JP6435568B2 - Detection of Acinetobacter baumannii - Google Patents

Description

  The present invention relates to the field of detection by Acinetobacter baumannii (hereinafter A. baumannii). More specifically, the present invention relates to a probe for detecting A. baumannii, a primer, a method for detecting A. baumannii using these, and the like.

  Over 30 species have been identified in the genus Acinetobacter, many of which can be isolated from humans, but many infections have been reported to be caused by A. baumannii. A. baumannii is an aerobic gram-negative bacterium that does not have flagella, is immobile and causes opportunistic infections. A. baumannii has been reported to be susceptible to mutation because it has a mechanism for taking in DNA fragments from other organisms and incorporating them into its own DNA. Since A. baumannii infection can cause serious symptoms, especially when immunity is reduced, early detection is highly clinically significant.

  The most widely used method for identifying bacterial species is the culture method, but it is difficult to detect A. baumannii by its biochemical properties, and various genetic test methods are used for identification. There is a need. However, since the methods reported so far require a large number of steps and time, it is difficult to detect A. baumannii quickly. In addition, International clone type II (hereinafter sometimes abbreviated as “IC II”), which has been reported to have drug resistance in recent years, and other A. baumannii are separated by the sequence method, and quickly No detectable method has been reported.

Journal of Microbiological Methods 94 (2013) 121-124 Journal of Clinical Microbiology 44 (3), 827-832 (2006)

  An object of the present invention is to provide a means for fractionating and detecting A. baumannii (in particular, A. baumannii International clone Type II, which has been reported to have high drug resistance) in a simple, rapid and specific manner.

  As a result of intensive studies to solve the above problems, the present inventors have used the presence of a specific sequence region of the blaOXA-51-like gene as an indicator, without detecting other species of the genus Acinetobacter, and It was found that A. baumannii can be detected accurately without leakage. Further, the present inventors have found that International clone II can be detected separately from other A. baumannii by using a specific position of the blaOXA-51-like gene as an index. Based on these findings, the present inventors have made further trial and error and have completed the present invention. A typical present invention is shown below.

A: Nucleic acid probe for detection of A. baumannii IC II < 1 >
A nucleic acid probe designed to recognize the 107th and 108th bases of the base sequence shown in SEQ ID NO: 1.
Item 2.
Item 2. The nucleic acid probe according to Item 1, consisting of 14 to 28 nucleotides.
Item 3.
Item 3. The nucleic acid probe according to Item 1 or 2, consisting of all or part of the base sequence represented by SEQ ID NO: 10.
Item 4.
Item 4. The nucleic acid probe according to any one of Items 1 to 3, which is fluorescently labeled.
B: A. baumannii IC II detection kit <5 >
The nucleic acid probe according to any one of Items 1 to 4, and
A primer consisting of a base sequence of 16 to 36 bases in sequence of the base sequence shown in SEQ ID NO: 2, and a primer consisting of a base sequence of 16 to 36 bases in sequence of the base sequence shown in SEQ ID NO: 3,
A detection kit for Acinetobacter baumannii.
Item 6.
Item 6. The kit according to Item 5, wherein the primer consisting of a base sequence of 16 to 36 bases continuous with the base sequence shown in SEQ ID NO: 2 is a primer consisting of the base sequence shown in SEQ ID NO: 4, 5, or 6.
Item 7.
Item 7. The primer according to Item 5 or 6, wherein the primer consisting of a base sequence of 16 to 36 bases that is continuous with the base sequence shown in SEQ ID NO: 3 is a primer consisting of the base sequence shown in SEQ ID NO: 7, 8, or 9. kit.
Item 8.
Item 8. The kit according to any one of Items 5 to 7, further comprising α-type DNA polymerase.
Item 9.
C: A. baumannii IC II detection method A method for detecting Acinetobacter baumannii International clone Type II, which comprises the step (I) of binding the nucleic acid probe according to any one of items 1 to 4 to a nucleic acid in a sample. 10.
Prior to step (I), a primer comprising a base sequence of 16 to 36 bases in the base sequence shown in SEQ ID NO: 2 and a base of 16 to 36 bases in the base sequence of SEQ ID NO: 3 A primer consisting of a base sequence,
Item 10. The method according to Item 9, comprising the step (II) of amplifying a nucleic acid in a sample using
Item 11.
Item 11. The method according to Item 10, wherein the nucleic acid amplification in step (II) is performed using α-type DNA polymerase.
D: Primer set for detection of A. baumannii Item 12.
A primer consisting of a base sequence of 16 to 36 bases in sequence of the base sequence shown in SEQ ID NO: 2, and a primer consisting of a base sequence of 16 to 36 bases in sequence of the base sequence shown in SEQ ID NO: 3,
Primer set containing
Item 13.
Item 13. The primer set according to Item 12, which is for detection of Acinetobacter baumannii.
Item 14.
The primer consisting of a base sequence of 16 to 36 bases in a continuous sequence of the base sequence shown in SEQ ID NO: 2 is a primer consisting of the base sequence shown in SEQ ID NO: 4, 5 or 6. Primer set.
Item 15.
15. The primer according to claim 12, wherein the primer consisting of a base sequence of 16 to 36 bases consecutive in the base sequence shown in SEQ ID NO: 3 is a primer consisting of the base sequence shown in SEQ ID NO: 7, 8 or 9. The primer set according to crab.

  By using the present invention, it is possible to detect the presence of A. baumannii or A. baumannii IC II in a sample more quickly, conveniently and accurately than before. Therefore, by using the present invention, it becomes possible to perform diagnosis of A. baumannii infection more quickly, simply and accurately.

1: A. baumannii IC II detection probe
A. baumannii IC II differs from other A. baumannii in the base sequence of the 107th and 108th bases in the gene sequence encoding the OXA-51-like β-lactamase protein shown in SEQ ID NO: 1. Specifically, in A. baumannii IC II, the 107th and 108th bases are thymine and adenine, respectively, while in other A. baumannii IC107, the 107th base is adenine, The 108th base is cytosine or adenine. Therefore, the presence of A. baumannii IC II can be distinguished from other A. baumannii by using the difference between these bases as an index.

  Detection of A. baumannii IC II using the difference in base as an index is preferably performed using a nucleic acid probe that is a polynucleotide designed to recognize the base. Such a nucleic acid probe can be designed based on the base sequence shown in SEQ ID NO: 1 and can be prepared using known techniques and devices. The length of the nucleic acid probe is not particularly limited as long as the difference in the base can be recognized. The lower limit of the length of the nucleic acid probe is preferably 14 bases, more preferably 15 bases, still more preferably 16 bases. The upper limit of the length of the nucleic acid probe is preferably 26 bases, more preferably 25 bases, still more preferably 24 bases, still more preferably 23 bases, particularly preferably 22 bases.

  Specific examples of suitable nucleic acid probes include all or part of the base sequence shown in SEQ ID NO: 10, and the 107th and 108th bases of the base sequence shown in SEQ ID NO: 1 described above. Mention may be made of polynucleotides (or oligonucleotides) to be recognized.

  The nucleic acid probe is preferably labeled in order to facilitate detection of its presence. The kind of the label of the nucleic acid probe is not particularly limited as long as the nucleic acid probe can be detected, and can be appropriately selected according to the detection mode. For example, the nucleic acid probe can be labeled with one or more substances selected from the group consisting of magnetic substances, electron carriers, enzymes, biotin, fluorescent substances, haptens, antigens, antibodies, radioactive substances, luminophores, and the like. These labeling substances can be bound to any position of the nucleic acid probe, but are preferably bound to the 3 'end or 5' end.

  Examples of the magnetic material include iron oxide, chromium dioxide, cobalt, and ferrite. Examples of the electron carrier include ferrocene, PQQ, and redox compound. Examples of the enzyme include peroxidase, beta-galactosidase, luciferase, and alkaline phosphatase. Examples of the fluorescent substance include Cy5 (registered trademark), Cy3 (registered trademark), FITC, rhodamine, lanthanide phosphor, Texas Red, FAM, JOE, CalFluor Red 610 (registered trademark), and Quasar 670 (registered trademark). Can be mentioned. Examples of the hapten include digoxigenin. Examples of radioactive isotopes include 32P, 35S, 3H, 14C, 125I, and 131I. Examples of the luminophore include ruthenium and aequorin.

  From the viewpoint that the nucleic acid probe can be detected more easily and in a short time, the nucleic acid probe is preferably labeled with a fluorescent substance. By fluorescently labeling the nucleic acid probe, whether or not the nucleic acid in the sample is A. baumannii IC II nucleic acid based on the change in fluorescence intensity when the nucleic acid in the sample and the nucleic acid probe bind / melt. It can be judged. Preferred fluorescent materials for use for such purposes are exemplified in the next paragraph.

  4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid, acridine and derivatives (acridine, acridine isothiocyanate), AlexaFluor® 350, Alexa Fluor® 488, AlexaFluor® ) 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probe), 5- (2′-aminoethyl) aminonaphthalene-1 -Sulfonic acid (EDANS), 4-amino-N- [3-vinylsulfonyl) phenyl] naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N- (4- Nilino-1-naphthyl) maleimide, anthranilamido, BODIPY® CR-6G, BOPIPY® 530/550, BODIPY® FL, brilliant yellow, coumarin and derivatives (coumarin, 7-amino-4 -Methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151)), Cy2 (registered trademark), Cy3 (registered trademark), Cy3.5 (registered trademark), Cy5 (registered trademark) ), Cy5.5®, cyanocin, 4 ′, 6-diaminidino-2-phenylindole (DAPI), 5 ′, 5 ″ -dibromopyrogallol-sulfonephthalein (BromopyrogalRed), 7-diethylamino-3- (4'-isocyanatophenyl) -4 Methylcoumarin, diethylenetriaminetetraacetic acid, 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, 5-chloride [Dimethylamino] naphthalene-1-sulfonyl (DNS, dansyl chloride), 4- (4′-dimethylaminophenylazo) benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), Eclipse ™ (EpochBiosciences Inc.), eosin and derivatives (eosin, eosin isothiocyanate), erythrosine and derivatives (erythrosine B, erythrosine isothiocyanate), ethidium, fluorescein and derivatives (5-carbohydrate) Cyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2 ', 7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein , Fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescein (TET)), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone , Orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, R-phycoerythrin, o-phthaldialdehyde, OregonGreen (registered trademark) Propidium iodide, pyrene and derivatives (pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate), QSY (R) 7, QSY (R) 9, QSY (R) 21, QSY (R) 35 (Molecular Probe) , Reactive Red 4 (Cibacron® Brilliant Red 3B-A), rhodamine and derivatives (6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lysamine rhodamine B sulfonyl chloride, rhodamine ( Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)), N, N, N ′ , N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosoleic acid, terbium chelate derivatives. Among these, it is preferable to use at least one selected from the group consisting of FITC, BODIPY-FL, FAM, CR6G, Cy3, and Cy5 from the viewpoint of good handleability.

  In the nucleic acid probe, the base at either or both of the 5 'end and the 3' end (preferably, the 5 'end) is cytosine, and the fluorescent substance is preferably bound to the nucleic acid probe via the cytosine. Further, when a nucleic acid probe having a fluorescent substance bound to the 5 'end is used, the 3' end is preferably modified with a phosphate group. Since the PCR reaction does not proceed with the sample nucleic acid to which the nucleic acid probe is bound by modifying the 3 'end, the nucleic acid probe can be added in advance to the amplification reaction solution. By doing so, the amplification of the nucleic acid in the sample, the binding with the nucleic acid probe, and the detection of the nucleic acid probe can be carried out substantially continuously in one step. If necessary, the nucleic acid probe may be added to the reaction solution before starting the amplification reaction. The amount of the probe added to the nucleic acid amplification reaction solution is not particularly limited, but for example, it can be added in the range of 10 to 1000 nmol.

  The mode for detecting the nucleic acid derived from A. baumannii IC II through detection of the nucleic acid probe is arbitrary, and can be appropriately selected according to the labeling substance used. When a fluorescent substance is used as the labeling substance, for example, the nucleic acid probe can be detected as follows. When using a labeled probe (for example, a guanine quenching probe) that shows a signal in a released state and does not show a signal when forming a double strand with a complementary nucleic acid, the nucleic acid in the sample In a state where the probe and the probe are dissociated, fluorescence is emitted. However, when a double strand is formed due to a decrease in temperature or the like, the fluorescence is reduced (or quenched). All the fluorescent materials described above have such characteristics. Therefore, for example, by increasing the temperature of the reaction solution containing the sample nucleic acid and the nucleic acid probe, rapidly decreasing the temperature, then gradually increasing the temperature, and measuring the increase in fluorescence intensity as the temperature increases, A The presence or absence of nucleic acid derived from baumannii IC II can be detected. Furthermore, in order to determine the genotype in the region to which the nucleic acid probe is bound, the Tm (melting temperature) value is determined by analyzing the change in fluorescence intensity accompanying the temperature change as described above, and this can be used as an index it can.

2: Primer set for detection of A. baumannii
A. baumannii can be distinguished from many other Acinetobacter species by the presence of a region called blaOXA-51-like gene in genomic DNA. However, among the bacteria belonging to A. baumannii, there are mutations in the blaOXA-51-like gene region. It is not possible to detect the genus accurately. Therefore, as a means for enabling more accurate detection of A. baumannii, a primer comprising a base sequence of 16 to 36 bases (hereinafter referred to as “F primer”) of the base sequence represented by SEQ ID NO: 2 And a primer set comprising a base sequence consisting of 16 to 36 bases of the base sequence shown in SEQ ID NO: 3 (hereinafter sometimes referred to as “R primer”). Is preferred.

  The base sequence shown in SEQ ID NO: 2 matches the 48th to 86th base sequences of the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 3 is complementary to the 169th to 218th base sequences of the base sequence shown in SEQ ID NO: 1. The present inventors have A. baumannii having a blaOXA-51-like gene in which a long-chain polynucleotide is inserted in a region upstream of the 48th base in the base sequence shown in SEQ ID NO: 1, It was found that such a gene cannot be amplified with a primer set complementary to any region of the blaOXA-51-like gene.

  The lower limit of the length of the F primer and R primer is preferably 17 bases, more preferably 18 bases, still more preferably 19 bases, still more preferably 20 bases, still more preferably 21 bases. . The upper limit of the length of the F primer and R primer is preferably 35 bases, more preferably 34 bases, still more preferably 33 bases, still more preferably 32 bases, still more preferably 31 bases. More preferably, it is 30 bases, more preferably 29 bases.

  Specific examples of suitable F primers include a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 4-6. Specific examples of suitable R primers include a polynucleotide comprising the base sequence shown in any of SEQ ID NOs: 7 to 9. The F primer and the R primer can be prepared using an arbitrary method and / or a DNA synthesizer.

  Detection of A. baumannii using a primer set including an F primer and an R primer can be performed by carrying out any nucleic acid amplification method using the genomic DNA of A. baumannii as a template and the primer set. Examples of nucleic acid amplification methods include PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence based amplification: Nature, Vol. 350, page 91 (1991)), TMA (Transcription-mediated amplification: J. Clin. Microbiol. Volume 31, page 3270 (1993)), SDA (Strand Displacement Amplification: Nucleic acid research Volume 20, page 1691 (1992)), LCR (Ligase Chain Reaction: International Publication 89/12696) No.), RCA (Rolling Circle Amplification: International Publication No. 90/1069), LAMP (loop-mediated isothermal amplification method: J Clin Microbiol. Volume 42: 1,956 (2004)), ICAN (isothermal and chimeric) primer-initiated amplification of nucleic acids: Kekkaku. 78, 533 (2003)). Specific conditions for carrying out these nucleic acid amplification methods can be appropriately set with reference to a manual or the like. A preferred nucleic acid amplification method is PCR.

In the PCR method, a sample nucleic acid, 4 types of deoxynucleoside triphosphates, a pair of primers, and a heat-resistant DNA polymerase are used, and a cycle consisting of three steps of denaturation, annealing, and extension is repeated to sandwich the pair of primers. This is a method of exponentially amplifying a region of a sample nucleic acid to be obtained. That is, one double-stranded nucleic acid of a sample is separated into two single-stranded nucleic acids in the heat denaturation step, and each primer and a region on the single-stranded sample nucleic acid complementary to each primer are separated in the subsequent annealing step. In the subsequent extension step, a DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended from each primer as a starting point by the action of DNA polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified 2 ⁿ times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, by using the gene amplification method, it is possible to detect even a very small amount of sample nucleic acid (which can be a single molecule), which could not be detected in the past.

  Specific conditions for each step in the PCR method include, for example, the first heat denaturation step at 80 to 100 ° C. for 10 seconds to 15 minutes, repeated heat denaturation at 80 to 100 ° C. for 1 to 300 seconds, and annealing. The elongation reaction step is carried out at 40 to 80 ° C. for 1 to 300 seconds and at 60 to 85 ° C. for about 1 to 300 seconds.

  The mixing ratio of the primer set to be added to the PCR reaction solution is arbitrary, but for example, it can be added so as to be 0.1 to 2 μmol / l. Moreover, the abundance ratio (mole) of the F primer and the R primer can be F primer: R primer = 1: 0.25 to 1: 4.

  The composition of the PCR reaction solution is not particularly limited. For example, each primer oligonucleotide is 0.5-50 pmol, x10 buffer is 2.5 μl, 2 mM dNTP is 2.5 μl, and salts are 25 μl of the reaction solution. It is preferable that 0.05 to 10 μl in a 25 mM concentration solution and about 0.1 to 3 U of DNA polymerase.

  When the PCR method is carried out in the presence of the probe described above, it is preferable to use α-type DNA polymerase as the DNA polymerase. This is to prevent the probe bound to the template DNA from being degraded by the 5 '→ 3' exonuclease activity possessed by Pol I type DNA polymerase such as Taq DNA Polymerase. Therefore, from such a viewpoint, it is not preferable to use Pol I type DNA polymerase as the DNA polymerase, and α type DNA polymerase is preferably used. Furthermore, the α-type DNA polymerase is also preferable from the viewpoint that the DNA is amplified with high accuracy due to its 3 ′ → 5 ′ exonuclease activity.

  Usually, since α-type DNA polymerase has 3 ′ → 5 ′ exonuclease activity, the nucleic acid amplification rate tends to be lower than that of Pol I-type enzyme. However, although KOD DNA Polymerase is an α-type DNA polymerase, it has a high DNA synthesis activity, a DNA synthesis rate of 100 bases / second or more, and an excellent elongation efficiency. Therefore, it is preferable to use KOD DNA Polymerase (registered trademark, Toyobo Co., Ltd.) among the α-type DNA polymerases for carrying out the nucleic acid amplification reaction.

  In addition, a mutant DNA polymerase having a deoxyribonucleic acid synthesis rate of 100 bases / second or more by mutating α-type DNA polymerase, or a DNA polymerase composition whose performance has been improved by combining a wild type and a mutant type is used. You can also For example, Prime STAR HS DNA polymerase (manufactured by Takara Bio Inc., registered trademark), PfuTurbo DNA polymerase (manufactured by Agilent Technologies), and the like can also be used.

  The addition rate of the DNA polymerase in the reaction solution is not particularly limited, but is, for example, 1 to 100 U / mL, preferably 5 to 50 U / mL, and more preferably 20 to 30 U / mL.

  A method for detecting a nucleic acid amplified by a nucleic acid amplification method such as PCR is arbitrary, and a known method can be appropriately selected. For example, it can be detected using a labeled oligonucleotide that specifically recognizes the amplification product, and can also be easily detected by subjecting the reaction solution after completion of the reaction to agarose gel electrophoresis as it is. By detecting the amplification product as such, the presence of genomic DNA derived from A. baumannii can be confirmed. The above 1. The nucleic acid probe described in 1) can also be used. Above 1. In addition to distinguishing A. baumannii from other bacteria or other Acinetobacter species, it is also possible to detect only A. baumannii IC II. The nucleic acid probe may be added in advance to the reaction solution before starting the PCR reaction, or may be added after the amplification reaction is completed, but it is preferable to add the nucleic acid probe in advance because the working efficiency is good.

  The sample containing the nucleic acid to be amplified using the primer set is preferably a sample that is considered (or suspected) to contain genomic DNA derived from A. baumannii. Examples of such samples include, but are not limited to, for example, pus, pleural effusion, pharyngeal wiping fluid, nasal wiping fluid, and blood collected from subjects suspected of being infected with A. baumannii, and bacterial colonies in which they are cultured. Suspension, culture solution, etc. are mentioned. A method for collecting a sample, a method for preparing a nucleic acid such as DNA or RNA, and the like are not limited, and conventionally known methods can be employed.

3. A. baumannii detection kit The primer set described in the above can be combined with an appropriate probe or the like to form an A. baumannii detection kit. As the probe, the above-mentioned 1. It is also possible to use the nucleic acid probe described in 1. Such a kit can further include an α-type DNA polymerase. The kit may further contain optional components and / or a use manual.

The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following examples.
1. Preparation of primers and probes Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 4 to 11 were synthesized by the phosphoramidite method using a DNA synthesizer type 392 manufactured by PerkinElmer. Hereinafter, the oligonucleotide shown in SEQ ID NO: 4 may be abbreviated as “oligo 4”, and the same applies to other oligonucleotides. Oligonucleotide synthesis was performed according to the above product manual, and oligonucleotide deprotection was performed overnight with ammonia water at 55 ° C. Oligonucleotide purification was performed on an OPC column manufactured by PerkinElmer. Oligos 4-6 and 11 are sense strand primers, and oligos 7-9 are antisense strand primers. Any combination of these sense strand primers and antisense strand primers can be used as a primer set. Oligo 10 was a probe, and cytosine at the 5 ′ end was fluorescently labeled with fluorescein isothiocyanate (FITC), and the 3 ′ end was labeled with a phosphate group. The position recognized by each oligonucleotide in relation to the base sequence represented by SEQ ID NO: 1 (encoding bla-oxa-51-like protein) is shown in Table 1 below.

2. Detection of A. baumannii International Clone II using PCR method PCR reaction solutions shown in Table 2 below were prepared, and PCR was performed under the conditions shown in Table 3 below. In Table 2, the extracted DNA solution contains DNA isolated from five different clinical samples and confirmed to be derived from A. baumannii International Clone II by sequencing. Moreover, any of oligo 4-6 and 11 was used for the forward primer, and any of oligo 7-9 was used for the reverse primer.

  After the reaction according to the above conditions, the amount of fluorescence was measured while the temperature of the reaction solution was increased from 40 ° C. to 75 ° C. at an increase rate of 0.09 ° C./sec. Based on the measurement results, a melting temperature analysis was performed to determine the amount of change in fluorescence intensity. The results are shown in Table 4 below. In all the tests, the temperature (Tm) at which the value represented by the formula [−dF (fluorescence intensity change amount) / dT (temperature change amount)] is the largest was around 60 ° C.

  As shown in Table 4, based on changes in the amount of fluorescence generated when the probe bound to the PCR product was dissociated in all tests, except when DNA solution 3 was detected using the oligo 11 and oligo 7 primer sets. Thus, it was confirmed that the Acinetobacter baumannii International clone Type II strain could be detected. When the nucleic acid of DNA solution 3 was examined, a sequence of about 1.3 kbp was inserted between the region recognized by oligo 11 and the region recognized by oligos 4-6 in the base sequence represented by SEQ ID NO: 1. It was found that this caused no nucleic acid amplification.

3. Discrimination between Acinetobacter baumannii and other strains belonging to the genus Acinetobacter Regarding the DNA solution extracted from Acinetobacter baumannii isolated from various clinical materials, 2. PCR was performed using the oligo 4 and oligo 7 primer sets under the same conditions as in the above test. Melting temperature analysis was performed to determine the amount of change in fluorescence intensity and the peak temperature (Tm) of the value represented by the formula [-dF (amount of change in fluorescence intensity) / dT (amount of change in temperature)]. The results are shown in Table 5 below.

  As shown in Table 5, it was confirmed that nucleic acids derived from the genus Acinetobacter other than A. baumannii were not amplified. It was also confirmed that A. baumannii International clone II was distinguishable from other A. baumannii based on the difference in Tm. This difference in Tm is due to the presence of a mutation in the region recognized by the probe (specifically, the 107th and 108th bases of SEQ ID NO: 1).

  According to the present invention, it is possible to amplify the nucleic acid in the sample in about 17 minutes, and then detect the amplification product in about 9 minutes. Therefore, in a total of about 26 minutes, A. baumannii and other nucleic acids can be detected. It is possible to discriminate between fungi and / or A. baumannii IC II from other strains. Furthermore, since nucleic acid amplification and amplification product detection can be carried out continuously with one apparatus, the identification can actually be performed in one operation step. On the other hand, in order to discriminate between A. baumannii and other bacteria, or to distinguish between A. baumannii IC II and other strains using the conventional sequencing method, amplification product is amplified for about 1.5 hours. The purification requires about 0.5 hours, the sequencing reaction takes about 2.5 hours, and the sequence analysis takes about 2 hours, so that 6.5 hours is required as a whole, and many separated work steps are required. Thus, by using the present invention, it is possible to identify A. baumannii and / or A. baumannii IC II much more quickly, simply, and accurately compared to the conventional method. .

Claims (7)

It consists of all or part of the base sequence shown in SEQ ID NO: 10, consists of 14 to 25 nucleotides, and is designed to recognize the 107th and 108th bases of the base sequence shown in SEQ ID NO: 1. Nucleic acid probes , and
A primer consisting of a base sequence of 16 to 36 bases in a continuous sequence of the base sequence shown in SEQ ID NO: 2 and a primer consisting of a base sequence of 16 to 36 bases in a sequence of the base sequence shown in SEQ ID NO: 3; A kit for detecting Acinetobacter baumannii . The kit according to claim 1, wherein the nucleic acid probe is fluorescently labeled. Primer consisting of consecutive 16 bases or more 36 bases following nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 2, a primer having the nucleotide sequence represented by SEQ ID NO: 4, 5, or 6, according to claim 1 or 2 Kit. Any one of claims 1 to 3 , wherein the primer consisting of a base sequence of 16 to 36 bases in sequence of the base sequence shown in SEQ ID NO: 3 is a primer consisting of the base sequence shown in SEQ ID NO: 7, 8 or 9. the kit according to either. The kit according to any one of claims 1 to 4 , further comprising α-type DNA polymerase. A primer consisting of a base sequence of 16 to 36 bases in a continuous sequence of the base sequence shown in SEQ ID NO: 2 and a primer consisting of a base sequence of 16 to 36 bases in a sequence of the base sequence shown in SEQ ID NO: 3. Using to amplify the nucleic acid in the sample; and
It consists of all or part of the base sequence shown in SEQ ID NO: 10, consists of 14 to 25 nucleotides, and is designed to recognize the 107th and 108th bases of the base sequence shown in SEQ ID NO: 1. a nucleic acid probe as engineering of binding to nucleic acids was amplified
A method for detecting Acinetobacter baumannii International clone Type II. Amplification of nucleic acid is performed using the α-type DNA polymerase The method according to claim 6.