KR101518561B1 - Development of species-specific PCR method for detection of Acinetobacter species - Google Patents

Abstract

The present invention relates to a primer, a probe, and a method for detecting an Ashtonobacter species using the same. More specifically, the present invention relates to a first primer pair consisting of a base sequence of SEQ ID NOS: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10; A sixth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12; A seventh primer pair consisting of the nucleotide sequences of SEQ ID NOS: 13 and 14; An eighth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16; A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And a tenth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20, and a primer capable of specifically detecting an Acinetobacter species including a pair of primers selected from the group consisting of a pair of primers consisting of a pair of primers consisting of a nucleotide sequence of SEQ ID NOs: The present invention relates to a method for detecting a fungus species. The primer for detecting Asnithobacter according to the present invention and the method for detecting Asnithobacter using the primer include Acinetobacter calcoaceticus), Acinetobacter baumannii (A. baumannii), Petey Acinetobacter (A. pittii), Acinetobacter au Alice Cormier (A. nosocomialis), Acinetobacter Earl destination (A. ursingii), Acinetobacter A. lwoffii , A. bereziniae , A. haemolyticus , A. parvus and A. cinereus ( A. there is a very useful effect in the presence or absence and species name to be quickly and accurately to the specific schindleri).

Description

[Background Art] [0002] Development of a species-specific PCR technique for the detection of an Acinetobacter species [

The present invention relates to a species specific detection primer for identification of an Acinetobacter species, and to a method for specific PCR of an Acinetobacter species using the primer.

Acinetobacter fungi are widely prevalent in natural environments such as soil and water. Especially in patients who have been living in a hospital environment for a long time and have weak immunity, Acinetobacter species are found in conjunctivitis, dermatitis, pneumonia, meningitis, sepsis, endocarditis, intraperitoneal infection, Infection, and urinary tract infection.

In the Acinetobacter sp . There are 27 genotypes assigned to species names and 11 genomic species that can be distinguished by DNA-DNA hybridization method. A. calcoaceticus (genospecies type 1), A. baumannii (genospecies type 2), A. pittii (genospecies type 3) and A. nosocomialis (genospecies 13 TU) are genetically closely related and routinely used in laboratories It is difficult to distinguish between phenotypic methods. Thus, these species have been referred to as A. calcoaceticus - A. baumannii complex (ACB complex).

Other species than A. baumannii are thought to be involved in hospital-acquired infections, but their role is still unclear. Although these strains are usually susceptible to a large number of antibiotics, they must be distinguished from A. baumannii strains in order to investigate their frequency and role as pathogenic organisms.

Biochemical and automated microbiological identification devices such as API 20NE, Vitek 2, Phoenix and MicroScan Walk-Away, which are commonly used in routine laboratories, can not distinguish these species, and 25% of the Acinetobacter strains belonging to the ACB complex belong to A. baumannii I am wrongly sympathized.

Due to the limitations of this phenotypic identification method, various molecular biological species identification methods have been developed. Among them, molecular biological techniques such as DNA-DNA hybridization and amplified rRNA restriction analysis (ARDRA) are labor-intensive, difficult to read, and rarely used on a daily basis. Recently, identification of the Acinetobacter strain as a species level has been proposed using the sequencing of the RNA polymerase beta-subunit gene ( rpoB ), flanking spacer regions, and 16S-23S rRNA gene spacer regions.

Although one of the methods most commonly being used to discriminate differences of homology and 16S rDNA sequence of this strain, though 16S rDNA all Acinetobacter It is not polymorphic enough to clearly distinguish species. Acinetobacter , including A. baumannii Since genomic species are increasingly important pathogens, recent studies have focused on the development of reliable identification methods. rpoB sequence is considered to be one of the most useful tools for taxonomic classification and identification of various bacterial species including Acinetobacter species.

Therefore, the resolution of the rpoB sequence is high, but sequences because the analysis of difficulties routinely used in hospital laboratories technique species from the rpoB gene devise specific PCR primers to quickly and Acinetobacter species that are commonly isolated from clinical, PCR techniques, The present invention has been completed in order to easily detect the species level.

Korea Patent No. 10-2008-0040773

Accordingly, it is an object of the present invention to provide a primer and a probe for detecting an Ashentai bacterium designed to effectively detect an Ashantobar species.

It is another object of the present invention to provide a microarray for detecting Asnithobacteria characterized in that the probe is integrated on a substrate.

It is another object of the present invention to provide a method for detecting an Asn, S. bacterium using the primer or probe.

In order to accomplish the object of the present invention as described above, the present invention provides a primer comprising a first primer pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4; A third primer pair consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10; A sixth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12; A seventh primer pair consisting of the nucleotide sequences of SEQ ID NOS: 13 and 14; An eighth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16; A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And a tenth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20, and a pair of primers selected from the group consisting of SEQ ID NOS: 19 and 20.

In one embodiment of the present invention, the first primer pair Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus , wherein the second pair of primers is a primer for amplifying DNA of Acinetobacter baumannii , and the third pair of primers is DNA of Acinetobacter pittii Wherein the fourth primer pair is an Acinetobacter and primers for amplifying the DNA of nosocomialis), the DNA of said fifth pair of primers is a primer for amplifying the DNA of the Acinetobacter ropi (Acinetobacter lwoffii), the sixth primer pair Acinetobacter Earl destination (Acinetobacter ursingii) and primers for amplifying the seventh pair of primers is a primer for amplifying the DNA of the Acinetobacter Beretta jiniah (Acinetobacter bereziniae), the eighth primer pair Acinetobacter by Mori T carcass (Acinetobacter and primers for amplifying the DNA of haemolyticus), the ninth pair of primers is a primer for amplifying the DNA of the Acinetobacter Parr booth (Acinetobacter parvus), the primer pair of claim 10 Acinetobacter swikdeul Larry (Acinetobacter Schindler 's DNA.

In one embodiment of the present invention, the first primer pair is a primer capable of amplifying at least 200 pg of DNA of Acinetobacter calcoaceticus , and the second pair of primers is a primer capable of amplifying at least 200 pg of DNA of Acinetobacter calcoaceticus , Wherein the third primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter baumannii and the third primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter pittii , A primer capable of amplifying at least 20 pg of DNA of Acinetobacter nosocomialis and a fifth primer pair capable of amplifying at least 200 pg of DNA of Acinetobacter lwoffii , The sixth primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter ursingii , The ema pair is a primer capable of amplifying at least 200 pg of DNA of Acinetobacter bereziniae and the eighth primer pair is Acinetobacter haemolyticus DNA, wherein the ninth primer pair is a primer capable of amplifying at least 2 ng of DNA of Acinetobacter parvus , and the tenth primer pair is a primer capable of amplifying at least 2 pg of DNA of Acinetobacter parvus , Acinetobacter Schindler 's DNA can be amplified by at least 20 pg. The primer can be a primer for detecting Acinetobacter species.

The present invention provides a microarray for detecting an Acinetobacter bacterium whose primer or probe is immobilized on a substrate.

In addition, the present invention provides a method for detecting a strain of Ashitto genotype comprising the step of detecting an Ashtonobacter species by a polymerase chain reaction (PCR) using a primer for detecting the Ashitto genotype.

In one embodiment of the present invention, the Acinetobacter species is Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter baumannii (A. baumannii), Petey Acinetobacter (A. pittii), Acinetobacter au Alice Cormier (A. nosocomialis), Acinetobacter Earl destination (A. ursingii), Acinetobacter A. lwoffii , A. bereziniae , A. haemolyticus , A. parvus and A. cinereus ( A. schindleri ). < / RTI >

In one embodiment of the present invention, the sensitivity of the polymerase chain reaction (PCR) may be between 98% and 100%.

Acinetobacter (Acinetobacter) species according to the invention-specific primer pairs are Acinetobacter species species-specific as there is an effect that it is possible to accurately and rapidly identified and detected, and high sensitivity to the hospital pathogen of Acinetobacter It can be used conveniently and conveniently in a hospital clinical laboratory.

Figure 1 shows A. calcoace t icus Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Figure 2 shows A. baumannii Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
3 shows A. pittii Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Fig. 4 shows the results of A. nosocomialis Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
5 shows A. ursingii Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Figure 6 shows A. lwoffii Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Figure 7 A. bereziniae Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Fig. 8 shows the expression of A. haemolyticus Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Figure 9 shows A. parvus Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
Figure 10 A. schindleri Lt; RTI ID = 0.0 > PCR-specific < / RTI > primers.
11 is a diagram showing a specific PCR result of A. calcoaceti cus.
12 is a diagram showing A. baumannii- specific PCR results.
13 is a diagram showing A. pittii- specific PCR results.
Fig. 14 shows the results of A. nosocomialis- specific PCR.
15 shows the results of specific PCR using A. usingii .
16 is a diagram showing A. lwoffii- specific PCR results.
17 is a diagram showing the A. bereziniae specific PCR result.
18 is a diagram showing the results of A. haemolyticus- specific PCR.
19 is a diagram showing A. parvus- specific PCR results.
20 is a diagram showing A. schindler specific PCR results.
21 shows the result of gel electrophoresis of PCR products obtained by amplifying step-diluted A. calcoaceticus with A. calcoaceticus - specific PCR (lanes 1 to 7) and gyrB Multiplex 2 PCR (lanes 8 to 14) Fig. Where lane M is the molecular weight marker (100 bp ladder).
22 shows the result of gel electrophoresis of PCR products obtained by amplifying step-diluted A. baumannii with A. baumannii - specific PCR (lanes 1 to 7) and gyrB Multiplex 1 PCR (lanes 8 to 14) Fig. Where lane M is the molecular weight marker (100 bp ladder).
Figure 23 shows the result of gel electrophoresis of the PCR product amplified with A. pittii - specific PCR (lane 1 through 7) and gyrB Multiplex 2 PCR (lanes 8-14 ) with step diluted A. pittii Fig. Where lane M is the molecular weight marker (100 bp ladder).
24 shows the result of gel electrophoresis of the PCR product obtained by amplifying step-diluted A. nosocomialis with A. nosocomialis - specific PCR (lanes 1 to 7) and gyrB Multiplex 1 PCR (lanes 8 to 14) Fig.
25 is a diagram showing the result of gel electrophoresis of a PCR product obtained by amplifying step-diluted A. u r singii with A. u r singii - specific PCR (lanes 1 to 7). Where lane M is the molecular weight marker (100 bp ladder).
26 is a graph showing gel electrophoresis results of a PCR product obtained by amplifying step-diluted A. lwoffii with A. lwoffii -specific PCR (lanes 1 to 7). Where lane M is the molecular weight marker (100 bp ladder).
FIG. 27 is a graph showing gel electrophoresis results of a PCR product obtained by amplifying step-diluted A. bereziniae with A. bereziniae -specific PCR (lanes 1 to 7). Where lane M is the molecular weight marker (100 bp ladder).
FIG. 28 is a diagram showing gel electrophoresis results of PCR products obtained by amplifying step-diluted A. haemolyticus with A. haemolyticus -specific PCR (lanes 1 to 7). FIG. Where lane M is the molecular weight marker (100 bp ladder).
FIG. 29 is a diagram showing gel electrophoresis results of a PCR product obtained by amplifying stepwise diluted A. parvus with A. parvus -specific PCR (lanes 1 to 7). FIG. Where lane M is the molecular weight marker (100 bp ladder).
30 shows a result of gel electrophoresis of PCR products obtained by amplifying step-diluted A. schindleri with A. schindleri -specific PCR (lanes 1 to 7). Where lane M is the molecular weight marker (100 bp ladder).

The present invention has devised a primer and a probe capable of specifically detecting a susceptible species of an Asnithobacter sp. That is a pathogenic infection to a hospital, and developed a method for detecting an Asnithobacter species using the primer or the probe.

Therefore, the present invention provides a primer capable of detecting seven strains of Acinetobacter species. Specifically, the present invention provides a primer pair comprising a first primer pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2; A second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4; a third primer pair consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6; A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8; A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10; A sixth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12; A seventh primer pair consisting of the nucleotide sequences of SEQ ID NOS: 13 and 14; An eighth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16; A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And a tenth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20, and a pair of primers selected from the group consisting of SEQ ID NOS:

The invention to the target in the present invention, Acinetobacter species is Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter baumannii (A. baumannii), Acinetobacter repetition (A. pittii), Acinetobacter au Cormier A. nosocomialis , A. ursingii , A. lwoffii, A. bereziniae , A. arnitoba , and A. auris . haemolyticus , A. parvus , and A. schindleri species.

The primer for detecting an Asnithobacter species according to the present invention is a primer that can be complementarily hybridized to a gene of a corresponding Asnithobacter species. Preferably, the primer of the Asnithobacter species is amplified Lt; / RTI >

The term primer in the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, a short nucleic acid capable of forming a base pair with a complementary template and serving as a starting point for template strand duplication ≪ / RTI > That is, the primers can be primers that are capable of initiating template-directed DNA synthesis under appropriate conditions in suitable buffers (e. G., Four other nucleoside triphosphates and polymerases such as DNA, RNA polymerase or reverse transcriptase) Refers to single stranded oligonucleotides.

In addition, the primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such primers may also be modified (e. G., Added, deleted, substituted) using a number of means known in the art within the scope of not affecting the species-specific detection of the Acinetobacter strain, It need not be completely complementary, but it should be sufficiently complementary to hybridize with the template. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid, The nucleic acid may be a nucleic acid or a nucleic acid that contains one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. The nucleic acid sequences of the present invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

In the present invention, the primer capable of detecting the Ashinomar fungus species is Acinetobacter < RTI ID = 0.0 > calcoaceticus), Acinetobacter baumannii (A. baumannii), Petey Acinetobacter (A. pittii), Acinetobacter au Alice Cormier (A. nosocomialis), Acinetobacter Earl destination (A. ursingii), Acinetobacter A. lwoffii , A. bereziniae , A. haemolyticus , A. parvus and A. cinereus ( A. . schindleri) may be a primer for specific amplification of a particular region of DNA of a species. In addition, the primer of the present invention may be a pair of forward and reverse primers having 10 to 40, preferably 20 to 30 nucleotide sequences complementary to the gene.

More preferably, the primer of the present invention comprises a first primer pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2, a second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4, a nucleotide sequence of SEQ ID NOS: 5 and 6 A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8, a fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10, a sixth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12, An eighth primer pair consisting of a base sequence of SEQ ID Nos: 15 and 16; a seventh primer pair consisting of SEQ ID NOS: 13 and 14; A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And a tenth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20, respectively.

Specifically, A. calcoaceticus has a pair of primers having the nucleotide sequences of SEQ ID NOS: 1 and 2, A. baumannii has a pair of primers having the nucleotide sequences of SEQ ID NOS: 3 and 4, and A. pittii has the nucleotide sequences of SEQ ID NOS: 5 and 6 A. nosocomialis is a pair of primers having the nucleotide sequences of SEQ ID NOS: 7 and 8, A. lwoffii is a pair of primers having the nucleotide sequences of SEQ ID NOS: 9 and 10, A. ursingii is a pair of primers having the nucleotides of SEQ ID NOS: 11 and 12 A. bereziniae is a pair of primers having the nucleotide sequences of SEQ ID NOs: 13 and 14, A. haemolyticus is a pair of primers having the nucleotide sequences of SEQ ID NOs: 15 and 16, A. parvus is a pair of primers having the nucleotide sequences of SEQ ID NOs: A pair of primers having the nucleotide sequences of SEQ ID NOS: 17 and 18, A. schindleri A specific region of the gene can be specifically amplified by a pair of primers having the nucleotide sequences of SEQ ID NOS: 19 and 20, respectively.

According to one embodiment of the present invention, in the pair of primers for detecting Escherichia coli species, the first primer pair Acal-F and Acla-R consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2 described below are specific for A. calcoaceticus And the specific gene was amplified to about 549 bp.

(Acal-F): 5-TCG TAT CTC AAT TAC ACC GTT CAC CT-3 '

SEQ ID NO: 2 (Acal-R): 5-CGC CTT CTG CCA GTT TCA CCA TA-3 '

In addition, the second primer pair Abau-F and Abau-R consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 described below was designed to specifically detect A. baumannii and amplified the specific gene to about 529 bp.

SEQ ID NO: 3 (Abau-F): 5-CAT AAA CTG AGA TAA CTG GCT TGA ACC-3 '

SEQ ID NO: 4 (Abau-R): 5-CGT CGA CTT CAC CTT TAC CGT TAC-3 '

In addition, the third primer pair Apitti-F / Apitti-R consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6 described below was designed to specifically detect A.pitti and amplified the specific gene to about 147 bp.

SEQ ID NO: 5 (Apitti-F): 5-TGG GCA GTT ACC AGA TTG ACC TA-3 '

SEQ ID NO: 6 (Apitti-R): 5-AAC CAG CAG CTT CCA TTT GAC G-3 '

In addition, the fourth primer pair Anosoco-F / Anosoco-R consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8 described below was designed to specifically detect A. nosocomialis and amplified the specific gene to about 394 bp.

Anosoco-F: 5-GCC GCT CGT GTA CGT GTA ATC-3 '

(Anosoco-R): 5-CAT CGT GTG GCA TAT CTT CAA C-3 '

In addition, the fifth primer pair Alwoffii-F / Alwoffii-R consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10 described below was designed to specifically detect A. lwoffii and amplified the specific gene to about 302 bp.

SEQ ID NO: 9 (Alwoffii-F): 5-CCG TGT CGG TCT GGT TCG TGT A-3 '

SEQ ID NO: 10 (Alwoffii-R): 5-CCG GCG TTT CAA TTG GAC ATA C-3 '

In addition, the sixth primer pair Aursingii-F / Aursingii-R consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12 described below was designed to specifically detect A. ursingii and amplified the specific gene to about 319 bp.

Aursingii-F: 5-AGC GTG TCA TCG TAT CTC AG-3 '

SEQ ID NO: 12 (Aursingii-R): 5-CCG CGT AAA CGT TCA GGC ACA AGA-3 '

In addition, the seventh primer pair Abere-F / Abere-R consisting of the nucleotide sequences of SEQ ID NOS: 13 and 14 described below was designed to specifically detect A. bereziniae and amplified the specific gene to about 539 bp.

SEQ ID NO: 13 (Abere-F): 5-ACG GTT CTC CGG GCG TCT-3 '

SEQ ID NO: 14 (Abere-R): 5-AAC ACC ACC TTC AGC GAT TTT A-3 '

In addition, the eighth primer pair Ahaemo-F / Ahaemo-R consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16 described below was designed to specifically detect A. haemolyticus and amplified the specific gene to about 584 bp.

SEQ ID NO: 15 (Ahaemo-F): 5-TGC ACA GCA GGT AGT ATC A-3 '

SEQ ID NO: 16 (Ahaemo-R): 5-GTA ATT TCT TCT GCA CCT AA-3 '

In addition, the ninth primer pair Apar-F / Apar-R consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18 described below was designed to specifically detect A. parvus and amplified the specific gene to about 239 bp.

SEQ ID NO: 17 (Apar-F): 5-CAT GGT GAA GAT CGC TGA AAA-3 '

SEQ ID NO: 18 (Apar-R): 5-CCC ACT GGA GAA AGG TCA TAA C-3 '

In addition, the tenth primer pair Aschin-F / Aschin-R consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20 described below was designed to specifically detect A. schindleri and amplified the specific gene to about 480 bp.

SEQ ID NO: 19 (Aschin-F): 5-ATC GCG GAT ACA TTA CGT GCC-3 '

SEQ ID NO: 20 (Aschin-R): 5-CCA CTG GCT TCG CAT TGA TT-3 '

Thus, the pair of primers for detecting the Acinetobacter species of the present invention can be classified into 10 kinds of strains of Acinetobacter calcoaceticus , A. baumannii , A. pittii , A. nosocomialis , A. urisii , A. lwoffii, A. bereziniae , A. . haemolyticus, A. parvus And A. schindleri Not only do they have specificity for each species, but also specific genes can be amplified to different sizes, so that each of the 10 strains of Asnithobacter can be detected. Furthermore, the primer of the present invention can be effectively used for the detection of an infection with Ashenovo bacterium, identification of an infectious species of Asnithobacterium, investigation of an epidemic of an Acinetobacter strain, efficacy and risk analysis of an Ashtonobacter vaccine.

In addition, the present invention provides a probe capable of detecting an Asnithobacter species.

The probe capable of detecting the Acinetobacter species according to the present invention preferably comprises a first primer pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2, a second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4, 5 and 6, a fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8, a fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10, a third primer pair consisting of the nucleotide sequences of SEQ ID NOS: A seventh primer pair consisting of the nucleotide sequence of SEQ ID NOS: 13 and 14; an eighth primer pair consisting of the nucleotide sequence of SEQ ID NOS: 15 and 16; A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And a tenth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20, respectively.

Also, the first primer pair is a primer for amplifying A. calcoaceticus DNA, the second primer pair is a primer for amplifying A. baumannii DNA, and the third primer pair is a primer for amplifying A. pittii DNA Wherein the fourth primer pair is a primer for amplifying DNA of A. nosocomialis , the fifth primer pair is a primer for amplifying DNA of A. lwoffii , and the sixth primer pair is amplifying DNA of A. ursingii Wherein the seventh primer pair is A. bereziniae , the eighth primer pair is A. haemolyticus , the ninth primer pair is A. parvus , and the tenth primer pair is a primer for amplifying A. schindleri DNA .

In the present invention, the term " probe " means a nucleic acid fragment corresponding to several to several hundred bases capable of specifically binding to DNA or RNA. Also, the probe is labeled, The presence or absence of existence can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a short-chain DNA probe, a double-stranded DNA probe, an RNA probe, etc., and may be labeled with biotin, FITC, rhodamine, DIG or the like or labeled with radioactive isotopes.

The present invention also provides a microarray for detecting an Escherichia coli strain, wherein the probe or primer of the present invention is immobilized on a substrate.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the probe or primer contains the polynucleotide of the present invention.

Methods for preparing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art.

In the present invention, the term " probe polynucleotide " means a polynucleotide capable of hybridization, and means an oligonucleotide capable of specifically binding to a complementary strand of a nucleic acid. Such probes can include the peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991).

Immobilization of the probe polynucleotide associated with the Acinetobacter strain according to the present invention on a substrate can also be easily performed using conventional techniques. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. Such detection may be achieved, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, for example, a substance such as Cy3 and Cy5, and then hybridizing on the microarray, The hybridization result can be detected by detecting the generated signal.

In addition, the present invention provides a method of detecting an Ashtonobacter species using a primer or a probe for detecting an Ashtona lacticum species according to the present invention.

Examples of the method for detecting the Acinetobacter species of the present invention include, but are not limited to, a multiplex polymerase chain reaction, a polymerase chain reaction, a reverse transcription polymerase chain reaction Reverse transcriptase PCR), DNA sequencing, hybridization by microarray, restriction fragment length polymerase (PCR), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF) arbitrarily primed PCR, sequence tagged site (EST), expressed sequence tag (EST), sequence characterized amplified regions (SCAR), inter-simple sequence repeat amplification (ISSR), amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence, single-strand conformation polymorphism (PCR-SSCP), Northern blot and Southern blot. .

In the present invention, the term polymerase chain reaction (PCR) is one of representative nucleic acid amplification techniques (NAT) in which a specific DNA region is amplified in vivo using an enzyme in a desired portion. It was developed in 1985 by Mullis et al., And any part of the DNA molecule can be amplified by this method if it knows its boundary sequence. PCR is basically composed of three steps of denaturation, binding, and extension, and DNA is amplified by repeating this process. The denaturation step, which is the first step of the PCR, denatures the template DNA into a single strand, and the second step, the binding step, binds the two types of single stranded DNAs between the desired regions. In the third step, the synthesis step, the DNA is synthesized from the primer by a high-temperature resistant DNA polymerase, and the above cycle is repeated 25 to 30 times.

A primer or a probe which can be used in the method of detecting an Acinetobacter species of the present invention comprises a first pair of primers for detection of A. calcoaceticus comprising the nucleotide sequences of SEQ ID NOS: 1 and 2; A second primer pair for detection of A. baumannii consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4; A third primer pair for A. pittii detection consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6; A. nosocomialis consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8 A fourth primer pair for detection; A fifth primer pair for A. lwoffii detection consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10; A sixth primer pair for detection of A. ursingii consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12; A bereziniae comprising the nucleotide sequence of SEQ ID NOs: 13 and 14 A seventh pair of primers for detection; A. haemolyticus consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16 An eighth primer pair for detection; A ninth primer pair for the detection of A. parvus consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; A. schindleri consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20 A tenth primer pair for detection, and a tenth primer pair for detection.

Specifically, in one embodiment of the present invention, a method of specifically detecting ten species of the Acinetobacter strain by the PCR method using the primer according to the present invention is exemplified. Acinetobacter calcoaceticus , A. baumannii , A. pittii , A. nosocomialis , A. lwoffii , A. urisii, A. bereziniae , A. haemolyticus , A. parvus And A. schindleri Ten pairs of Asnithobacter species can be specifically detected using a pair of primers capable of amplifying 10 species-specific DNA sequences.

In addition, when the PCR is carried out using the primer of the present invention, the species specificity for discriminating the Ashinotobacter species and the sensitivity of the PCR detection are high, so that the diagnostic value can be enhanced.

In one embodiment of the present invention, Acinetobacter As a result of the sensitivity of the PCR for the species-specific PCR, A. baumannii The sensitivity of the PCR-only PCR specificity was 98.86% and the sensitivity of the other PCRs was 100%.

In the present invention Acinetobacter baumannii Anosoco-F / Anosoco-R primers that differentiate between Abau-F / bau-R and Acinetobacter nosocomialis isolates are able to detect up to 10 times less gemnomic DNA than the gyrB Multiplex 1 PCR primers designed by Higgins et al. (2007) Acal-F / Acal-R and Acinetobacter, which differentiate Acinetobatec calcoaceticus species. The Apittii -F / Apittii-R primer, which differentiates pittii species, was able to detect 10 times less gemnomic DNA than the gyrB Multiplex 2 PCR primer designed by Higgins et al. (2010).

Acinetobacter Acinetobacter Calcoaceticus (type 1) and A. pittii (type 3) are similar, and Acinetobacter baumannii (type 2) and A. nosocomialis (type 13) are similar and there is a limit to biochemically differentiate. Acinetobacter , currently isolated from hospital patients Species are determined mainly by Vitek GNI card or MicroScan or commercial kit. Identification by phenotypic and biochemical characteristics has limitations on identification of genotypes having almost the same characteristics. These commercialized biochemical microbial identification kits include Acinetobacter A. calcoaceticus - Acinetobacter , belonging to the A. baumannii complex (ACB complex) The strains often fail to isolate and identify correctly. Many researchers have recognized the limitations of automatic microbial synergism and have used Acinetobacter genospecies.

Dolzani et al. Used the restriction fragment length polymorphism (RFLP) method. The PCR amplification products were treated with various restriction enzymes and reacted for 4 hours. There are ineffective drawbacks. Vaneechoutte et al. Used the amplified ribosomal DNA-restriction analysis (ARDRA) method because of the complexity of comparing PCR products with several restriction enzymes after two hours of reaction, Has ineffective disadvantages over PCR in terms of time, labor, and economy. Koeleman et al. Used Random Amplified Polymorphic DNA (RAPD) assay, which is used for epidemiologic studies of bacteria. The Acinetobacter spp. Differed from the others in that it was very dense and difficult to distinguish. Especially, too many subtypes were found. There is a limit to the rapid identification of Acinetobacter species due to the complexity of comparing these strains with the standard strains. The Acinetobacter species-specific PCR of the present invention is a more effective method because it can detect and identify the species by a single experiment.

In addition, according to an embodiment of the present invention, the lowest detection limit can be obtained, and the accuracy of the detection of the strain of Asnstor bacterium can be improved.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not limited by the following examples.

< Example  1>

Target strain and strain culture

In the present invention, the 34 strains of the standard strain and the reference strains and 382 strains of the clinical isolates were studied. Among the 34 strains, the standard strains and reference strains are as follows. Acinetobacter calcoaceticus KCTC 2357, Acinetobacter baumannii KCTC 2508, Acinetobacter haemolyticus KCTC 12404, Acinetobacter johnsonii KCTC 12405, Acinetobacter junii KCTC 12406, Acinetobacter lwoffii KCCM 40172, Acinetobacter bereziniae KCTC 12683, Acinetobacter guillouiae KCTC 12684, Acinetobacter soli KCTC 22184, Acinetobacter baylyi KCTC 12413, Acinetobacter tandoi KCTC 12417, Acinetobacter parvus KCTC 12408, Acinetobacter ursingii KCTC 12410, Acinetobacter schindleri KCTC 12409, Acinetobacter grimontii KCTC 12416, Acinetobacter pittii CCUG 61664, Acinetobacter nosocomialis CCUG 61663 , Acinetobacter gyllenbergii CCUG 51248, Acinetobacter beijerinckii CCUG 51249, Vibrio fluvialis KCCM 40827, Vibrio vulnificus KCCM 41665, Vibrio mimicus KCCM 42257, Staplylococcus epidermidis KCTC 1917, Staplylococcus aureus KCTC 29213, Staplylococcus pyogenes KCTC 3208, Aeromonas hybrophila KCTC 2358, Shigella sonnei KCTC 2518, Salmonella pneumoniae KCTC 1925, Enterococcus casseliflavus ATCC 700327, Enterococcus faecalis ATCC 29212, Klebsiella oxytoca ATCC 700324, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922. Clinical isolates were identified as Acinetobacter species from Vitek 2 GNI card (BioMeriex Vitek Inc., Hazelwood, Mo., USA) among strains isolated from patient samples from Chosun University Hospital from April 2004 to April 2012 The clinical isolates were 382 strains. Standard strains, reference strain and clinical isolates of Acinetobacter species were inoculated on LB broth and cultured at 37 ℃ for 24 hours.

< Example  2>

The bacterial genome ( genomic ) DNA Extraction of

1.5 ml of bacterial culture was harvested using centrifugal force of 10,000 x g, and G-spin ^TM Bacterial genomic DNA was extracted using genomic DNA Extraction Kit (iNtRON Co., Seoul, Korea) according to the manufacturer's instructions. In other words, 50 μl of pre-incubation solution and 3 μl of lysozyme solution were added to harvested bacteria, mixed well and incubated at 37 ° C for 1 hour. Add 250 μl of G-buffer solution, mix well, incubate at 65 ° C for 15 minutes, add 250 μl of binding solution, mix well and vortex. These cell lysates were placed in a G-spin ^TM column and centrifuged at 10,000 xg for 1 min. 500 μl of washing buffer A was added to the column and centrifuged again for 1 minute. After adding 500 μl of washing buffer B for 1 min, the G-spin ^™ column was placed in a new eppendorf tube, and 100 μl of elution buffer was added. The mixture was allowed to stand at room temperature for 1 min and then centrifuged at 10,000 × g for 1 min Bacterial genomic DNA was extracted and stored at 4 ° C for subsequent experiments.

< Example  3>

Using molecular biology Of clinical isolates  Sympathy

Vitek GNI card, 293 isolates identified as Acinetobacter species were re - identified by molecular biology. In other words, gyrB Multiplex 1 PCR (amplification product 294 bp, 490 bp) was performed to amplify a part of gyrB gene from each clinical isolate. The primers used were sp2F (5-GTT CCT GAT CCG AAA TTC TCG-3), sp4F (5-CAC GCC GTA AGA GTG CAT TTA-3) and sp4R (5-AAC GGAGCT TGT CAG GGT TA- The PCR conditions were as follows. The initial denaturation was carried out at 94 ° C for 2 minutes and three cycles of denaturation (94 ° C for 1 minute), binding (60 ° C for 1 minute) and extension (72 ° C for 1 minute) 72 ° C, 7 minutes) was performed. The strains identified as A. baumannii in the above gyrB Multiplex 1 PCR result are not further tested and are not A. baumannii Acinetobacter The strains were tested for the following. PCR was performed to amplify 16S rDNA (about 1.4 kbp) and a portion of the rpoB gene (350 bp). The primers used for the amplification of 16S rDNA were 27F (5-AGA GTT TGA TCM TGG CTC AG-3) and 1492R (5-TAC GGY TAC CTT GTT ACG ACT T-3). The initial denaturation was carried out at 94 ° C for 5 minutes and three cycles of denaturation (94 ° C for 1 minute), binding (55 ° C for 30 seconds) and elongation (72 ° C for 45 seconds) were repeated 30 times, 72 ° C, 10 min) was performed. The primers used for amplification of the rpoB gene were 696F (5-TAY CGY AA-GAY TTG AAA GAA G-3) and 1093R (5-CMA CAC CYT TGT TMC CRT GA-3). The PCR conditions were as follows. The initial denaturation was carried out at 94 ° C for 5 minutes and three cycles of denaturation (94 ° C for 30 seconds), binding (56 ° C for 30 seconds), and elongation (72 ° C for 1 minute) 72 ° C, 10 min) was performed. PCR amplification products were subjected to nucleotide sequence analysis by Solent Co., Daejeon, Korea using Dye Terminator Cycle Sequencing Kit and ABI PRISM 3730 DNA analyzer (PE Applied Biosystems, Foster, CA, USA). These analyzed nucleic acid sequences were used to determine bacterial species using the Blast search program provided by the National Institutes of Health. At this time, the existing Acinetobacter spp. The isolates were identified as having the highest homology with the standard strain with> 98% homology.

In the present invention, 293 strains isolated from hospital patients were identified as ACB complex or Acinetobacter spp . Through Vitek GNI card. GyrB Multiplex 1 PCR was performed and 210 strains were identified as A. baumannii It appeared positive and was identified as A. baumannii . The remaining 83 were gyrB Multiplex 1 PCR A. baumannii After 16 s rDNA PCR and rpoB PCR were performed, the amplified products were submitted to Solgent company for sequencing analysis. The sequence was analyzed by NCBI or EZTAXON database. As a result, 62 nosocomials , A. pittii 4 weeks, A. calcoaceticus 1 week , A. lwoffii 3 weeks, A. bereziniae 3 weeks, A. guillouiae 3 weeks, A. junii 4 weeks, A. johnsonii 2 weeks, A. radioresistens 1 week (Table 1, 2). In Table 1, Neg means PCR negative.

Further , 89 strains of clinical isolates identified by gastric molecular biology ( gyrB Multiplex 1 PCR, 16S rDNA PCR, and rpoB 350 PCR) were used by the present inventors. The 89 species of A. baumannii , A. calcoaceticus , A. pittii , A. nosocomialis , A. bereziniae , A. gyllenbergii , A. junii , and A. junii were 54 , A. ursingii is one week (see Table 2).

< Example  4>

Polymerase chain reaction Primer  Design and production

In the database of GenBank, A. calcoaceticus , A. baumannii , A. pittii , A. nosocomialis , A. urisii, A. lwoffii , A. bereziniae , A. haemolyticus , A. parvus And rpoB nucleotide sequences of A. schindleri . These nucleic acid sequences were analyzed using the MegAlign computer program (Lasergene, DNASTAR Inc., Madison, USA) and were analyzed for A. calcoaceticus , A. baumannii , A. pittii , A. nosocomialis, A. urisii , A. lwoffii , A. bereziniae , A. haemolyticus , A. parvus And A. schindleri 's rpoB Based on the nucleotide sequence, and using the PrimerSelect program (DNASTAR Inc.) A. calcoaceticus, A. baumannii, A. pittii, A. nosocomialis, A. urisingii, A. lwoffii, A. bereziniae, A. haemolyticus, A. parvus And by A. each design a PCR primer pair capable of detecting schindleri Acal-F and R-Acal, Abau-F and R-Abau, Apittii-F and R-Apittii, Aconoso-F and R-Aconoso, Aursingii- F and Aursingii-R, Alwoffii-F and Alwoffii-R, Abere-F and Abere-R, Ahaemo-F and Ahaemo-R, Apar-F and Apar-R and Aschin-F and Aschin- 3 and 4). The primer pairs designed at this time were assigned to Bioneer Company (Daejeon, Korea) to produce primer pairs. The expected sizes of the PCR amplification products are 549 bp, 529 bp, 147 bp, 394 bp, 319 bp, 302 bp, 539 bp, 584 bp, 239 bp and 480 bp, respectively (see Tables 3 and 4).

< Example  5>

Acinetobacter  Species-specific Polymerase chain reaction Primer  Species-Specificity and Sensitivity Measurement

A. calcoaceticus , A. baumannii , A. pittii , A. nosocomialis , A. urisingii, A. lwoffii , A. bereziniae The strains used to determine the species-specificity and sensitivity of Acinetobacter species-specific PCR primers for detection and identification are as follows. 19 strains belonging to the genus Acinetobacter and 15 strains belonging to the genus other than Acinetobacter , and 293 strains identified by the molecular biology identification method in this study, and the clinical isolates identified by the molecular biology method in the previous study 445 weeks were used.

A. baumannii -specific PCR, A. calcoaceticus -specific PCR, A. pittii -specific PCR, and A. nosocomialis -specific PCR were used to measure 445 strains and to measure species-specificity and sensitivity. However, A. lwoffii -specific PCR and A. ursingii -specific PCR, A. bereziniae -specific PCR is species-specificity and clinical isolates can be measured when the sensitivity instead of 382 weeks, 41 weeks and type strain or strain collections of reference strains representing each strain 34 weeks) were evaluated.

A. haemolyticus -specific PCR , A. parvus -specific PCR And A. schindleri -specific PCR When species-specificity and sensitivity are measured, There were no positive clinical isolates and 29 strains were tested and evaluated. Acinetobacter 22 strains belonging to the genus, Acinetobacter . A total of 29 strains were used, including 7 strains of reference strains or reference strains belonging to other genus.

Gradient PCR was performed with Acinetobacter species-specific PCR primers to determine the optimal temperature for each PCR. The optimal binding temperature was selected after PCR was performed at 12 different binding temperatures by evenly distributing the temperature range from 52 ° C to 72 ° C. PCR reaction mixture was prepared by adding 2μl of template DNA, 1μl of forward primer (10pM / μl) and 1μl of reverse primer (10pM / μl) to AccuPower PCR PreMix (Bioneer, Daejeon, Korea) and mixing with distilled water. AccuPower PCR PreMix contains 5 nmoles of 4 deoxynucleotide triphosphates, 0.8 mole of KCl, 0.2 mole of Tris-HCl (pH 9.0), 0.03 mole of MgCl _2, and 1 unit of Taq polymerase. PCR reaction mixture was performed under the conditions shown in Tables 5 and 6 with Gene Amp PCR System 9600 (Perkin-Elmer Centus Co., CT, USA).

As a result of each PCR, Acinetobacter The sensitivity of the species-specific PCR was determined by A. baumannii The sensitivity of the PCR-only PCR specificity was 98.86% and the sensitivity of the other PCRs was 100%.

<5-1> Acal -F and Acal -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results are shown in Fig. The optimal binding temperature for the pair of Acal-F and Acal-R primers designed in the present invention is 68 ° C and the size of the amplified product is 549 bp. A. Species for calcoaceticus - specific strains of the A. calcoaceticus and Acientobacter PCR was performed on the genomic DNA of the isolates of other species and clinical isolates, and 8 out of 8 isolates of A. calcoaceticus (100%) were positive. Acientobacter other than A. calcoaceticus (100%) of the strains of 416 strains determined to be strains were negative (see Table 5, Fig. 11). 1 to 7, lane M is a 100 bp molecular weight marker and the annealing temperature of each PCR is lane 1, 52 ° C; Lane 2, 54 DEG C; Lane 3, 56 &lt; 0 &gt;C; Lane 4, 58 캜; Lane 5, 60 캜; Lane 6, 62 占 폚; Lane 7, 64 DEG C; Lane 8, 66 DEG C; Lane 9, 68 &lt; 0 &gt;C; Lane 10, 70 캜; Lane 11, 71 DEG C; Lane 12, 72 ° C. 11, lane P is a positive control of Acinetobacter calcoaceticus KCTC 2357; Lanes 1-3 are A. calcoaceticus Clinical isolates; Lane 4 is Acinetobacter baumannii KCTC 2508; Lane 5 is Acinetobacter pittii CCUG 61664; Lane 6 is Acinetobacter nosocomialis CCUG 61663; Lane 7 is Acinetobacter ursingii KCTC 12410; Lane 8 is Acinetobacter lwoffii KCCM 40172; Lane 9 is Acinetobacter bereziniae KCTC 12683 was analyzed.

<5-2> Abau -F and Abau -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results are shown in Fig. The optimal binding temperature for the Abau-F and Abau-R primer pairs designed in the present invention is 66 ° C and the exact size of the amplified product is 529 bp. As a result of evaluating the species-specificity of A. baumannii by PCR with the genomic DNA of the strains tested for 416 PCR, 262 (98.86%) of 265 strains determined to be A. baumannii were positive and 3 (1.14 %) Were negative. Acinetobacter other than A. baumannii All of the 151 strains identified as mycobacteria were negative (100%) (see Table 5, FIG. 12).

12, lane M is a marker having a molecular weight of 100 bp; Lane N is a negative control without DNA template; Lane P is Acinetobacter baumanni Positive control of KCTC 2508; Lanes 1-3 are A. baumannii Clinical isolates; Lane 4 is Acinetobacter calcoaceticus KCTC 2357; Lane 5 is Acinetobacter pittii CCUG 61664; Lane 6 is Acinetobacter nosocomialis CCUG 61663; Lane 7 is Acinetobacter ursingii KCTC 12410; Lane 8 is Acinetobacter lwoffii KCCM 40172; Lane 9 is Acinetobacter bereziniae KCTC 12683.

<5-3> Apittii -F and Apittii -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 3) show that the optimal binding temperature for the pair of Apittii-F and Apittii-R primers designed in the present invention is 68 ° C and the size of the amplified product is 147 bp. A. Species for pittii - specific strains of the A. pittii and Acientobacter PCR was carried out on the genomic DNA of the standard strains of other species and clinical isolates. Ten out of 10 strains of A. pittii (100%) were positive. As shown in Table 5, Acinetobacter other than A. pittii (100%) of the strains of 416 strains determined to be strains were negative (see Table 5, FIG. 13).

13, lane M is a marker having a molecular weight of 100 bp; Lane N is a negative control without a DNA template; Lane P is Acinetobacter pittii CCUG 61664 is a positive control; Lanes 1-3 are A. pittii Clinical isolates; Lane 4 is Acinetobacter calcoaceticus KCTC 2357; Lane 5 is Acientobacter baumannii KCTC 2508; Lane 6 is Acinetobacter nosocomialis CCUG 61663; Lane 7 is Acinetobacter ursingii KCTC 12410; Lane 8 is Acinetobacter lwoffii KCCM 40172; Lane 9 is Acinetobacter bereziniae KCTC 12683.

<5-4> Anosoco -F and Anosoco -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 4) show that the optimum binding temperature of the Anosoco-F and Anosoco-R primer pairs designed in the present invention is 66 ° C and the exact size of the amplified product is 394 bp. A. Species for nosocomialis - specific strains of the A. nosocomialis and Acientobacter PCR was performed on the genomic DNA of the standard isolates and clinical isolates of other species. As a result, 64 out of 64 isolates determined as A. nosocomialis were positive (100%). As shown in Table 5, Acinobacter other than A. nosocomialis All 352 strains were 100% negative.

14, lane M is a marker having a molecular weight of 100 bp; Lane N is a negative control without template DNA; Lane P is Acinetobacter nosocomialis Positive control of CCUG 61663; Lanes 1-3 are A. nosocomialis Clinical isolates; Lane 4 is Acinetobacter calcoaceticus KCTC 2357; Lane 5 is Acientobacter baumannii KCTC 2508; Lane 6 is Acinetobacter pittii CCUG 61664; Lane 7 is Acinetobacter ursingii KCTC 12410; Lane 8 is Acinetobacter lwoffii KCCM 40172; Lane 9 is Acinetobacter bereziniae KCTC 12683.

<5-5> Ausingii -F and Ausingii -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 5) show that the optimum binding temperature for the pair of Ausingii-F and Ausingii-R primers designed in the present invention is 68 ° C and the size of the amplified product is 319 bp. A. Species for usingii - specific strains of the A. usingii and Acientobacter PCR was performed on the genomic DNA of the standard strains of other species and clinical isolates. Two out of two strains determined as A. usingii (100%) were positive (see FIG. 15). As shown in Table 5 A. non usingii other Acientobacter All of the 73 isolates were negative (100%).

In Fig. 15, lane M indicates lane M indicates a molecular weight of 100 bp marker; Lane N is a negative control without template DNA; Lane P is Acinetobacter positive control of ursingii KCTC 12410; Lane 1 is a clinical isolate of A. ursingii ; Lane 2 is Acinetobacter calcoaceticus KCTC 2357; Lane 3 is Acientobacter baumannii KCTC 2508; Lane 4 is Acinetobacter pittii CCUG 61664; Lane 5 is Acinetobacter nosocomialis CCUG 61663; Lane 6 is Acinetobacter lwoffii KCCM 40172; Lane 7 is Acinetobacter bereziniae KCTC 12683; Lane 8 is A. junii KCTC 12406 ^T ; Lane 9 is A. guillouiae KCTC 12684.

<5-6> Alwoffii -F and Alwoffii -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (FIG. 6) show that the optimum binding temperature of the Alwoffii-F and Alwoffii-R primer pairs designed in the present invention is 66 ° C and the size of the amplified product is 302 bp. A. Species for lwoffii - specific strains of the A. lwoffii and Acientobacter PCR was performed on the genomic DNA of the isolates of other species and clinically isolated isolates. Four out of four isolates identified as A. lwoffii were positive (100%). As shown in Table 5, other non- A. lwoffii Acientobacter (100%) of the 71 strains of the strains determined to be strains were negative (see Table 5, Fig. 16).

16, lane M indicates lane M indicates a molecular weight of 100 bp marker; Lane N is a negative control without template DNA; Lane P is Acinetobacter positive control of lwoffii KCCM 40172; Lanes 1-3 are A. lwoffii Clinical test strains; Lane 4 is Acinetobacter calcoaceticus KCTC 2357; Lane 5 is Acientobacter baumannii KCTC 2508; Lane 6 is Acinetobacter pittii CCUG 61664; Lane 7 is Acinetobacter nosocomialis CCUG 61663; Lane 8 is Acinetobacter ursingii KCTC 12410; Lane 9 is Acinetobacter bereziniae KCTC 12683.

<5-7> Abere -F and Abere -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 7) show that the optimal binding temperature for the Abere-F and Abere-R primer pairs designed in this study is 64 ° C and the exact amplification product size is 539 bp. A. Species for bereziniae - specific strains of the A. bereziniae and Acientobacter PCR was performed on the genomic DNA of the other isolates and 17 isolates (100%) of 17 isolates identified as A. bereziniae . As shown in Table 5, Acinetobacter other than A. bereziniae (100%) of all the strains of the strain identified as the strain (Table 5, Fig. 17).

17, lane M indicates lane M indicates a molecular weight of 100 bp marker; Lane N is a negative control without template DNA; Lane P is a positive control, Acinetobacter bereziniae KCTC 12683; Lanes 1-3 are clinical isolates of A. bereziniae ; Lane 4 is Acinetobacter calcoaceticus KCTC 2357; Lane 5 is Acientobacter baumannii KCTC 2508; Lane 6 is Acinetobacter pittii CCUG 61664; Lane 7 is Acinetobacter nosocomialis CCUG 61663; Lane 8 is Acinetobacter ursingii KCTC 12410 ; Lane 9 is Acinetobacter lwoffii KCCM 40172.

<5-8> Ahaemo -F and Ahaemo -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 8) show that the optimum binding temperature for the pair of Ahaemo-F and Ahaemo-R primers designed in this study is 60 ° C and the size of the amplified product is 584 bp. A. Species for haemolyticus - specific strains of the A. haemolyticus and Acientobacter PCR was performed on the genomic DNA of the standard strains of other species and clinically isolated strains. As a result, 1 out of 1 strain (100%) of A. haemolyticus was positive. In addition, other than A. haemolyticus , Acientobacter All of the 29 strains determined as mycobacteria were negative (100%) (see Fig. 18).

In FIG. 18, lane M indicates lane M indicates a molecular weight of 100 bp marker; Lane N is a negative control without template DNA; Lane P is a positive control, A. haemolyticus KCTC 12404; Lane 1 is A. baumannii KCTC 2508; Lane 2 is A. calcoaceticus KCTC 2357; Lane 3 is A. pittii CCUG 61664; Lane 4 is A. nosocomialis CCUG 61663; Lane 5 is A. ursingii KCTC 12410; Lane 6 is A. lowffii KCCM 40172; Lane 7 is A. bereziniae KCTC 12683; Lane 8 is A. parvus KCTC 12408; Lane 9 is A. schindleri KCTC 12409.

<5-9> Apar -F and Apar -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 9) show that the optimum binding temperature for the pair of Apar-F and Apar-R primers designed in this study is 63 ° C and the exact amplification product size is 239 bp. A. parvus Species specificity for A. parvus and Acientobacter PCR was performed on the genomic DNA of the isolates of other species and clinically isolated isolates. One out of 100 isolates of A. parvus was positive (100%). In addition, other non- A. parvus Acientobacter (100%) of the strains of 29 strains determined to be strains were negative (see FIG. 19).

In FIG. 19, lane M is a marker with a molecular weight of 100 bp; Lane N is a negative control without template DNA; Lane P is a positive control, A. parvus KCTC 12408; Lane 1 is A. baumannii KCTC 2508; Lane 2 is A. calcoaceticus KCTC 2357; Lane 3 is A. pittii CCUG 61664; Lane 4 is A. nosocomialis CCUG 61663; Lane 5 is A. ursingii KCTC 12410; Lane 6 is A. bereziniae KCTC 12683; Lane 7 is A. haemolyticus KCTC 12404; Lane 8 is A. schindleri KCTC 12409; Lane 9 is A. lowffii KCCM 40172.

<5-10> Aschin -F and Aschin -R primer  Verification of species-specificity and sensitivity of pairs

Gradient PCR results (see FIG. 10) show that Aschin-F and Aschin-R The optimal binding temperature for the primer pair is 63 ° C and the exact amplification product size is 480 bp. A. Species for schindleri - specific strains of the A. schindleri and Acientobacter PCR was performed on the genomic DNA of the standard strains of other species and clinically isolated strains. As a result, one strain (100%) of A. schindleri strain was positive. In addition, Acindobacter other than A. schindleri (100%) of the 29 strains determined as mycobacterial strains were negative (see Fig. 20).

20, lane M indicates lane M indicates a molecular weight of 100 bp marker; Lane N is a negative control without template DNA; Lane P is a positive control group, A. schindleri KCTC 12409; Lane 1 is A. baumannii KCTC 2508; Lane 2 is A. calcoaceticus KCTC 2357; Lane 3 is A. pittii CCUG 61664; Lane 4 is A. nosocomialis CCUG 61663; Lane 5 is A. ursingii KCTC 12410; Lane 6 is A. lowffii KCCM 40172; Lane 7 is A. bereziniae KCTC 12683; Lane 8 is A. haemolyticus KCTC 12404; Lane 9 is A. parvus KCTC 12408.

< Example  6>

Acinetobacter  Species-specific Polymerase chain reaction  Lowest detection limit measurement

To determine the minimum detection limit of the specific Acinetobacer species-specific PCR of the present invention, the Acinetobacer standard strain genomic DNA was diluted 10 to 10 times from 2 ng to 2 fg, and each dilution was used as a template. PCR was performed using AccuPower PCR PreMix and Gene Amp PCR System 9600 as described above. 2 μl of the PCR amplification product was subjected to electrophoresis at 100 V for 30 minutes using 1.5% agarose gel and Tris-acetate buffer (0.04 M Tris-actate, 0.001 M EDTA, pH 8.0). The amplified product was stained with ethidium bromide and developed by UV transillumination.

<6-1> Acal -F and Acal -R PCR The lowest detection limit of

As Acal-F and Acal-R primers, Acinetobacter The minimum amount of amplification of genomic DNA of calcoaceticus standard strain (KCTC 2357) was 200 pg, and the minimum amount of amplification of genomic DNA of Acinetobacter calcoaceticus standard strain (KCTC 2357) with gyrB Multiplex 2 PCR primer was 2 ng (see FIG. 21) .

<6-2> Abau -F and Abau -R PCR The lowest detection limit of

As Abau-F and Abau-R primers, Acinetobacter The minimal amount of baumannii standard strain (KCTC 2508) capable of amplifying the genomic DNA was 20 pg, and the gyrB Multiplex 1 PCR primer was used as the primer for Acinetobacter The minimum amount of amplification of the baumannii standard strain (KCTC 2508) genomic DNA was 200 pg (see FIG. 22).

<6-3> Apittii -F and Apittii -R PCR The lowest detection limit of

As Apittii-F and Apittii-R primers, Acinetobacter The minimal amount of pittii standard strain (CCUG 61664) that can amplify the genomic DNA was 20 pg, and the gyrB Multiplex 1 PCR primer, Acinetobacter The minimum amount of amplification of pittii standard strain (CCUG 61664) genomic DNA was 200 pg (see FIG. 23).

<6-4> Anosoco -F and Anosoco -R PCR The lowest detection limit of

As Anosoco-F and Anosoco-R primers, Acinetobacter The minimum amount of amplification of genomic DNA of the standard strain of nosocomialis (CCUG 61663) was 20 pg and the minimum amount of amplification of the genomic DNA of Acinetobacter nosocomialis standard strain (CCUG 61663) by the gyrB Multiplex 1 PCR primer was 200 pg (see FIG. 24) .

<6-5> Ausingii -F and Ausingii -R PCR The lowest detection limit of

As Ausingii-F and Ausingii-R primers, Acinetobacter The minimum amount of amplification of the ursingii standard strain (KCTC 12410) genomic DNA was 20 pg (see FIG. 25).

<6-6> Alwoffii -F and Alwoffii -R PCR The lowest detection limit of

As Alwoffii-F and Alwoffii-R primers, Acinetobacter The minimum amount of amplification of the lwoffii standard strain (KCCM 40172) genomic DNA was 200 pg (see FIG. 26).

<6-7> Abere -F and Abere -R PCR The lowest detection limit of

As Abere-F and Abere-R primers, Acinetobacter The minimum amount of amplification of bereziniae standard strain (KCTC 12683) genomic DNA was 200 pg (see FIG. 27).

<6-8> A. haemo -F and A. haemo -R PCR The lowest detection limit of

A. haemo- F and As the A. haemo - R primer, Acinetobacter The minimum amount of amplification of haemolyticus standard strain (KCTC 12404) genomic DNA was 2 pg (see FIG. 28).

<6-9> A. parvus -F and A. parvus -R PCR The lowest detection limit of

As A. parvus- F and A. parvus- R primers, Acinetobacter The minimum amount of parvus standard strain (KCTC 12408) capable of amplifying genomic DNA was 2 ng (see FIG. 29).

<6-10> A. schin -F and A. schin -R PCR The lowest detection limit of

As A. schin- F and A. schin- R primers, Acinetobacter schindler The minimum amount of amplification of the standard strain (KCTC 12409) genomic DNA was 20 pg (see FIG. 30).

Therefore, it was found from the above results that the primers of the present invention shown in Table 1 are primers capable of specifically amplifying strains belonging to the genus Ashtona bacterium. Using the primers of the present invention, gene amplification methods such as PCR It was found that the presence of Ashinobacterium, a pathogenic strain of the hospital, and the presence of Ashinobacterium can be determined quickly and accurately.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Chosun University <120> Primer and probe for detection of Acinetobacter and method for          detecting Acinetobacter using the same <130> PN1306-216 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Acal-F <400> 1 tcgtatctca attacaccgt tcacct 26 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Acal-R <400> 2 cgccttctgc cagtttcacc ata 23 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Abau-F <400> 3 cataaactga gataactggc ttgaacc 27 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Abau-R <400> 4 cgtcgacttc acctttaccg ttac 24 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Apittii-F <400> 5 tgggcagtta ccagattgac cta 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Apittii-R <400> 6 aaccagcagc ttccatttga cg 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Anosoco-F <400> 7 gccgctcgtg aacgtgtaat c 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Anosoco-R <400> 8 catcgtgtgg catatcttca ac 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Alwoffii-F <400> 9 ccgtgtcggt ctggttcgtg ta 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Alwoffii-R <400> 10 ccggcgtttc aattggacat ac 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aursingii-F <400> 11 agcgtgtcat cgtatctcag 20 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Aursingii-R <400> 12 ccgcgtaaac gttcaggcac aaga 24 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Abere-F <400> 13 acggttctcc gggcgtct 18 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Abere-R <400> 14 aacaccacct tcagcgattt ta 22 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ahaemo-F <400> 15 tgcacagcag gtagtatca 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ahaemo-R <400> 16 gtaatttctt ctgcacctaa 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Apar-F <400> 17 catggtgaag atcgctgaaa a 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Apar-R <400> 18 cccactggag aaaggtcata ac 22 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aschin-F <400> 19 atcgcggata cattacgtgc c 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aschin-R <400> 20 ccactggctt cgcattgatt 20

Claims (7)

A first primer pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2;
A second primer pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4;
A third primer pair consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6;
A fourth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8;
A fifth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10;
A sixth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 11 and 12;
A seventh primer pair consisting of the nucleotide sequences of SEQ ID NOS: 13 and 14;
An eighth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 15 and 16;
A ninth primer pair consisting of the nucleotide sequences of SEQ ID NOS: 17 and 18; And
A pair of primers for detecting Acinetobacter fungus comprising a tenth pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 19 and 20. The method according to claim 1,
The first primer pair is a primer for amplifying DNA of Acinetobacter calcoaceticus ,
The second pair of primers is a primer for amplifying DNA of Acinetobacter baumannii ,
The third pair of primers is a primer for amplifying DNA of Acinetobacter pittii ,
The fourth primer pair is a primer for amplifying DNA of Acinetobacter nosocomialis ,
The fifth primer pair is a primer for amplifying DNA of Acinetobacter lwoffii ,
The sixth primer pair is a primer for amplifying DNA of Acinetobacter ursingii ,
The seventh primer pair is a primer for amplifying DNA of Acinetobacter bereziniae ,
The eighth primer pair is a primer for amplifying DNA of Acinetobacter haemolyticus ,
The ninth primer pair is a primer for amplifying DNA of Acinetobacter parvus ,
The primer pair of claim 10 Acinetobacter swikdeul Larry Acinetobacter (Acinetobacter) strains a pair of primers for detecting, characterized in that the primer for amplifying the DNA of (Acinetobacter schindleri). 3. The method of claim 2,
The first primer pair is a primer capable of amplifying DNA of Acinetobacter calcoaceticus at a minimum of 200 pg,
The second primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter baumannii ,
The third primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter pittii ,
The fourth primer pair is a primer capable of amplifying DNA of Acinetobacter nosocomialis at a minimum of 20 pg,
The fifth primer pair is a primer capable of amplifying a DNA of Acinetobacter lwoffii at least 200 pg,
The sixth primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter ursingii ,
The seventh primer pair is a primer capable of amplifying at least 200 pg of DNA of Acinetobacter bereziniae ,
The eighth primer pair is a primer capable of amplifying at least 2 pg of DNA of Acinetobacter haemolyticus ,
The ninth primer pair is a primer capable of amplifying at least 2 ng of DNA of Acinetobacter parvus ,
Wherein the tenth primer pair is a primer capable of amplifying at least 20 pg of DNA of Acinetobacter schindleri . The pair of primers for detecting Acinetobacter fungus. 7. A microarray for detecting Acinetobacter species in which the primer pair according to any one of claims 1 to 3 is immobilized on a substrate. A method for detecting an Acinetobacter species comprising the step of detecting an Acinetobacter species by a polymerase chain reaction (PCR) using a pair of primers for detecting an Acinetobacter species according to claim 1. 6. The method of claim 5,
The Acinetobacter species is Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter baumannii (A. baumannii), Petey Acinetobacter (A. pittii), Acinetobacter au Alice Cormier (A. nosocomialis), Acinetobacter Earl destination (A. ursingii), Acinetobacter A. lwoffii , A. bereziniae, A. haemolyticus , A. parvus and A. cinereus ( A. The present invention relates to a method for detecting an Asnithobacter species. 6. The method of claim 5,
Wherein the sensitivity of the polymerase chain reaction (PCR) is 98% to 100%.

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