KR101373756B1 - Primers for molecular identification of Staphylococcus aureus and method for identifying Staphylococcus aureus using the same - Google Patents

Abstract

The present invention relates to a technique for identifying genotype of Staphylococcus aureus, which is a major cause of food poisoning and acute sepsis, using RNA polymerase beta subunit ( rpoB ) gene. More particularly, the present invention for amplifying a second nucleotide sequence 1-3538 of a method of analysis to identify the genotype of Staphylococcus aureus 1-3538 a second nucleotide sequence of the rpoB gene of Staphylococcus aureus, rpoB gene of Staphylococcus aureus A primer for determining a primer and a nucleotide sequence, a composition and kit for genotyping of Staphylococcus aureus comprising the primer, and an rpoB polynucleotide specific for the genotype of major Staphylococcus aureus. The present invention can be usefully used as a molecular identification method that can replace the Staphylococcus aureus genotyping method using the existing MLST method.

Description

Primers for molecular identification of Staphylococcus aureus and method for identifying Staphylococcus aureus using the same}

The present invention is one of the main causes of food poisoning by using the RNA polymerase beta subunit ( rpoB ) gene, which is the cause of acute sepsis ( Staphylococcus) Aureus ) relates to a technique for identifying genotypes. More particularly, the present invention provides primers for amplifying the second nucleotide sequence 1-3538 of the method for identifying the Staphylococcus aureus by analyzing the base sequence of the rpoB gene of 1-3538 second Staphylococcus aureus, rpoB gene of Staphylococcus aureus, and A primer for determining a nucleotide sequence, a composition and kit for identifying Staphylococcus aureus comprising the primer, and an rpoB polynucleotide specific for major Staphylococcus aureus. The present invention can be usefully used as a molecular identification method that can replace the staphylococcus aureus identification method using the conventional multilocus sequence typing (MLST) method.

Staphylococcus aureus is one of the most frequent pathogens, causing food poisoning, skin or tissue infections, toxin shock, pneumonia, bacteremia, and group infections like hospital infections or food poisoning. 20% to 40% of Staphylococcus aureus may be present on human skin or mucous membranes, increasing the risk of opportunistic infections (Wertheim, 2005, Lancet Infect Dis 5: 751-762), and 47% of US meat stores sell Staphylococcus aureus. , 52% of which are reported to be antibiotic resistant (Waters, 2011, Clin Infect Dis 52: 1227-1230). This high frequency means that there is a high risk of infection through food distribution channels, so Staphylococcus aureus is a serious hazard to food safety and public health. Therefore, there is a need for a system that can identify and identify Staphylococcus aureus accurately and quickly.

Staphylococcus spp . 16S rRNA genes are widely used for the identification of microorganisms (Waters, 2011, Clin Infect Dis 52: 1227-1230; Bialkowska-Hobrzanska, 1990, Eur. J. Microbiol. Infect. Dis. 9: 588-594; De Buyser , 1992, J. Gen. Microbiol. 138: 889-899). In addition, the 16S-23S rRNA intergenic spacer region (Maes, 1997, J. Clin. Microbiol. 35: 2477-2481), heat shock protein 60, hsp60 gene (Goh, 1996, J. Clin. Microbiol. 34 : 818-823; Goh, 1997, J. Clin.Microbiol . 35: 3116-3121), femA Gene (Vannuffel, 1999, Res. Microbiol. 150: 129-141), sodA Gene (Poyart, 2001, J. Clin Microbiol 39:.. 4296-4301), tuf Gene (Martineau, 2001, J. Clin. Microbiol. 39: 2541-2547) and gap The gene (Yugueros, 2000, J. Clin. Microbiol. 38: 4351-4355; Yugueros, 2001, J. Clin. Microbiol. 39: 3693-3695) has been used, but the same species is Staphylococcus aureus. It did not apply to identifying differences between isolates.

In addition, PFGE (pulse-field gel electrophoresis) has been developed and used to study the differences between S. aureus isolates, but there is a problem in that reproducibility of results between laboratories is inferior due to variables in genomic extraction and restriction enzyme processing. (Hallin, 2006, J Clin Microbiol 45: 127-133). In addition, by using the nucleotide sequence of seven housekeeping genes by MLST (Multilocus sequence typing), the isolates are classified into a sequence type (ST) to build a database with objective data and high reproducibility ( http: //www.mlst.net ), 7 gene amplification requires 7 times of polymerase chain reaction, 7 times of amplification product purification, and 14 times of sequence determination using both primers. There are disadvantages.

Recently, a partial sequence of 751bp corresponding to the high mutated region of the rpoB gene of the Staphylococcus microorganism was used to distinguish the phylogenetic relationship between the Staphylococcus microorganisms (Drancourt, 2002 J. Clin. Microbiol. 40: 1333-1338), no attempt has been made to distinguish genotypes between isolates of the same species of Staphylococcus aureus. In addition, the identification method using the rpoB gene is a partial nucleotide sequence including a high mutation region of the rpoB gene, there is a limit that can be identified only for some microorganisms belonging to the Staphylococcus.

Accordingly, the present inventors have established a primer that can amplify the 1-3538th sequence of the rpoB gene of all Staphylococcus aureus and 1-3538 in order to establish a technology that can more accurately and easily distinguish between genotypes between Staphylococcus aureus A primer was designed to determine the first nucleotide sequence, and a database was constructed by analyzing the 1-3538 nucleotide sequences of the rpoB gene specific to domestic human and poultry-derived Staphylococcus aureus. In addition, when an unknown bacterium (target strain) suspected of Staphylococcus aureus is isolated from a food poisoning patient or the surrounding environment, the primers are used to amplify and determine the 1-353th base sequence of the rpoB gene of the target strain. By comparing the nucleotide sequence of the rpoB gene of the Staphylococcus aureus on the already secured database, it was confirmed that the genotype of the target strain can be accurately determined. In addition, the Staphylococcus aureus genotyping method using the 1-353th nucleotide sequence of the rpoB gene showed more accurate results compared to the case of using the existing MLST method, but it was more economical than the MLST method (one polymerase chain reaction and one purification). , 6 sequencing) was confirmed to be useful as a molecular identification method that can replace MLST to complete the present invention.

The present invention provides a technique for identifying Staphylococcus aureus genotypes using the 1-353 nucleotide sequences of the RNA polymerase beta subunit ( rpoB ) gene. In addition, the present invention uses a primer capable of amplifying and determining the 1-336th rpoB gene of the Staphylococcus aureus genotype, and the rpoB polynucleotide and nucleotide sequence information of the genotype strains of the major Staphylococcus aureus identified using the primer. It provides a technique for effectively identifying the genotype of Staphylococcus aureus.

More specifically, it is an object of the present invention to provide a method for identifying genotype of Staphylococcus aureus, comprising amplifying a 1-3538 nucleotide sequence of the rpoB gene.

Another object of the present invention is to provide a primer for amplifying the 1-3538th sequence of the rpoB gene genotype of Staphylococcus aureus.

Another object of the present invention is to provide a primer for determining the 1-3538th sequence of the rpoB gene genotype of Staphylococcus aureus.

Another object of the present invention to provide a composition for identification of genotype of Staphylococcus aureus comprising the primer.

Another object of the invention to provide a kit for genotyping of Staphylococcus aureus comprising the composition.

Another object of the present invention is to provide rpoB polynucleotides specific for genotypes of Staphylococcus aureus, identified using the primers.

It is still another object of the present invention to provide a composition for genotyping of Staphylococcus aureus comprising rpoB polynucleotide specific for genotypes of Staphylococcus aureus.

As one embodiment for achieving the above object, the present invention is to identify the genotype of Staphylococcus aureus comprising the step of amplifying the 1-353 nucleotide sequence of the RNA polymerase beta subunit ( rpoB ) gene of Staphylococcus aureus It is about a method.

As a preferred embodiment for this purpose,

(1) amplifying the 1-3538 nucleotide sequences of rpoB gene of Staphylococcus aureus;

(2) determining the 1-353th nucleotide sequence of the amplified rpoB gene; And

(3) determining the genotype of Staphylococcus aureus by comparing the base sequence obtained in step (2) with the base sequence of the rpoB gene of the standard strain;

The present invention relates to a method for identifying genotype of Staphylococcus aureus.

Preferably, the step (1) and step (2) may be performed using a primer derived from the conserved region in the 1-3538 nucleotide sequence of the rpoB gene of Staphylococcus aureus.

Also preferably, the step (1) may be to amplify the 1 to 3538 nucleotide sequence of the rpoB gene using a primer pair having the nucleotide sequence of SEQ ID NO: 1 and 2.

Also preferably, the step (2) is to determine the 1 to 3,538th nucleotide sequence of the rpoB gene using at least one primer having a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 3 to SEQ ID NO: 8 It may be.

Also preferably, in step (3), the nucleotide sequence of the rpoB gene of the standard strain may be selected from the group consisting of the nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 27.

As another aspect, the present invention relates to a primer derived from a conserved region in the 1-3538 nucleotide sequences of rpoB gene of Staphylococcus aureus.

As another aspect, the present invention relates to a primer having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 in the base sequence of the rpoB gene of Staphylococcus aureus.

In still another aspect, the present invention relates to a primer for amplifying 1-353 nucleotide sequences of rpoB gene of Staphylococcus aureus having the nucleotide sequences of SEQ ID NOs: 1 and 2.

In still another aspect, the present invention relates to a primer for determining the 1-3538 nucleotide sequence of rpoB gene of Staphylococcus aureus, which has a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 9 to 27.

As another aspect, the present invention relates to a composition for genotyping of Staphylococcus aureus comprising any one or more of the above primers.

As another aspect, the present invention relates to a kit comprising the genotyping composition of the Staphylococcus aureus.

As another aspect, the present invention relates to a composition for genotyping of Staphylococcus aureus, comprising rpoB polynucleotide of Staphylococcus aureus having a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 27 will be.

As another aspect, the present invention relates to a rpoB polynucleotide of Staphylococcus aureus having a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23 will be.

Hereinafter, the present invention will be described in more detail.

In order to establish a method for identifying genotype of Staphylococcus aureus by analyzing the 1-353th sequences of the rpoB gene of Staphylococcus aureus, the present inventors have multiplexed the rpoB genome sequence of 23 strains of Staphylococcus aureus strains registered with GenBank. We identified the mutations of the rpoB gene showing differences between the isolates and the conserved portions that could be useful for primer design. Based on this, we designed a pair of primers to amplify rpoB 1-3538 th, and to determine the sequence Primers were designed in conserved parts. In addition, using these primers, 1-3 strains of rpoB genes of 6 strains of Staphylococcus aureus strains, 2 strains purchased from ATCC, and 4 strains isolated from Biopoa Co. The database was constructed by analyzing. In addition, by amplifying and determining the rpoB 1-3538 nucleotide sequence of the target strain to be identified and compared with the nucleotide sequence of the database, a new method for identifying the Staphylococcus aureus genotype was developed.

In order for a gene to be a chronological molecular marker that reflects the systemic relationships of bacteria, the target gene must be functionally essential and conserved in all bacteria, the mutation of the homologous sequence due to acquired effects does not occur, And should show intraspecies conservation within the genus such as interspecies variation appropriate for presentation. Since the rpoB gene encodes essential enzymes involved in the transcription of the bacterium, it is essential and conserved in all strains, and variation and homology in other strains have been reported (Kim et al. , J Clin Microbiol 37: 1714-1720, 1999; Lee et al., J Clin Microbiol 38: 2557-2562, 2000).

When analyzing the 1-3538th nucleotide sequence of the rpoB gene as in the present invention and used for genotyping, it is applicable to all known Staphylococcus aureus isolates because the primer derived from the conserved portion is used, Table 1 and Figure As shown in 1, genotypes can be identified exactly. As can be seen in the specific examples of the present invention, in the present invention, it was possible to distinguish genotypes of strains that could not be distinguished by conventional methods (eg, Mu3, 04-02981, COL, ATCC19685, ATCC25923 strains), and existing MLST. As the genotyping method, the monarchs classified into 14 genotypes (MLST sequence type, ST) are divided into 15 genotypes (rpoB sequence type, RST) by the method of the present invention, while the discrimination ability is improved compared to the MLST method. It was economical.

Therefore, the present invention is rpoB Provided are primers that can amplify or determine the 1-353 nucleotide sequence of the gene.

In the present invention, the primer may be a primer derived from a conserved region in the 1-3538th sequence of the rpoB gene of Staphylococcus aureus. Specifically, the conserved region is a region excluding the mutated portion, and among the nucleotide sequences of SEQ ID NO: 9, 42, 127, 255, 453, 497, 546, 570, 609, 843, 858, 885, 894, 972, 975, and 1011. , 1065, 1122, 1122, 1131, 1140, 1142, 1143, 1176, 1388, 1390, 1401, 1411, 1417, 1422, 1429, 1434, 1441, 1506, 1554, 1566, 1658, 1797, 1845, 1890, 1974 , 2013, 2040, 2073, 2181, 2192, 2210, 2304, 2393, 2624, 2682, 2697, 2736, 2829, 2844, 2847, 2859, 2874, 2982, 3144, 3159, 3195, 3225, 3494 and 3495th base Refers to the nucleotide sequence region excluding the sequence.

Therefore, preferably, the primer of the present invention is 42, 127, 255, 453, 497, 546, 570, 609, 843, 858, 885, 894, 972, 975, 1011, 1065, 1122, 1122, 1131, 1140, 1142, 1143, 1176, 1388, 1390, 1401, 1411, 1417, 1422, 1429, 1434, 1441, 1506, 1554, 1566, 1658, 1797, 1845, 1890, 1974, 2013, 2040, Base sequences other than 2073, 2181, 2192, 2210, 2304, 2393, 2624, 2682, 2697, 2736, 2829, 2844, 2847, 2859, 2874, 2982, 3144, 3159, 3195, 3225, 3494, and 3495 Or a primer having a sequential sequence of 7 to 50 bp complementary thereto.

In addition, rpoB of the Staphylococcus aureus strain in the present invention A primer capable of amplifying the 1-353 nucleotide sequence of the gene is a primer capable of amplifying the r-3B nucleotide sequence of the rpoB gene 1-3538 of all S. aureus strains, and preferably SEQ ID NO: 1 (SA-rpoBF) and sequence Primer pair having a nucleotide sequence of No. 2 (SA-rpoBR).

In addition, a primer, a primer for determining the base sequence of rpoB gene 1-3538 second all Staphylococcus aureus strains, SEQ ID NO: 3 (SA-rpoB- for determining the base sequence of the rpoB gene 1-3538 second in the present invention S1R), SEQ ID NO: 4 (SA-rpoB-S1F), SEQ ID NO: 5 (SA-rpoB-S2F), SEQ ID NO: 6 (SA-rpoB-S3F), SEQ ID NO: 7 (SA-rpoB-S4F), and SEQ ID NO: 8 It may be a primer having a base sequence selected from the group consisting of the base sequence of (SA-rpoB-S5F).

The primers of the present invention are the first primers for amplifying and determining the nucleotide sequences of the 1-336 sequences of rpoB genes of Staphylococcus aureus strains, and multiply the rpoB genomic sequences of 23 strains of Staphylococcus aureus strains registered in GenBank. Based on the conserved portion confirmed by the rpoB is a primer that is highly calculated and prepared to amplify and determine the nucleotides 1-3538. That is, in the present invention, primers were designed based on the conserved portions of the rpoB gene among the Staphylococcus aureus strains, and the primers of the present invention can be applied to all Staphylococcus aureus strains known to date.

The present invention also provides a polynucleotide having the 1-3538th nucleotide sequence of rpoB specific for frequent Staphylococcus aureus.

Preferably, the rpoB polynucleotide provided in the present invention has a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 27. The polynucleotide has been identified using primers for amplification and determination of the rpoB gene of the present invention, it is possible to build a unique staphylococcus aureus genotyping database.

In particular, the nucleotide sequences of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23 were found to be new base sequences in Genbank search.

Therefore, more preferably, the rpoB polynucleotide provided in the present invention has Staphylococcus aureus having a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23 RpoB polynucleotide.

As a result of comparing the nucleotide sequences of each of the nucleotide sequences of SEQ ID NOs: 9 to 27 analyzed in the present invention, all of them had different nucleotide sequences, indicating interspecies variation, and all of the base sequences on the multiple sequences. Since 3538 bp of base was encoded without inserting or deleting a sequence, it can be confirmed that the base sequence (1-3538) of rpoB of the present invention is suitable as a chronological molecular label. In addition, since the nucleotide sequence of the polynucleotide and the genotype of the strain including the same are known in advance, the genotype of the strain to be analyzed is determined by comparing with the rpoB nucleotide sequence of the strain to be analyzed based on the nucleotide sequence, and the phylogenetic relationship is analyzed. Can be used to

Accordingly, the present invention provides a method for identifying genotype of Staphylococcus aureus using the primers and rpoB polynucleotide sequences.

Specifically, the method for identifying genotype of Staphylococcus aureus includes amplifying the base sequence 1-3538 of the rpoB gene.

Preferably, the method of identifying genotype of Staphylococcus aureus comprises the following steps:

(1) amplifying sequences 1-3538 of the rpoB gene of Staphylococcus aureus;

(2) determining the 1-353th nucleotide sequence of the amplified rpoB gene;

(3) determining the genotype by comparing the base sequence obtained in step (2) with the base sequence of the rpoB gene of the standard strain.

The step (1) is to amplify an unknown Staphylococcus aureus strain, i.e., the 1-3538th sequence of the rpoB gene of the target strain to be identified.

Preferably, the step is rpoB RpoB as primers to amplify the second nucleotide sequence of the gene 1-3538 It can be performed by PCR amplification using primers derived from the conserved region of the gene.

The step rpoB More preferably, As a primer capable of amplifying the 1-353th nucleotide sequence of the gene, it may be performed by PCR amplification using a primer pair having a nucleotide sequence of SEQ ID NO: 1 (SA-rpoBF) and SEQ ID NO: 2 (SA-rpoBR). . PCR amplification can use all assays known to those skilled in the art.

Step (2) is a primer for determining the 1-353th base sequence of the rpoB gene amplified in step (1), preferably SEQ ID NO: 3 (SA-rpoB-S1R), SEQ ID NO: 4 (SA- rpoB-S1F), SEQ ID NO: 5 (SA-rpoB-S2F), SEQ ID NO: 6 (SA-rpoB-S3F), SEQ ID NO: 7 (SA-rpoB-S4F), and SEQ ID NO: 8 (SA-rpoB-S5F) It can be carried out by the nucleotide sequence determination method using a primer selected from the group consisting of the sequence.

Step (3) is a step of determining the genotype by comparing the base sequence obtained in step (2) with the base sequence of the rpoB gene of the standard strain, preferably, the base sequence of the rpoB gene of the standard strain is SEQ ID NO: 9 To SEQ ID NO: 27 is selected from the group consisting of. The polynucleotide having the nucleotide sequences of SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23 using primers for amplification and determination of the rpoB gene of the present invention As a result, the genome of the 1-3538th base sequence and the genotype of the strain is known in advance, so that the genotype of the target strain can be determined and the lineage relationship can be analyzed by comparing the rpoB base sequence of the target strain based on the base sequence. have. Molecular biological methods using the difference between the rpoB 1-3538 nucleotide sequences of the standard strain and the target strain can be applied to the present invention.

In a preferred example, and after analysis of the rpoB DNA sequence of the target strain this comparison rpoB nucleotide sequence of type strain to be, identified with the the rpoB sequences of the strains built previously aligned database in order to determine the genotype of the target strain You can add the rpoB sequence of the bacteria and perform the sequence alignment again to complete the schematic. When the phylogenetic tree is completed, branches are formed in close proximity to the corresponding species, so the phylogeny can be determined by the phylogenetic tree. In order to complete the schematic, a neighbor-joining method using MEGA software or the like may be used, and the support of the branch may be determined by performing 100 to 1000 repetition of bootstrap analysis.

According to the genotyping method, when an unknown bacterium suspected of Staphylococcus aureus is isolated from a food poisoning patient or the surrounding environment, amplification and determination of the rpoB 1-3538 nucleotide sequences with a specific primer are performed. Genotyping can be determined by comparing with the nucleotide sequence of these cells, and it is made more precisely than the conventional species identification method, so that the treatment policy of patients infected with Staphylococcus aureus can be determined quickly, and the effective prevention by understanding the causes and pathways of transmission. Method can be derived.

The present invention also provides a composition for genotyping of Staphylococcus aureus comprising the primer described above.

Preferably, the present invention provides a composition for genotyping of Staphylococcus aureus, comprising a primer having a nucleotide sequence complementary to the base sequence of the conserved portion of the nucleotide sequence of the rpoB gene of Staphylococcus aureus. More preferably, the present invention is the nucleotide sequence of SEQ ID NO: 9 Among 42, 127, 255, 453, 497, 546, 570, 609, 843, 858, 885, 894, 972, 975, 1011, 1065, 1122, 1122, 1131, 1140, 1142, 1143, 1176, 1388, 1390 , 1401, 1411, 1417, 1422, 1429, 1434, 1441, 1506, 1554, 1566, 1658, 1797, 1845, 1890, 1974, 2013, 2040, 2073, 2181, 2192, 2210, 2304, 2393, 2624, 2682 , Nucleotide sequences other than the 2697, 2736, 2829, 2844, 2847, 2859, 2874, 2982, 3144, 3159, 3195, 3225, 3494, and 3495th nucleotide sequences or complementary sequences of 7 to 50 bp The branch provides a composition for identification of genotype of Staphylococcus aureus, comprising a primer.

Also preferably, the present invention provides a genotyping composition for Staphylococcus aureus, comprising a primer for amplifying the 1-353 nucleotide sequence of Staphylococcus aureus rpoB gene having the nucleotide sequence of SEQ ID NO: 1 and 2. More preferably, the composition is SEQ ID NO: 3 (SA-rpoB-S1R), SEQ ID NO: 4 (SA-rpoB-S1F), SEQ ID NO: 5 (SA-rpoB-S2F), SEQ ID NO: 6 (SA-rpoB-S3F) And, SEQ ID NO: 7 (SA-rpoB-S4F), SEQ ID NO: 8 (SA-rpoB-S5F) may further comprise a primer for determining the 1-3538 base sequence of the rpoB gene having a nucleotide sequence.

Also preferably, the present invention provides a composition for genotyping of Staphylococcus aureus, comprising a primer for determining the 1-353th nucleotide sequence of the rpoB gene selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 3 to 8.

The present invention also provides a kit for genotyping of Staphylococcus aureus comprising the composition. Preferably, the kit may be a PCR-RFLP (restriction fragment length polymorphism) kit, an allele-specific PCR kit, a real time PCR kit, a DNA chip kit or a Southern blotting kit. In addition to the primers, the kit may further comprise a DNA polymerase, a DNA staining reagent or a suitable carrier.

The technique of identifying the Staphylococcus aureus genotype by analyzing the 1st to 3538th nucleotide sequences of the rpoB gene of Staphylococcus aureus of the present invention, can obtain more objective and accurate results compared to the conventional genotyping method, all the Staphylococcus aureus strains It can be applied to a wide range of applications, and can be useful for the identification of Staphylococcus aureus genotypes because of its simple process and time and cost savings.

Figure 1 shows the results of the phylogenetic analysis (Neighbor-joining method; Tamura-Nei distance; bootstrapping 500 repeats) using the 1-3538th nucleotide sequence of the rpoB gene coding portion of Staphylococcus aureus strains.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. Primer  Design and Identification of Staphylococcus Aureus Genotypes

1-1. 1-353th of Staphylococcus Aureus rpoB  For amplification and sequencing Primer  design

The rpoB sequences of 23 strains of Staphylococcus aureus registered in GenBank (strains with Accession no. In Table 1) were multi-arranged, and the variation of the rpoB gene showing differences between strains based on ED98 and related to rifampin and vancomycin resistance. A primer having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and a base sequence determining primer having a nucleotide sequence of SEQ ID NO: 3 to SEQ ID NO: 8 to amplify the 1-3538th part of the rpoB gene avoiding the frequent mutation Was designed.

Based on the information in Table 1 provided by the present inventors, the mutant portion can be usefully used for genotyping using DNA chips, allele-specific PCR, RFLP and Southern blotting, and other conserved portions. It can be used for the design of new primers for sequencing and sequencing of Staphylococcus aureus strains. The present inventors designed primer pairs of SA-rpoBF (SEQ ID NO: 1) and SA-rpoBR (SEQ ID NO: 2) as optimal primers capable of amplifying rpoB 1-353 genes, and based on the conserved portion, SEQ ID NO: 3 (SA-rpoB-S1R), SEQ ID NO: 4 (SA-rpoB-S1F), SEQ ID NO: 5 (SA-rpoB-S2F), SEQ ID NO: 6 (SA-rpoB-S3F), SEQ ID NO: 7 (SA-rpoB- S4F), a primer for determining the 1-353th base sequence of the rpoB gene having a nucleotide sequence of SEQ ID NO: 8 (SA-rpoB-S5F) was designed.

1-2. 1-353th of Staphylococcus Aureus rpoB  Sequence Determination and Genotyping

Six strains of Staphylococcus aureus strains supplied by the Antimicrobial Bacterial Bank (CCARM), two strains purchased from ATCC, and four strains isolated from Biopoa Co., Ltd., totaled 12 weeks, were cultured all night in LB broth, and 1 ml of 1.5ml microcentrifuge After dispensing into the tube and centrifuged at 3,500xg for 10 minutes, the medium was removed. The 1-3538th DNA was extracted from the remaining bacterial pellets using the BioPOA GenomEx kit, and then rpoB was amplified by PCR. PCR reaction solution was 10 × buffer 5µl, dNTPs (2.5mM each) 1µl, primers of SEQ ID NO: 1 and SEQ ID NO: 2 (1.0μmol / ul), respectively, Taq DNA polymerase (5U / µl; MACROGEN Co., Seoul , ROK) 0.5 μl, DW 40.5 μl, template DNA (50 ng / μl) 1 μl, temperature was 94 −4 min, (94 ° C.-30 sec, 54 ° C.-20 sec, 72 ° C.-4 min 30 sec) × 40 72 degreeC-5min was reacted once. As a result, a specific amplification product of 3,538bp was identified in all Staphylococcus aureus genotypes, and these sequences were determined by using a PCR amplification product purification kit (BioPOA Co., Yongin) (MACROGEN Co.). As primers for nucleotide sequence determination, primers 1-353 for nucleotide sequence determination of the rpoB gene having nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 8 were used. It was. The determined sequences were multiarrayed using the MEGA 5 program and then systematically analyzed by the Neighbor-joining method (Tamura-Nei distance; 100 repeats of bootstrapping), and the rpoB base of Staphylococcus aureus strain based on ED98 using the Bioedit program. The rpoB sequence type (RST) was encoded according to the number and type of mutations of the sequence and is shown in FIG. 1 and Table 1. FIG. In the RST code, the middle number indicates the number of nucleotide sequences that differ from ED98, and the last number is an identification number of each genotype and is assigned to a unique nucleotide sequence.

In Table 1 below, 23 weeks registered in GenBank and 12 weeks newly determined rpoB gene sequence in the present invention, a total of 35 shares were classified into 19 RST. In the present invention, it was possible to distinguish genotypes of strains that could not be distinguished by conventional methods (eg, Mu3, 04-02981, COL, ATCC19685, ATCC25923 strains), and 14 genotypes by genotyping by conventional MLST methods. Monarchs classified into MLST sequence type (ST) were divided into 15 genotypes (rpoB sequence type, RST) by the method of the present invention, which improved the ability to distinguish them from the MLST method. In addition, all the analysis was completed by one PCR reaction, one amplification product purification, and six sequencing per strain of Staphylococcus aureus, compared to MLST (7 PCR reactions, 7 amplification product purifications, and 14 sequencing sequences). ) It was economic.

RST Strain ST Acesion no . Variation
(SNP; single nucleotide polymorphism) RST-0-0 ED98 ST5 CP001781 - 11022 - - 11159 - - 11214 - - RST-1-1 Mu3 ST5 AP009324 A3494G JH1 ST105 CP000736 ECT R2 ST5 FR714927 N315 ST5 BA000018 CCARM3A172 - - RST-2-1 Mu50 ST5 BA000017 C1441T, A3494G RST-2-2 04-02981 ST225 CP001844 C255T, A3494G RST-5-1 JH9 ST105 CP000703 G1411T, G1417T, G1429T, G1434G, A3494G RST-5-2 11069 - - G127A, T609C, C1422T, C3225T, A3494G RST-7-1 JKD6008 ST239 CP002120 T609C, C1890T, A1974T, A2013G, A2073T, C3225T, A3494G TW20 ST239 FN433596 TCH1516 ST8 CP000730 Newman ST254 AP009351 NCTC8325 ST8 CP000253 FPR3757 ST8 CP000255 CCARM3803 - - RST-9-1 COL ST250 CP000046 T609C, C1890T, A1974T, A2013G, A2073T, C2393T, C2624T, C3225T, A3494G RST-9-2 CCARM3416 - - T609C, G1388A, T1390C, C1890T, A1974T, A2013G, A2073T, C3225T, A3494G CCARM3690 - - RST-13-1 MSSA476 ST1 BX571857 T609C, C1422T, T1797C, C1890T, A1974T, T2682C, T2736C, T2847C, T2874C, T2982C, C3195A, A3494G, C3495T MW2 ST1 BA000033 RST-14-1 CCARM3A049 - - T609C, C1422T, C1658T, T1797C, C1890T, A1974T, T2682C, T2736C, T2847C, T2874C, T2982C, C3195A, A3494G, C3495T RST-14-2 JKD6159 ST93 CP002114 A546G, T858C, A885G, T975C, C1122T, T1142C, C1176T, C1422T, C1890T, A1974T, T2682C, T2874C, A3144G, A3494G RST-15-1 SO385 ST398 AM990992 A546G, A843G, T858C, A972G, T975C, T1011A, C1122T, T1143C, C1176T, C1422T, C1890T, T2040C, T2847C, T2874C, A3894G RST-17-1 CCARM3895 - - A546G, T858C, A885G, A894G, T975C, C1122T, C1422T, G1845A, C1890T, A1974T, A2181T, C2304T, T2829C, C2859T, T2874C, A3159T, A3494G RST-17-2 ATCC19685, - - T497A, A546G, T858C, A885G, T975C, C1122T, T1143C, C1176T, C1422T, A1506G, A1554G, C1890T, A1974T, A2210T, T2697C, C2859T, A3494G ATCC25923 - - RST-18-1 TCH60 ST30 CP002110 T497A, A546G, T858C, A885G, T975C, C1122T, T1143C, C1176T, C1422T, A1506G, A1554G, C1890T, A1974T, T2192C, A2210T, T2697C, C2859T, A3494G RST-18-2 MRSA252 ST36 BX571856 T497A, A546G, T858C, A885G, T975C, C1122T, A1131G, T1143C, C1176T, C1422T, A1506G, A1554G, C1890T, A1974T, A2210T, T2697C, C2859T, A3494G RST-18-3 RF122 ST151 AJ938182 A546G, T858C, A885G, T975C, C1122T, C1140T, T1142C, C1176T, A1401G, C1422T, C1890T, A1974T, T2682C, A2844G, T2847C, T2874C, A3144G, A3494G RST-21-1 ED133 ST1452 CP001996 T42C, C453T, A546G, A570G, T858C, A885G, T975C, T1065A, C1122T, T1142C, C1176T, C1422T, A1566T, G1845A, C1890T, A1974T, T2682C, T2847C, T2874C, A3159T, A3494G

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Claims (17)

(1) amplifying the 1 to 3538 nucleotide sequences of rpoB gene of Staphylococcus aureus;
(2) determining the 1 to 3538 nucleotide sequences of the amplified rpoB gene; And
(3) determining the genotype by comparing the nucleotide sequence obtained in step (2) with the nucleotide sequence of the rpoB gene of the standard strain selected from the group consisting of the nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 27,
Method for identifying genotype of Staphylococcus aureus.
The method according to claim 1, wherein step (1) and step (2) are 42, 127, 255, 453, 497, 546, 570, 609, 843, 858, 885, 894, 972, 975, 1011, 1065, 1122, 1122, 1131, 1140, 1142, 1143, 1176, 1388, 1390, 1401, 1411, 1417, 1422, 1429, 1434, 1441, 1506, 1554, 1566, 1658, 1797, 1845, 1890, 1974, 2013, 2040, 2073, 2181, 2192, 2210, 2304, 2393, 2624, 2682, 2697, 2736, 2829, 2844, 2847, 2859, 2874, 2982, 3144, 3159, 3195, 3225, 3494 and A base sequence other than the 3495 th sequence or complementary thereto and having a sequence having a sequence of 7 to 50 bp.
The method of claim 1, wherein step (1) is to amplify the 1 to 3538 nucleotide sequence of the rpoB gene using a primer pair having a nucleotide sequence of SEQ ID NO: 1 and 2.
The method of claim 1, wherein step (2) is to determine the 1 to 3538 nucleotide sequence of the rpoB gene using one or more primers having a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 3 to SEQ ID NO: 8 How to do.
delete The method according to claim 1, wherein the step (3) is performed by assigning the 1 to 3538 nucleotide sequences of the rpoB gene of Staphylococcus aureus into the nucleotide sequences of the rpoB gene of the standard strain, and then completing the phylogeny and determining the phylogenetic tree. .
Nucleotide sequence of SEQ ID NO: 9 Among 42, 127, 255, 453, 497, 546, 570, 609, 843, 858, 885, 894, 972, 975, 1011, 1065, 1122, 1122, 1131, 1140, 1142, 1143, 1176, 1388, 1390 , 1401, 1411, 1417, 1422, 1429, 1434, 1441, 1506, 1554, 1566, 1658, 1797, 1845, 1890, 1974, 2013, 2040, 2073, 2181, 2192, 2210, 2304, 2393, 2624, 2682 , Nucleotide sequences other than the 2697, 2736, 2829, 2844, 2847, 2859, 2874, 2982, 3144, 3159, 3195, 3225, 3494, and 3495th nucleotide sequences or complementary sequences of 7 to 50 bp With primer.
A primer pair for amplifying 1 to 3538 nucleotide sequences of rpoB gene of Staphylococcus aureus, which has nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
A primer for determining the 1 to 3538 nucleotide sequence of the rpoB gene of Staphylococcus aureus having a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3 to SEQ ID NO: 8.
Genotyping composition for Staphylococcus aureus comprising the primer of claim 7.
Genotyping composition for Staphylococcus aureus comprising the primer pair of claim 8.
The genotype-identifying composition of Staphylococcus aureus according to claim 11, further comprising one or more primers having a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 3 to 8.
Genotyping composition for Staphylococcus aureus comprising the primer of claim 9.
Kit for genotyping of Staphylococcus aureus comprising the composition of any one of claims 10-13.
The kit for identifying genotypes of Staphylococcus aureus according to claim 14, wherein the kit is a PCR-RFLP (restriction fragment length polymorphism) kit, an allele-specific PCR kit, a real-time PCR kit, a DNA chip kit or a Southern blotting kit.
A composition for genotyping of a microorganism of Staphylococcus aureus, comprising a rpoB polynucleotide of Staphylococcus aureus having a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 27.
A rpoB polynucleotide of Staphylococcus aureus having a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23.

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