Neis
seria gonorrhoeae and transformation of resistance
to other strains. J. Antimicrob. Chemother. 28:60
1-604., Deguchi, T., M. Yasuda, M. Asano, K. Tada,
H. Iwata, H. Komeda, T. Ezaki, I. Saito, and Y. Ka
wada. 1995. DNA gyrase mutations in quinolone-resi
stant clinical isolates of Neisseria gonorrhoeae.
Antimicrob. Agents Chemother. 39: 561-563., Deguch
i, T., M. Yasuda, M. Nakano, S. Ozeki, T. Ezaki,
I. Saito, and Y. Kawada. 1996. Quinolone-resistant
Neisseria gonorrhoeae: correlation of alterations
in the GyrA subunit of DNA gyrase and the ParCsub
unit of topoisomerase IV with antimicrobial suscep
tibility profiles.Antimicrob. Agents Chemother. 4
0: 1020-1023., Tanaka, M., H. Fukuda, K. Hirai, M.
Hosaka, T. Matsumoto, and J. Kumazawa. 1994. Redu
ced uptake and accumulation of norfloxacin In resi
stant strains of Neisseria gonorrhoeae isolated in
Japan. Genitourin. Med. 70: 253-255.). The new quinolones act directly on the DNA gyrase and topoisomerase IV of N. gonorrhoeae and exert antibacterial activity. These target enzymes are DNA gy
rase consists of GyrA protein and GyrB protein encoded by gyrA gene and gyrB gene, respectively, and topoisomerase IV
Is Pa encoded by parC gene and parE gene, respectively.
It consists of rC protein and ParE protein. In N. gonorrhoeae, DNA gyra
se is the main target (Tress, DL, AL Sandul,
V. Peto-Mesola, MR Aplasca, HB Leng, WL Wh
ittington, and JS Knapp. 1999. Alterations with
in the resistance-determining regions of GyrA and
ParC of Neisseria gonorrhoeae isolated in the Far
East ant the United States. Int. J. Antomicrob. A
gents. 12: 325-332.), changes in the GyrA protein due to mutations in the gyrA gene, especially changes in serine at amino acid 91 and aspartic acid at amino acid 95 play a central role in resistance to new quinolones (Belland, RJ. , SG Morrison,
C. Ison, and WM Huang. 1994. Neisseria gonorrho
eae acquires mutations in similar of gyrA and pa
rC in fluoroquinolone-resistant isolates. Mol. Mic
robiol. 14: 371-380., Deguchi, T., M. Yasuda, M. Na
kano, S. Ozeki, T. Ezaki, I. Saito, and Y. Kawada.
1996. Quinolone-resistant Neisseria gonorrhoeae:
correlation of alterations in the GyrA subunit of
DNA gyrase and the ParC subunit of topoisomerase I
V with antimicrobial susceptibility profiles. Anti
microb. Agents Chemother. 40: 1020-1023.). Therefore, the analysis of these amino acid changes is important and essential for understanding the resistance mechanism of N. gonorrhoeae resistant to new quinolone and for the epidemiological investigation of the spread and transmission of N. gonorrhoeae. Further, by analyzing changes in these amino acids, it is possible to evaluate the susceptibility of N. gonorrhoeae to the new quinolone drug in the sample, and prevent inappropriate treatment such as unnecessary administration of the N. quinolone drug to highly resistant N. gonorrhoeae.

On the other hand, for patients with N. gonorrhoeae infection, they are either submitted to a susceptibility test for N. gonorrhoeae separated from N. gonorrhoeae culture at the first visit, or submitted to a gene diagnostic method for detecting N. gonorrhoeae. It was Since the culture test has more than 3 days for the detection and susceptibility test for Neisseria gonorrhoeae, the treatment with antibacterial agents must be started without looking at the results at the first visit. In addition, regarding genetic diagnosis currently available, it takes about half a day to obtain results, but it is not possible to obtain information on susceptibility only by detecting N. gonorrhoeae. Even when using these conventional gene diagnostic methods, it is necessary to start treatment with an antibacterial agent without seeing the results of the detection of Neisseria gonorrhoeae at the time of initial diagnosis, as in the case of culture tests. This makes it extremely difficult to select the next suitable antibacterial agent.

[0005]

As described above, in the treatment of Neisseria gonorrhoeae infection, rapid detection of Neisseria gonorrhoeae is important,
In addition, it is extremely important to evaluate the susceptibility of N. gonorrhoeae to new quinolone agents in order to select an appropriate treatment method. However, under the present circumstances, there is no method capable of treating a large number of specimens in a short time for clinical application. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method capable of rapidly detecting N. gonorrhoeae and rapidly detecting the susceptibility of N. gonorrhoeae to a new quinolone agent.

[0006]

[Means for Solving the Problems] The present inventors conducted studies using clinical samples in order to solve the above problems. That is,
Amplification of target DNA containing polymorphic site (region encoding amino acid 91 and amino acid 95) in N. gonorrhoeae gyrA gene and melting temperature analysis using a nucleic acid probe designed for analysis of the polymorphic region Detection of N. gonorrhoeae in each sample and
We tried to detect polymorphism in gyrA gene. As a result, it was demonstrated that polymorphisms could be detected extremely rapidly, and the obtained polymorphism information was highly correlated with the susceptibility of N. gonorrhoeae to new quinolone drugs. At the same time, it was confirmed that Neisseria gonorrhoeae can be rapidly detected in clinical samples. The present invention has been completed based on the above-mentioned examination results and has the following configurations. [1] A method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone agent, which comprises the following steps: (a) specifically amplifying a target DNA containing a region encoding amino acid 91 in the Neisseria gonorrhoeae gyrA gene; (b) A step of analyzing melting temperatures of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion including the region of the target DNA. [2] A method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone agent, which comprises the following steps: (A) specifically amplifying a target DNA containing a region encoding the 95th amino acid in the Neisseria gonorrhoeae gyrA gene; (B) A step of analyzing melting temperatures of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion including the region of the target DNA.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for detecting Neisseria gonorrhoeae and assessing the sensitivity of a new quinolone agent to (1) Neisseria gonorrhoeae gyrA
Step of specifically amplifying a target DNA containing a specific polymorphic site in the gene, (2) the target DNA
And a melting temperature of a nucleic acid probe capable of specifically hybridizing to a portion of the target DNA containing the polymorphic site.

As a result of applying the present invention, the presence of N. gonorrhoeae is confirmed when the target DNA that is specifically amplified is detected. Further, when N. gonorrhoeae is present in the sample, the susceptibility of the N. gonorrhoeae to the new quinolone agent is evaluated by melting temperature analysis. Examples of specimens to which the present invention is applied include urethral secretions, urine, pharyngeal scrapings, and cervical secretions of suspected infected persons. These samples are prepared in advance with appropriate DNA.
It is preferable to perform an extraction process. For example, DNA can be extracted by combining cell disruption with a protease or a surfactant, chloroform extraction, ethanol precipitation and the like. Also, depending on the sample, heat treatment
It is also possible to extract DNA. Operation time can be shortened by DNA extraction by such heat treatment. For example, urethral secretions of a suspected infected person can be diluted with an appropriate buffer and then heat-treated to extract DNA.

The new quinolone drug is a drug known to have excellent antibacterial activity against Neisseria gonorrhoeae. A fluorine atom is added to the structure of a conventional quinolone agent (old quinolone agent), and the antibacterial activity is enhanced by converting other side chains, which is also called fluoroquinolone. Quinolones after norfloxacin are classified as new quinolones, and ciprofloxacin (ciprof
loxacin: CPFX), including levofloxacin.
Penicillin-based, tetracycline-based, and cephem-based antibacterial agents have been used for chemotherapy for gonorrhea infection, and resistant bacteria with reduced sensitivity to these antibacterial agents have appeared. The new quinolone agent has an excellent antibacterial activity against Neisseria gonorrhoeae including such various resistant bacteria, and is therefore an antibacterial agent that has been widely used for Gonorrhea infections.

New quinolone drugs are bacterial DNA gyrase and D
It exerts its antibacterial activity by inhibiting NAtopoisomerase IV. DNAgyrase is a tetramer composed of two molecules of GyrA protein and two molecules of GyrB protein, and it is known that in Gram-negative bacteria including Neisseria gonorrhoeae, a change in GyrA protein plays an important role in resistance.

In the present invention, a step (step (1)) of specifically amplifying a target DNA containing a specific polymorphic site in the Neisseria gonorrhoeae gyrA gene is performed. The specific polymorphic site is present in the region encoding amino acid 91 and amino acid 95 of the GyrA protein of N. gonorrhoeae (in the present specification, the former is the "first region" and the latter is the "second region"). Also called). It is also possible to specifically amplify the target DNA containing the first region and the second region. In this case, the melting temperature analysis is performed for each of the first region and the second region in the steps described below, and the obtained results are A comprehensive evaluation can be performed to evaluate the susceptibility of N. gonorrhoeae to new quinolone agents. The 91st amino acid of GyrA protein is the amino acid located at the 91st position from the N-terminus of GyrA protein,
Similarly, the 95th amino acid is the amino acid located at the 95th position from the N-terminus. Amino acid 91 of GyrA protein is serine (Ser) in the wild type, and it is known that there is a mutation (polymorphism) that changes to phenylalanine or tyrosine. On the other hand, the 95th amino acid of GyrA protein is aspartic acid (Asp) in the wild type, and it is known that there is a mutation (polymorphism) that changes to asparagine or glycine.

The method for specifically amplifying the target DNA is not particularly limited, and a known method can be appropriately selected and used. For example, well-known PCR method and strand displacement amplification method (Japanese Patent Publication No.
-114718 gazette) etc. can be utilized.
A method that allows amplification with high specificity and efficiency (eg PC
R method) is preferable, among which real time P method is used.
CR method (Molecular Cloning, Third Edition, 8.94-8.9
5, Cold Spring Harbor Laboratory Press, New York)
Is particularly preferably used. The real-time PCR method is a method that enables simultaneous and real-time amplification and quantification of a specific nucleic acid sequence, and a dedicated device is commercially available (for example, LightCycler (Roche), Ge
neAmp5700 system, ABI Prism7700 system (above, Perk
in-Elmer Biosystems))). By adopting the real-time PCR method, the amplification of the target DNA can be detected in real time.

The primer used when performing the PCR method (including the real-time PCR method) is not particularly limited as long as it can specifically amplify the target DNA. The length of the primer is not limited as long as it functions as a PCR primer, and is, for example, 15 to 30 bp, preferably 20 to 30 b.
p, more preferably 20 to 25 bp. There may be 1 to several mismatches, preferably 1 to 5 mismatches, and more preferably 1 to 3 mismatches between the primer and the region of the N. gonorrhoeae gyrA gene to which it hybridizes. As a specific example of the primer set, the partial DNA shown in FIG. 1 is used.
A primer DNA (SEQ ID NO:
1) and a primer DNA complementary to the partial DNA (code 2)
An example is the set of (SEQ ID NO: 2). still,
FIG. 1 shows a part of the DNA sequence of the N. gonorrhoeae gyrA gene. In the figure, the arrowhead indicates the mutation site in the region encoding amino acid 91, and the arrow indicates the mutation site in the region encoding amino acid 95. The length of the target DNA is, for example, 50 to 500 bp, preferably 150 to 300
bp, more preferably 200 to 250 bp.

After step (1), a step of analyzing the melting temperature of the target DNA and a nucleic acid probe capable of specifically hybridizing to the portion containing the polymorphic site of the target DNA is performed (step (2)). ). The melting temperature (Tm) is a temperature at which a double-stranded portion of a DNA or RNA molecule is generally denatured into a single strand and the double strand and the single strand are present at a ratio of 1: 1. In the invention, a single-stranded double-stranded nucleic acid formed by the target DNA amplified in step (1) and a nucleic acid probe capable of specifically hybridizing to a portion containing a specific polymorphic site of the target DNA It is the temperature at which the chain is denatured and the double strand and the single strand are present at a ratio of 1: 1.

The nucleic acid probe is a polymorphic site of the target DNA (a region encoding amino acid 91 or amino acid 95)
There is no particular limitation as long as it specifically hybridizes to the portion containing. When such a nucleic acid probe is hybridized to the target DNA, the target DNA
If there is a mutation at the polymorphic site, a mismatch will occur between the nucleic acid probe and
The binding force with and becomes weak, and the melting temperature (Tm) becomes low. Therefore, the mutation at the polymorphic site of the target DNA can be detected by analyzing the melting temperature of the nucleic acid probe and the target DNA. In other words, the gene sequence at the polymorphic site can be determined. As a nucleic acid probe,
It is preferable to use one having a sequence complementary to a part of the target DNA having a specific polymorphism (wild type or specific mutant type). However, 1 to several, preferably 1 to 5,
More preferably, it may contain about 1 to 3 mismatches. Both DNA probes and RNA probes can be used, but considering the high specificity, DNA
It is preferable to use a probe. The length of the nucleic acid probe is not particularly limited and is, for example, 5 to 100 bp, preferably 10 to 80 bp, more preferably 15 to 50 bp.

The melting temperature analysis can be performed using, for example, a fluorescent label bound to a nucleic acid probe as an index.
Preferably, it can be carried out by a method using a pair of nucleic acid probes each labeled with a different fluorescent substance.
The properties of each nucleic acid probe when using such a pair of nucleic acid probes are described below. (i) Can be hybridized specifically with a gap of 1 to 5 bases in the target DNA, (ii) One is a specific polymorphic site (a region encoding the 91st amino acid or a region encoding the 95th amino acid) ) And designed to have a higher melting temperature than the other, (iii) a first nucleic acid probe having a first fluorescent substance bound to the end located on the gap side when hybridized to the target DNA, The second nucleic acid probe has a second fluorescent substance that is excited by fluorescence of the first fluorescent substance and emits fluorescence when bound to an end located on the side of the gap when hybridized to the target DNA. The melting temperature analysis using a pair of nucleic acid probes as described above can be performed as follows. First, (2-1) adding the pair of nucleic acid probes to the sample, (2-
2) The first nucleic acid probe and the second nucleic acid probe are targets
The temperature is lowered to a temperature at which it hybridizes to DNA, and (2-3) the temperature is continuously or stepwise raised, and the sample is irradiated with light having the excitation wavelength of the first fluorescent substance, and emitted from the second fluorescent substance. Measure the amount of fluorescence.

Fluorescent substance bound to the first nucleic acid probe or the second nucleic acid probe (first fluorescent substance or second fluorescent substance)
The type of is not particularly limited, and for example, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Texas red, eosin isothiocyanate, cyanine dye and the like can be used. Labeling of the first nucleic acid probe and the second nucleic acid probe with a fluorescent substance can be performed by a method using terminal transferase (Molecular Cloning, Third Ed.
ition, 9.76-9.81, Cold Spring Harbor Laboratory Pr
ess, New York).

The first nucleic acid probe and the second nucleic acid probe are
It is designed to hybridize to the target DNA with a gap of 1 to 5 bases interposed. When both probes are hybridized to the target DNA, the fluorescent substance bound to each probe is present in close proximity with the gap. Thus, when the first fluorescent substance is irradiated with the excitation light, the first fluorescent substance emits fluorescence, and the energy of the fluorescence excites the second fluorescent substance. As a result, fluorescence peculiar to the second fluorescent substance is observed. Then, when the temperature is raised continuously or stepwise to approach the melting temperature of the nucleic acid probe covering the polymorphic site, the nucleic acid probe starts to be liberated from the target DNA. When such liberation occurs, the first fluorescent substance and the second fluorescent substance no longer exist close to each other, and the second fluorescent substance is excited by the fluorescent energy of the first fluorescent substance and no longer emits fluorescence. That is, the amount of fluorescence from the second fluorescent substance decreases near the melting temperature of the nucleic acid probe.

On the other hand, when there is a mismatch between the portion containing the polymorphic site of the amplified target DNA and the nucleic acid probe which hybridizes by covering the polymorphic site,
Release of the nucleic acid probe occurs at a lower temperature. Therefore, the temperature at which the amount of fluorescence from the second fluorescent material decreases shifts to the low temperature side. Therefore, it is possible to determine the type of polymorphism by measuring the temperature at which the amount of fluorescence from the second fluorescent substance decreases. Preferably, the temperature at which the rate of decrease in the amount of fluorescence from the second fluorescent substance is maximized is obtained, and the presence or absence of mutation at the polymorphic site can be determined based on this temperature.

As described above, the first nucleic acid probe and the second nucleic acid probe are designed so as to hybridize to the target DNA with a gap of 1 to 5 bases interposed therebetween, but preferably 1 to 3 bases, and more preferably Designs both nucleic acid probes so that they hybridize to the target DNA with a gap of 1 base. If the gap sandwiched between both nucleic acid probes is made as small as possible, the first fluorescent substance and the second fluorescent substance can be brought closer to each other in the hybridized state of both nucleic acid probes, so that the light from the first fluorescent substance can be efficiently emitted. Further, the second fluorescent substance can be irradiated, and the amount of fluorescence emitted from the second fluorescent substance can be increased.
Therefore, the detection sensitivity can be improved.

As a specific example of a pair of nucleic acid probes (consisting of a first nucleic acid probe and a second nucleic acid probe), the partial DNA shown in FIG. 1 for detecting a polymorphism in the region encoding the 91st amino acid ( DNA complementary to code 3)
(3'-end is labeled with a fluorescent substance, SEQ ID NO:
3) and a pair of DNA probes consisting of DNA complementary to partial DNA (symbol 4) (SEQ ID NO: 4 labeled at the 5'end with a fluorescent substance). In addition, as a detection of the polymorphism in the region encoding the 95th amino acid, a DNA complementary to the partial DNA (symbol 5) shown in FIG.
(3'-end is labeled with a fluorescent substance, SEQ ID NO:
5) and a pair of DNA probes consisting of DNA complementary to partial DNA (code 6) (SEQ ID NO: 6 labeled at the 5 ′ end with a fluorescent substance).

A step of amplifying the target DNA with the first nucleic acid probe and the second nucleic acid probe (step
It can be added before (1)). By doing so, the amplification of the target DNA can be monitored using the fluorescence from the first nucleic acid probe or the second nucleic acid probe. Therefore, it becomes possible to detect the mutation in the target DNA and the gonococcus at the same time. For example, a method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone drug can be constructed by including the following steps. (c) a step of adding a PCR primer capable of specifically amplifying a target DNA containing a region encoding the 91st amino acid in the N. gonorrhoeae gyrA gene to a sample, (d) with a gap of 1 to 5 bases in the target DNA A pair of nucleic acid probes that are specifically hybridizable, one designed to cover the region and have a higher melting temperature than the other, and are located on the gap side when hybridized to the target DNA. A first nucleic acid probe having a first fluorescent substance bound thereto, and a terminal located on the side of the gap when hybridized to the target DNA, which emits fluorescence when excited by fluorescence of the first fluorescent substance. 2nd with 2 fluorescent substances bound
A step of adding a pair of nucleic acid probes consisting of a nucleic acid probe to a sample; (e) a step of performing a real-time PCR method; (f) a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA (G) increasing the temperature continuously or stepwise, irradiating the specimen with light having the excitation wavelength of the first fluorescent substance, and measuring the amount of fluorescence emitted from the second fluorescent substance; (h) A step of obtaining a temperature at which the reduction rate of the fluorescence amount is maximum and determining the sequence of the region encoding the 91st amino acid in the gyrA gene from the temperature.

Similarly, it is possible to construct a method for detecting N. gonorrhoeae and evaluating the sensitivity to a new quinolone agent by including the following steps. (C) a step of adding a PCR primer capable of specifically amplifying target DNA containing a region encoding amino acid No. 95 in N. gonorrhoeae gyrA gene to a sample, (D) with a gap of 1 to 5 bases in the target DNA A pair of nucleic acid probes that are specifically hybridizable, one designed to cover the region and have a higher melting temperature than the other, and are located on the gap side when hybridized to the target DNA. A first nucleic acid probe having a first fluorescent substance bound thereto, and a terminal located on the side of the gap when hybridized to the target DNA, which emits fluorescence when excited by fluorescence of the first fluorescent substance. 2nd with 2 fluorescent substances bound
A step of adding a pair of nucleic acid probes comprising a nucleic acid probe to a sample; (E) a step of executing a real-time PCR method; (F) a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA (G) irradiating the specimen with light having an excitation wavelength of the first fluorescent substance and measuring the amount of fluorescence emitted from the second fluorescent substance, while (G) continuously or stepwise raising the temperature. (H) A step of obtaining a temperature at which the reduction rate of the fluorescence amount is maximum and determining the sequence of the region encoding the 95th amino acid in the gyrA gene from the temperature.

By using a commercially available LightCycler (Roche Diagnostics Co., Ltd.), etc., the target DN
The step of amplifying A (step (1)) and the step of performing melting temperature analysis (step (2)) can be continuously performed. In addition, analysis in a short time is possible.

By combining the above-mentioned primer for specifically amplifying the target DNA and a pair of nucleic acid probes, a kit for detecting Neisseria gonorrhoeae and evaluating the sensitivity to a new quinolone agent can be constructed. That is, another aspect of the present invention relates to a kit having the following constitution ((I) or (II)). (I) A pair of primers capable of specifically amplifying a target DNA containing a region encoding the 91st amino acid in N. gonorrhoeae gyrA gene, and capable of specifically hybridizing with a gap of 1 to 5 bases in the target DNA A pair of nucleic acid probes, one of which is designed to cover the region and has a higher melting temperature than the other, wherein the first end is located on the gap side when hybridized to the target DNA. A first nucleic acid probe bound with a fluorescent substance and a second fluorescent substance that emits fluorescence when excited by the fluorescence of the first fluorescent substance are bound to the end located on the gap side when hybridized to the target DNA. A pair of nucleic acid probes consisting of a second nucleic acid probe. (II) A pair of primers capable of specifically amplifying a target DNA containing a region encoding the 95th amino acid in N. gonorrhoeae gyrA gene, and capable of specifically hybridizing with a gap of 1 to 5 bases in the target DNA. A pair of nucleic acid probes, one of which is designed to cover the region and has a higher melting temperature than the other, and has a first fluorescence at the end located on the gap side when hybridized to the target DNA. A first nucleic acid probe having a substance bound thereto, and a second fluorescent substance which is excited by fluorescence of the first fluorescent substance and emits fluorescence at the end located on the gap side when hybridized with the target DNA. A pair of nucleic acid probes consisting of two nucleic acid probes. These kits can appropriately contain enzymes, reagents and the like necessary for amplification of target DNA.

[0026]

The present invention will be described in more detail with reference to examples. In this example, LightCycler (Roche Diagnostics KK) was used to detect N. gonorrhoeae and evaluate N. gonorrhoeae susceptibility to new quinolone agents. (Materials and Methods) (1) Strains and Samples Used 91 samples were prepared as test subjects. Among them, 55 specimens are control Neisseria gonorrhoeae. For the susceptibility of control Neisseria gonorrhoeae to new quinolone drugs, see National Com
mittee for Clinical Laboratory Standards (NCCLS) method (National Committee for Clinical Laboratory S
tandards. 1993. Method for dilution antimicrobial
susceptibility test for bacteria that grow aerobic
ally, 3rded.Approved standard M7-A3. National Com
mittee for Clinical Laboratory Standards, Villanov
a, Pa.) and further changes in serine at amino acid 91 of the GyrA protein and aspartic acid at amino acid 95 are determined by conventional nucleotide sequencing methods (Deguchi, T., M. Yasuda, M.
Asano, K. Tada, H. Iwata, H. Komeda, T. Ezaki, I.
Saito, and Y. Kawada. 1995. DNA gyrase mutations
in quinolone-resistant clinical isolates of Neisse
ria gonorrhoeae. Antimicrob. Agents Chemother. 39:
561-563., Deguchi, T., M. Yasuda, M. Nakano, S. Oz
eki, T. Ezaki, I. Saito, and Y. Kawada. 1996. Quin
olone-resistant Neisseria gonorrhoeae: correlation
of alterations in the GyrA subunit of DNA gyrase
and the ParC subunit of topoisomerase IV with anti
microbial susceptibility profiles. Antimicrob. Age
nts Chemother. 40: 1020-1023.). Of these 25 strains were new quinolone drug sensitive strains with no mutation in the gyrA gene, and 27 strains had a change in either the serine of amino acid 91 of the GyrA protein or the change of aspartic acid of amino acid 95 of GyrA protein. It is a low-susceptibility strain, and the remaining 3 strains are new quinolone drug resistant strains with changes in both. Urinary secretions in 36 samples were 19
It was obtained from a gonococcal urethritis patient who visited the Toyota Memorial Hospital Urology Department from 1999 to 2000. N. gonorrhoeae was cultured from these specimens (Deguchi, T., M. Yasuda, M. Asan.
o, K. Tada, H. Iwata, H. Komeda, T. Ezaki, I. Sait
o, and Y. Kawada. 1995. DNA gyrase mutations in qui
nolone-resistant clinical isolates of Neisseria go
norrhoeae. Antimicrob. Agents Chemother. 39: 561-56
3., Deguchi, T., M. Yasuda, M. Nakano, S. Ozeki,
T. Ezaki, I. Saito, and Y. Kawada. 1996. Quinolone-
resistant Neisseria gonorrhoeae: correlation of al
terations in the GyrA subunit of DNA gyrase and th
e ParC subunit of topoisomerase IV with antimicrob
ial susceptibility profiles. Antimicrob. Agents Ch
emother. 40: 1020-1023.). Further, some of these urethral secretions were suspended in Tris-EDTA buffer and frozen and stored at -80 ° C until used for analysis by LightCycler.

(2) Susceptibility test The minimum inhibitory concentration (MIC) of ciprofloxacin (CPFX) for 36 strains cultured and isolated from urethral secretions was determined by the NCCLS method (National Committee for Clinical Laboratory Sta.
ndards. 1993. Method for dilution antimicrobial su
sceptibility test for bacteria that grow aerobical
ly, 3rd ed.Approved standard M7-A3.National Commi
ttee for Clinical Laboratory Standards, Villanova,
Pa.).

(3) DAN Extraction DNA extraction from 55 strains of control samples was carried out by the usual sodium dodecyl sulfate treatment, phenol / chloroform extraction and ethanol precipitation (Deguchi,
T., M. Yasuda, M. Asano, K. Tada, H. Iwata, H. Ko
meda, T. Ezaki, I. Saito, and Y. Kawada. 1995. DNA
gyrase mutations in quinolone-resistant clinical i
solates of Neisseria gonorrhoeae. Antimicrob. Agen
ts Chemother. 39: 561-563., Deguchi, T., M. Yasuda,
M. Nakano, S. Ozeki, T. Ezaki, I. Saito, and Y. Ka
wada. 1996. Quinolone-resistant Neisseria gonorrho
eae: correlation of alterations in the GyrA subuni
t of DNA gyrase and theParC subunit of topoisomera
se IV with antimicrobial susceptibility profiles.
Antimicrob. Agents Chemother. 40: 1020-1023.). Lig
Extraction of DNA from urethral secretions for htCycler analysis was performed using 36 samples of urethral secretions that had been frozen and stored in Tris-EDTA buffer and stored directly in hot water for 5 minutes together with their Eppendorf tubes, which are their storage containers. Done by putting, D
No treatment was added to further purify NA.

(4) Amplification of Neisseria gonorrhoeae gyrA gene by LightCycler and analysis of mutation of gyrA gene Specific amplification by PCR of Neisseria gonorrhoeae gyrA gene by LightCycler was carried out with a pair of PCR primers.
Confirmation of gene amplification and detection of mutations in the gyrA gene can be performed using a pair of probes (fluorescein-labeled probe and LC R
ed640 labeled probe) was used for hybridization. Figure 2 shows the PCR primers (gyr
A-up (SEQ ID NO: 1) and gyrA-down (SEQ ID NO :)
2)), 91st amino acid detection probe (gyrA-Ser-Flu
(SEQ ID NO: 3) and gyrA-Ser-LC (SEQ ID NO:
4)), 95th amino acid detection probe (gyrA-Asp-Flu
(SEQ ID NO: 5) and gyrA-Asp-LC (SEQ ID NO: 6))
Indicates. PCR primers (gyrA-up and gyrA-down) are
It was designed to amplify a 225 base pair DNA fragment (target DNA) from the 174th position to the 398th position of the N. gonorrhoeae gyrA gene (see FIGS. 1 and 2). The specificity of the primers used here for the N. gonorrhoeae gyrA gene was confirmed by comparison with other gene information in the database.

Confirmation of amplification of N. gonorrhoeae gyrA gene and GyrA protein 91
In order to detect the mutation in the gyrA gene involved in the change in the amino acid serine, LC Red 640 was added to the 5'side covering this region.
Probe (gyrA-Ser-LC) with a gap of 1 base and a probe labeled with fluorescein on the 3'side (gyrA-Ser-LC)
-Ser-Flu) was designed (see FIGS. 1 and 2). Theoretical melting temperature of probe gyrA-Ser-LC and probe gyrA-Ser-Flu (Tm, temperature at which 50% of DNA is single-stranded and the remaining 50% of DNA is double-stranded) ) Is 66.8 ° C
It was 91.1 ° C.

Theoretically, when both probes hybridize to the amplified gyrA gene (target DNA),
Fluorescence of adjacent fluorescein excites LC Red 640,
Fluorescence is emitted from LC Red 640. As the amount of DNA amplified by PCR increases, the amount of hybridized probe increases and the emitted fluorescence increases. Therefore, the amplification of the gyrA gene (target DNA) by PCR can be monitored by the fluorescence intensity. Furthermore, the temperature gradually rises from the hybridized state of the probe, and when the temperature approaches the temperature of Tm, the probe gyrA-Ser-L
C begins to be released from the target DNA, the fluorescence of fluorescein does not excite LC Red 640, and the fluorescence from LC Red 640 disappears. At this time, if there is a mutation in the gyrA gene involved in the change of serine at the 91st amino acid, the probe gyrA-Ser-L
Since the binding between C and the gyrA gene is weak due to mismatched bases, probe release occurs at lower temperatures and the fluorescence disappears. Therefore, by observing the temperature at which the reduction rate of fluorescence from LC Red 640 is the largest, it is possible to know whether or not there is a mutation in the gyrA gene involved in the change in serine of the 91st amino acid.

In order to detect the change in the aspartic acid at the 95th amino acid, a probe (gyrA-Asp-Flu) labeled with fluorescein was placed on the 3'side covering this region and a gap of 1 base was placed on the 5'side. A probe (gyrA-Asp-LC) labeled with LC Red 640 was designed (see FIGS. 1 and 2). The theoretical Tm of the probe gyrA-Asp-LC and that of the probe gyrA-Asp-Flu were 68.2 ° C and 90.8 ° C, respectively. When both probes hybridize to the amplified gyrA gene (target DNA), the fluorescence of adjacent fluorescein excites LC Red 640, and LC Red 640 emits fluorescence. The temperature gradually rises from this state, and the probe gyrA-Asp-Flu is detected by the gyrA gene (target
Fluorescein fluorescence becomes LC Red 64 when released from
0 is no longer excited and the fluorescence from LC Red 640 disappears. At this time, if there is a mutation in the gyrA gene involved in the change of the 95th amino acid aspartic acid, the probe gyrA-Asp-F
Release of lu occurs at a lower temperature and the fluorescence disappears. Therefore, by observing the temperature at which the decrease rate of fluorescence from LC Red 640 is the largest, it is possible to know whether or not there is a mutation in the gyrA gene involved in the change in the aspartic acid of the 95th amino acid.

When carrying out the PCR reaction, LightCycler-DNA master hybridiza which is a commercially available reaction solution containing Taq polymerase, reaction buffer, deoxynucleotide and 10 mM MgCl _2.
To 2 μl of tionprobes (Roche Diagnostics KK), add MgCl ₂ to a final magnesium concentration of 4 mM, and add primers and probes respectively.
0.5 mM and 0.2 mM were added. In addition, a DNA lysate extracted from the control N. gonorrhoeae or put in boiling water for 5 minutes
2 μl of the urethral secretion sample from which A was extracted was added, and the final volume was adjusted to 20 μl with distilled water. This reaction solution was placed in a glass capillary tube and set in the Light Cycler. The conditions of PCR by LightCycler are denaturation 95 ° C. for 5 minutes for the first cycle, 5 seconds for the second and subsequent cycles, and annealing and extension are 55 ° C., 5 seconds, 72 ° C. and 10 seconds from the first cycle, respectively. did. The denaturation, annealing and extension were repeated for 46 cycles. After this process was completed, the mixture was once heated to 95 ° C., immediately cooled to 40 ° C. and kept for 5 seconds, and then reheated to 80 ° C. at a rate of 0.1 ° C./sec.

Fluorescence from LC Red 640 was monitored through the amplification process by PCR and the melting process by reheating from 40 ° C., and the amplification curve and melting curve of DNA were drawn by the software attached to LightCycler. The amplification curve was drawn with the PCR cycle number as the X-axis and the fluorescence intensity of LC Red 640 as the Y-axis. The melting curve was drawn with the temperature as the X axis and the fluorescence decrease rate as the Y axis.

(5) Sensitivity of detection of N. gonorrhoeae gyrA gene by LightCycler The sensitivity of detection of N. gonorrhoeae gyrA gene by LightCycler was examined using DNA extracted from one of the control strains of N. gonorrhoeae. DNA was quantified, adjusted to 10 mg / l, and 10-fold dilution was repeated to prepare a dilution series of DNA up to 1 × 10 ⁻⁵ mg / l. Using this DNA dilution series, L under the same conditions as above.
PCR was performed using the ight Cycler. DNA content in the reaction solution at that time, the 10 mg / l is 5x10 ⁶ copies of the genome of Neisseria gonorrhoeae, 1x10 ^- was an ^{amount 5} mg / l corresponds to 5 copies.

(Results) (1) 36 gonococcus strains sensitive to a new quinolone drug were cultured and separated from 36 specimens of urethral secretions, and MIC of CPFX for them was measured. The MIC was 0.002 μg / ml to 8 μg / ml. MIC from 0.002 μg / ml to 0.015 μg / ml
There are 15 sensitive strains, from 0.06 μg / ml to 0.5 μg / m
There were 10 low-susceptibility strains (1) and 11 resistant strains (1 μg / ml to 8 μg / ml).

(2) Detection sensitivity of N. gonorrhoeae gyrA gene detection by LightCycler In the examination using a dilution series of DNA extracted from one strain of N. gonorrhoeae as a control, N. gonorrhoeae was detected by LightCycler even when a DNA solution having the minimum concentration was used. It was possible to amplify the gyrA gene. Amplification of the gyrA gene was similarly confirmed using both the gyrA-Ser-LC and gyrA-Ser-Flu probes and the gyrA-Asp-Flu and gyrA-Asp-LC probes.
The detection sensitivity of N. gonorrhoeae using the LightCycler was shown to be 5 copies of N. gonorrhoeae, that is, 5 cells.
FIG. 3 shows amplification curves when the probes of gyrA-Ser-LC and gyrA-Ser-Flu were used.

(3) N. gonorrhoeae gy using control 55 strain
Amplification of rA Gene and Detection of Mutation FIG. 4 shows a typical example of the result of detection of gyrA gene mutation by LightCycler using N. gonorrhoeae, which is a control for the mutation of gyrA gene. A typical melting curve when a strain with no mutation of the gyrA gene and a strain with a mutation related to the change of serine of the 91st amino acid was examined using the probe gyrA-Ser-LC and the probe gyrA-Ser-Flu, It is shown in FIG. In the strains without mutation, the greatest decrease in fluorescence was 66.8 ° C, which is equal to the Tm of gyrA-Ser-LC, but in the mutant strain, it was 57.8 ° C, which was 9 ° C lower, which was clear from the melting curve. It was possible to recognize a large difference in temperature. B in FIG. 4 is 9
GyrA is a probe for strains that do not have a mutation in the gyrA gene that is associated with changes in the aspartic acid at amino acid 5 and those that have a mutation.
A typical melting curve when examined using -Asp-LC and the probe gyrA-Asp-Flu is shown. The highest decrease in fluorescence is found in the strain without mutation, which is equal to the Tm of gyrA-Asp-Flu.
It was 68.2 ℃, but it was lower by 5.6 ℃ in the mutant strain.
C., and a clear difference in temperature could be recognized in the melting curve. For the control 55 strains, mutations of the gyrA gene involved in changes in the amino acid serine at amino acid 91 and the aspartic acid at amino acid 95 were examined using LightCycler.The results of the presence or absence of the mutations obtained were previously obtained. It was completely consistent with the presence or absence of the mutation confirmed by the nucleotide sequencing method.

(4) Detection of Neisseria gonorrhoeae from 36 specimens of urethral secretions by LightCycler and detection of mutation of gyrA gene Using a urinary secretion sample obtained by extracting DNA for 5 minutes in boiling water, Gonorrhea gyrA by LightCycler The results of gene amplification and mutation detection are shown in FIG. FIG. 5 is an amplification curve of the Neisseria gonorrhoeae gyrA gene from 36 urethral secretions when the probe gyrA-Ser-LC and the probe gyrA-Ser-Flu were used. Amplification of the N. gonorrhoeae gyrA gene was confirmed in all the samples. That is, Neisseria gonorrhoeae was directly detected from the urethral secretion by the detection system using LightCycler. Probe gyrA-Asp
When -LC and the probe gyrA-Asp-Flu were used, the N. gonorrhoeae gyrA gene was also amplified in all samples, that is, N. gonorrhoeae was detected (data not shown). The result of melting temperature analysis after melting the gyrA gene (melting curve) is shown in FIG. When the analysis was performed using the probe gyrA-Ser-LC and the probe gyrA-Ser-Flu, the specimen with the largest fluorescence reduction rate at 57.8 ° C and the specimen with the largest fluorescence reduction rate at 66.8 ° C were observed. (A in FIG. 6). In the sample with the largest decrease in fluorescence at 57.8 ° C, N. gonorrhoeae in the sample has a mutation in the gyrA gene involved in the change of serine at amino acid 91,
It was determined that the sample with the largest decrease in fluorescence did not have a mutation in the gyrA gene related to the change in serine at the 91st amino acid. Probe gyrA-Asp-LC and probe gyrA-As
When examined using p-Flu, a sample with the largest decrease rate of fluorescence at 62.6 ° C and a sample with the largest decrease rate of fluorescence at 68.2 ° C were observed (B in Fig. 6). In the sample with the largest decrease rate of fluorescence at 62.6 ° C, N. gonorrhoeae in the sample has a mutation in the gyrA gene involved in the change in the aspartic acid of the 95th amino acid, and in the sample with the largest decrease rate of fluorescence at 68.2 ° C,
It was determined not to have a mutation in the gyrA gene related to the change in the 95th amino acid aspartic acid. That is, 15 shares are 91
GyrA, which has no mutation in the gyrA gene involved in the changes in amino acids 95 and 95, has 10 gyrA genes involved in the changes in amino acid 91 only.
It was judged that 11 strains had a mutation in the gene, and 11 strains had a mutation in the gyrA gene involved in changes in both the 91st and 95th amino acids. Detection of N. gonorrhoeae from urethral secretions by Light Cycler and analysis of mutations in the gyrA gene were performed using DNA extraction in boiling water, amplification by PCR and mutation by melting curve analysis.
I was able to get the results within minutes.

(5) Results of detection of mutations of the Neisseria gonorrhoeae gyrA gene in 36 specimens of urethral secretions by LightCycler, and CPFX against 36 Neisseria gonorrhoeae strains cultured and isolated from 36 specimens of urethral secretions.
Relationship with MIC: N. gonorrhoeae 15 cultured and isolated from 15 specimens determined not to have mutations in the gyrA gene associated with changes in amino acids 91 and 95
CPFX MIC for strains ranges from 0.002 μg / ml to 0.015 μg / ml
(The left column of FIG. 7), and these 15 strains were susceptible strains. CPFX for 10 strains cultured and isolated from 10 specimens that had a mutation in the gyrA gene related to the change in amino acid number 91 only
MIC was 0.06 μg / ml to 0.5 μg / ml (middle column of FIG. 7), and these 10 strains were hyposensitive strains. MIC of CPFX for 11 strains cultured and isolated from 11 specimens having mutations in the gyrA gene involved in changes in both 91st and 95th amino acids
Is 1 μg / ml to 8 μg / ml (right column of FIG. 7).
Eleven strains were resistant. From the above, by detecting the mutation of the Neisseria gonorrhoeae gyrA gene from the urethral secretions by LightCycler,
It was found that it is possible to determine the susceptibility of N. gonorrhoeae to CPFX.

From the above results, it was found that the method for detecting a gene mutation using LightCycler is a rapid and simple method and is a useful method having some advantages.
This method using LightCycler is a completely new detection system that can detect Neisseria gonorrhoeae directly from urethral secretions and can simultaneously detect mutations in the gyrA gene. This method is a highly sensitive detection method capable of detecting five Neisseria gonorrhoeae by using primers and probes specific to the gyrA gene of Neisseria gonorrhoeae. In addition, it was possible to directly detect the mutation of gyrA gene related to the susceptibility of N. gonorrhoeae to the new quinolone drug without culturing N. gonorrhoeae from clinical specimens, and the detection result accurately reflected the susceptibility to the new quinolone drug. In the usual diagnostic method for N. gonorrhoeae infection, N. gonorrhoeae must be separated from the secretion by a culture method, and at least 3 days are required for identification and susceptibility test. On the other hand, the method using LightCycler described above is an extremely rapid method because it can detect Neisseria gonorrhoeae and mutations in the gyrA gene within 60 minutes including the treatment of urethral secretions. In addition, in the conventional detection system using PCR, after PCR, PC
There is a risk that the reliability and reproducibility of the detection system will be impaired, for example, false positives due to contamination when handling DNA amplified by R, but real-time PCR
The above method using PCR can monitor the DNA amplified by PCR in real time, and it is not necessary to take out the DNA amplified by PCR from the glass capillary tube at the time of melting curve analysis after performing PCR. It can be said that this is a highly reliable detection method without risk of contamination, since analysis of gene mutations is being advanced. In addition, the above method prepares the PCR reaction solution and Light
All you have to do is set it on the Cycler, the procedure is extremely simple and you can get reliable data without any special training.

The present invention is not limited to the above description of the embodiments and examples of the invention. Various modifications are also included in the present invention within a range that can be easily conceived by those skilled in the art without departing from the scope of the claims.

[0043]

INDUSTRIAL APPLICABILITY According to the method of the present invention, it is possible to detect Neisseria gonorrhoeae from clinical specimens and search for resistance to new quinolone at the same time in an extremely short time, and it is possible to know test results at the time of initial diagnosis and before the start of treatment. That is, it is possible to confirm the gonococcal infection and to know the susceptibility to the new quinolone drug, to avoid improper administration of the new quinolone drug to the resistant gonococcus, and to perform more appropriate antibacterial chemotherapy from the first visit. become. The clinical application of the present invention is considered to greatly contribute to the optimization of chemotherapy for Neisseria gonorrhoeae infection.

[0044]

[Sequence list]                                SEQUENCE LISTING        <110> NAGOYA INDUSTRIAL SCIENCE RESEARCH INSTITUTE <120> A method for detecting Neisseria gonorrhoeae and for       evaluating fluoroquinolone resistance that. <130> Quinolone GyrA <140> <141> <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer       designed for amplifing a portion of gyrA gene of       N. gonorrhoeae <400> 1 cgcgatgcac gagctgaaaa a 21    <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer       designed for amplifing a portion of gyrA gene of       N. gonorrhoeae <400> 2 atttcggtat agcgcatggc tg 22    <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial       Sequence: Oligonucleotides probe for detecting       mutation in codon Ser-91 of gyrA gene <400> 3 gcatcgtcgg cgacgtcatc ggtaaatacc acccc 35    <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial       Sequence: Oligonucleotides probe for detecting       mutation in codon Ser-91 of gyrA gene <400> 4 acggcgattc cgcagtt 17    <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial       Sequence: Oligonucleotides probe for detecting       mutation in codon Asp-95 of gyrA gene <400> 5 gcagtttacg acaccatcgt c 21    <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial       Sequence: Oligonucleotides probe for detecting       mutation in codon Asp-95 of gyrA gene <400> 6 gtatggcgca aaatttcgct atgcgttatg tgctgataga cggac 45

[Brief description of drawings]

FIG. 1 is a diagram showing a partial DNA sequence of the N. gonorrhoeae gyrA gene. In the figure, the arrowhead indicates the mutation site in the region encoding amino acid 91, and the arrow indicates the mutation site in the region encoding amino acid 95. Symbols 1 and 2 represent partial DNAs to which a pair of nucleic acid probes for detecting the polymorphism at the 91st amino acid position hybridize. Similarly, symbols 4 and 5 represent partial DNAs to which a pair of nucleic acid probes for detecting the polymorphism at the 95th amino acid position hybridize.

FIG. 2 is a PC used in an embodiment of the present invention.
3 is a table showing R primers and probes. The position (Position) of each oligonucleotide is GenBank accessionn
Corresponds to umber U08817. LC Red640 and Light Cycler-R
The ed640 has an absorption maximum at 622 nm and an emission maximum at 638 nm.
It has a fluorescent label. The 3'end of the probe labeled with LC Red 640 is phosphorylated to prevent extension from the probe during the PCR reaction. Flu represents Fluorescein.

FIG. 3 is a LightCycler in an embodiment of the present invention.
It is the result of examining the detection sensitivity of N. gonorrhoeae gyrA gene detection. The amplification curve when using the probe of gyrA-Ser-LC and gyrA-Ser-Flu is shown, and the sample with a DNA amount of 10x10 ^-6 mg / l is 2μ
Amplification of N. gonorrhoeae gyrA gene was observed even when l was used,
It can be seen that it was possible to detect 5 copies of N. gonorrhoeae, that is, up to 5 N. gonorrhoeae. The seven curves in the graph are 10 mg / l, 10x10 ^-1 mg / l, 10x10 ^-2 mg / l, 1 from the left.
0x10 ^-3 mg / l, 10x10 ^-4 mg / l, 10x10 ^-5 mg / l and 10x10 ^-6
It is a result when a sample of mg / l is used.

FIG. 4 shows the results of detection of gyrA gene mutation by LightCycler using N. gonorrhoeae as a control in Examples. Gy related to the change of the 91st amino acid serine
Probes for strains without rA gene mutation and strains with mutation
A representative melting curve when examined using gyrA-Ser-LC and the probe gyrA-Ser-Flu is shown in A of FIG. Among the strains without mutation, gyrA-S has the highest fluorescence reduction rate.
The temperature was 66.8 ° C, which was equal to the Tm of er-LC, but it was 57.8 ° C, which is 9 ° C lower in the mutant strain, and a clear temperature difference can be observed in the melting curve. FIG. 4B shows a strain having no mutation in the gyrA gene involved in the change of the aspartic acid at the 95th amino acid and a strain having the mutation using the probe gyrA-Asp-LC and the probe gyrA-Asp-Flu. A representative melting curve of is shown. The highest fluorescence reduction rate was 68.2 ° C, which is equal to the Tm of gyrA-Asp-Flu, in the mutant-free strain, but was 62.6 ° C, which is 5.6 ° C lower in the mutant strain. The difference in temperature can be recognized.

[Fig. 5] Fig. 5 is a result of examination of detection of Neisseria gonorrhoeae from 36 specimens of urethral secretions by LightCycler in Examples. Shown is the amplification curve of the Neisseria gonorrhoeae gyrA gene from 36 urethral secretions using the probe gyrA-Ser-LC and probe gyrA-Ser-Flu. Amplification of the Neisseria gonorrhoeae gyrA gene can be confirmed in all the samples.

[Fig. 6] Fig. 6 shows the results of examining the detection of mutations of the Neisseria gonorrhoeae gyrA gene in urethral secretions by LightCycler in Examples. Probe gyrA-Ser-LC and probe gyrA-Se
In the melting curve when r-Flu was used, a sample with the largest decrease rate of fluorescence at 57.8 ° C and a sample with the largest decrease rate of fluorescence at 66.8 ° C were observed (A in Fig. 6). For urethral secretions with the highest fluorescence reduction rate at 57.8 ° C, 91
Urinary secretions having a mutation in the gyrA gene related to the change in the serine of amino acid No. 91 and the greatest decrease in fluorescence at 66.8 ° C do not have the mutation in the gyrA gene related to the change in serine of the amino acid No. 91. It was judged as something. Probe gyrA-Asp
-LC and the probe gyrA-Asp-Flu were examined, a sample with the largest fluorescence reduction rate at 62.6 ° C and a sample with the largest fluorescence reduction rate at 68.2 ° C were observed (Fig. 6).
B). In the sample with the largest decrease rate of fluorescence at 62.6 ° C, N. gonorrhoeae in the sample has a mutation in the gyrA gene involved in the change in the aspartic acid of the 95th amino acid, and in the sample with the largest decrease rate of fluorescence at 68.2 ° C, It was determined not to have a mutation in the gyrA gene related to the change in the 95th amino acid aspartic acid.

[Fig. 7] Fig. 7 shows the results of detection of mutations of Neisseria gonorrhoeae gyrA gene from urethral secretions by LightCycler and the relationship between MIC of CPFX against Neisseria gonorrhoeae cultured and isolated from urethral secretions. The MIC of CPFX for 15 N. gonorrhoeae strains cultured and separated from 15 specimens determined not to have mutations in the gyrA gene related to changes in amino acids 91 and 95 was 0.002 μg / ml to 0.015 μg
/ ml (left column), and these 15 strains were susceptible strains. 9
MI of CPFX for 10 strains cultured and isolated from 10 specimens having a mutation in the gyrA gene related to the change of amino acid No. 1 only
C was 0.06 μg / ml to 0.5 μg / ml (middle column), and these 10 strains were hyposensitive strains. The MIC of CPFX for 11 strains cultured and isolated from 11 specimens having mutations in the gyrA gene involved in changes in both 91st and 95th amino acids was 1 μg / ml to 8 μg / ml (right column). The strain was resistant. It was possible to determine the susceptibility of N. gonorrhoeae to CPFX by detecting mutations of N. gonorrhoeae gyrA gene in urethral secretions by Light Cycler.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 33/53 M G01N 21/64 33/566 33/53 33/573 Z 33/566 33 / 58 A 33/573 C12R 1:01 33/58 C12N 15/00 ZNAA // (C12Q 1/04 A C12R 1:01) F term (reference) 2G043 AA03 BA16 BA17 DA02 DA08 EA01 GA25 GB07 GB21 KA02 KA05 MA16 2G045 AA25 AA28 CB21 DA12 DA13 DA14 FB02 FB07 FB12 4B024 AA01 AA11 CA01 CA11 HA14 4B063 QA01 QA13 QA18 QA19 QQ06 QQ44 QR32 QR56 QR62 QS22 QS25 QS34 QS36 QX02

Claims (13)

[Claims] 1. A method for detecting N. gonorrhoeae and assessing susceptibility to a new quinolone agent, which comprises the steps of: (a) specifically amplifying a target DNA containing a region encoding amino acid 91 in the N. gonorrhoeae gyrA gene. Step; (b) Analyzing the melting temperature of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion including the region of the target DNA. 2. The method according to claim 1, wherein the step (a) is performed by a real-time PCR method. 3. The method according to claim 1 or 2, wherein
The step (b) includes the following steps: (b1) The target DNA can be specifically hybridized with a gap of 1 to 5 bases interposed, one of which covers the region and a melting temperature of which is higher than that of the other. A pair of nucleic acid probes designed to be high, the first nucleic acid probe having a first fluorescent substance bound to the end located on the gap side when hybridized to the target DNA, and the target DNA hybridized. A pair of nucleic acid probes consisting of a second nucleic acid probe having a second fluorescent substance that is excited by the fluorescence of the first fluorescent substance and emits fluorescence when bound to the end located on the gap side when soybean is added to the sample Step; (b2) lowering the temperature to a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA; (b3) A step of irradiating the specimen with light having an excitation wavelength of the first fluorescent material while continuously or stepwise raising the temperature, and measuring the amount of fluorescence emitted from the second fluorescent material. 4. The method according to claim 3, further comprising the following steps: (b4) After step (b3), a temperature at which the rate of decrease of the fluorescence amount is maximum is determined, and the temperature is determined from the temperature. Determining the sequence of the region encoding amino acid 91 in the gyrA gene. 5. A method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone agent, comprising the steps of: (c) specifically amplifying a target DNA containing a region encoding amino acid 91 in the Neisseria gonorrhoeae gyrA gene. (D) It is possible to specifically hybridize to the target DNA with a gap of 1 to 5 bases, one of which covers the region and has a higher melting temperature than the other. A pair of nucleic acid probes designed to, wherein the end located on the gap side when hybridized to the target DNA, a first nucleic acid probe bound with a first fluorescent substance, and hybridized to the target DNA A second fluorescent substance, which is excited by fluorescence of the first fluorescent substance and emits fluorescence, is bound to an end located on the gap side.
A step of adding a pair of nucleic acid probes consisting of a nucleic acid probe to a sample; (e) a step of performing a real-time PCR method; (f) a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA (G) increasing the temperature continuously or stepwise, irradiating the specimen with light having the excitation wavelength of the first fluorescent substance, and measuring the amount of fluorescence emitted from the second fluorescent substance; (h) A step of obtaining a temperature at which the reduction rate of the fluorescence amount is maximum and determining the sequence of the region encoding the 91st amino acid in the gyrA gene from the temperature. 6. A method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone agent, comprising the steps of: (A) specifically amplifying a target DNA containing a region encoding amino acid 95 in the Neisseria gonorrhoeae gyrA gene. Step; (B) a step of analyzing melting temperatures of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion including the region of the target DNA. 7. The method according to claim 6, wherein the step (A) is performed by a real-time PCR method. 8. A method according to claim 6 or 7, wherein
The step (B) includes the following steps: (B1) The target DNA can be specifically hybridized with a gap of 1 to 5 bases in between, one of which covers the region and a melting temperature of which is higher than that of the other. A pair of nucleic acid probes designed to be high, the first nucleic acid probe having a first fluorescent substance bound to the end located on the gap side when hybridized to the target DNA, and the target DNA hybridized. A second fluorescent substance, which is excited by fluorescence of the first fluorescent substance and emits fluorescence when bound to the end located on the side of the gap when soybean is bound,
Adding a pair of nucleic acid probes consisting of nucleic acid probes to the sample; (B2) lowering the temperature to a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA; (B3) continuously or A step of increasing the temperature stepwise, irradiating the specimen with light having the excitation wavelength of the first fluorescent substance, and measuring the amount of fluorescence emitted from the second fluorescent substance. 9. The method according to claim 8, further comprising the following steps: (B4) After the step (B3), a temperature at which the reduction rate of the fluorescence amount is maximum is determined, and the temperature is determined. More in the gyrA gene
Determining the sequence of the region encoding the 95th amino acid. 10. A method for detecting Neisseria gonorrhoeae and assessing sensitivity to a new quinolone agent, which comprises the following steps: (C) Target DNA containing a region encoding amino acid 95 in the Neisseria gonorrhoeae gyrA gene can be specifically amplified. (D) It is possible to specifically hybridize to the target DNA with a gap of 1 to 5 bases, and one of them covers the region and has a higher melting temperature than the other. A pair of nucleic acid probes designed to, wherein the end located on the gap side when hybridized to the target DNA, a first nucleic acid probe bound with a first fluorescent substance, and hybridized to the target DNA Sometimes a second fluorescent substance, which is excited by the fluorescence of the first fluorescent substance and emits fluorescence, is bound to the end located on the gap side. A step of adding a pair of nucleic acid probes comprising a nucleic acid probe to a sample; (E) a step of executing a real-time PCR method; (F) a temperature at which the first nucleic acid probe and the second nucleic acid probe hybridize to the target DNA (G) irradiating the specimen with light having an excitation wavelength of the first fluorescent substance and measuring the amount of fluorescence emitted from the second fluorescent substance, while (G) continuously or stepwise raising the temperature. (H) A step of obtaining a temperature at which the reduction rate of the fluorescence amount is maximum and determining the sequence of the region encoding the 95th amino acid in the gyrA gene from the temperature. 11. A method for detecting Neisseria gonorrhoeae and assessing susceptibility to a new quinolone agent, comprising the steps of: (x) encoding a first region encoding amino acid 91 and amino acid 95 in the Neisseria gonorrhoeae gyrA gene. Specifically amplifying a target DNA containing a second region; (y) analyzing melting temperatures of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion of the target DNA containing the first region (Z) a step of analyzing melting temperatures of the target DNA and a nucleic acid probe capable of specifically hybridizing to a portion of the target DNA including the second region. 12. A pair of primers capable of specifically amplifying a target DNA containing a region encoding the 91st amino acid in the Neisseria gonorrhoeae gyrA gene, and specifically hybridizing with a gap of 1 to 5 bases sandwiched in the target DNA. It is possible, one is a pair of nucleic acid probes designed to cover the region, and has a higher melting temperature than the other, at the end located on the gap side when hybridized to the target DNA, A first nucleic acid probe bound with a first fluorescent substance, and a second fluorescent substance that is excited by fluorescence of the first fluorescent substance and emits fluorescence at the end located on the gap side when hybridized to the target DNA. A kit for detecting N. gonorrhoeae and assessing the sensitivity of a new quinolone agent, comprising a pair of nucleic acid probes each comprising a bound second nucleic acid probe. 13. A pair of primers capable of specifically amplifying a target DNA containing a region encoding the 95th amino acid in the Neisseria gonorrhoeae gyrA gene, and specifically hybridizing with a gap of 1 to 5 bases sandwiched in the target DNA. It is possible, one is a pair of nucleic acid probes designed to cover the region, and has a higher melting temperature than the other, at the end located on the gap side when hybridized to the target DNA, A first nucleic acid probe bound with a first fluorescent substance, and a second fluorescent substance that is excited by fluorescence of the first fluorescent substance and emits fluorescence at the end located on the gap side when hybridized to the target DNA. A kit for detecting N. gonorrhoeae and assessing the sensitivity of a new quinolone agent, comprising a pair of nucleic acid probes each comprising a bound second nucleic acid probe.