JP2011120528A - Genotype classification method for staphylococcus aureus and primer set for use in the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a genotype classification method for Staphylococcus aureus, which is obtained by improving a technique described in Publication NO.2006-141249 and is suitable for the actual industry (medical spot) and a primer set for use in the same. <P>SOLUTION: The genotype classification method for Staphylococcus aureus includes a step for detecting the presence/absence of (1) a phage-derived open reading frame (ORF), (2) an ORF derived from genomic islands except Staphylococcal cassette chromosome mec; SCCmec, (3) a transposon-derived ORF, (4) SCCmec-derived ORF, and (5) ORF constituting a genomic islet on the Staphylococcus aureus genome in an arbitrary order and performing genotype classification by combination of these ORF presence/absence. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for classifying S. aureus by genotype (strain identification method) and a primer set used therefor.

  Methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) is a pathogen that is isolated in many hospitals. One hospital in Tokyo (925 beds) reported that MRSA was detected in 72 inpatients in one month, and another hospital in Kumamoto (550 beds) also had 35 in one month. MRSA was reported to be detected in hospitalized patients.

According to the Ministry of Health, Labor and Welfare's infectious disease outbreak survey, MRSA infections account for more than 20,000 cases per year based on reports from approximately 500 major hospitals nationwide.
Based on the above, MRSA is isolated in large numbers in hospitals, and is considered to be a pathogen that always requires epidemiological studies to prevent infection. In other words, MRSA is an important fungus that causes nosocomial infections. In order to reduce nosocomial infection, it is necessary to identify the infection route.

  When nosocomial infection is suspected, it is necessary to determine whether the strains isolated from different patients are identical in order to identify the infection route. Therefore, the characteristics of the genome possessed by the strain are detected to identify the strain, which is called genotyping classification.

  Conventionally, pulse field gel electrophoresis (hereinafter referred to as PFGE) has been frequently used as a method for classifying bacteria by genotype. PFGE is a method that cleaves bacterial genomic DNA with restriction enzymes and determines the genotype of the strain as an electrophoretic pattern of its fragments. It is a standard method for classification by genotype because of its high discrimination ability and reproducibility. Yes.

  However, PFGE takes about 3 days after the identification and identification of the strain at the earliest before results are obtained, and when the test results are obtained, the spread of infection is often already completed. In addition, since it is determined by a complicated band pattern, subjective elements tend to be included, and it is difficult to determine whether or not the genotype is the same except between the strains that migrated simultaneously. Therefore, it was virtually impossible to compare genotypes between PFGE results that were separated in time or distance (performed at different times or elsewhere).

Furthermore, PFGE is cumbersome and requires skilled workers, and requires special equipment such as a pulse field gel electrophoresis apparatus, which is expensive.
Thus, as a method of overcoming these PFGE drawbacks, a genotyping method by detecting the presence or absence of a specific ORF of methicillin-resistant Staphylococcus aureus and a primer set used therefor have been developed (see Patent Document 1). ). With the technique described in Patent Document 1, results with the same accuracy as PFGE can be obtained in about 3 hours, and objective genotyping can be performed.

  However, the technique described in Patent Document 1 requires four PCR tubes to analyze one strain (see [0062] and the like). Among the primers used for PCR, different primers may bind to each other to form a dimer, and thus four PCR tubes are necessary.

  For this reason, in the above technique, the number of MRSA strains that can be analyzed by a single PCR reaction is small. Therefore, the labor and time of analysis are extra, and the cost has not yet been reduced sufficiently.

  Furthermore, because it specializes in strain identification, it is not possible to distinguish between methicillin-resistant strains and sensitive strains, and in order to carry out the genotyping classification method described in Patent Document 1, another test (drug sensitivity test etc. ) To determine if S. aureus is methicillin resistant.

  In addition, as can be seen from Table 3 of Patent Document 1, the technique described in Patent Document 1 is based on MRSA called NewYork / Japan clone, which is SCCmec type Type II and is the most isolated in Japanese hospitals. It is thought that it was devised for the purpose of classifying neatly. For this reason, the technique described in Patent Document 1 is inferior in strain identification ability for MRSA and methicillin-sensitive Staphylococcus aureus other than New York / Japan clone.

  Furthermore, in the technique described in Patent Document 1, there is a problem that the band may become thin or no band may appear at all depending on the amount of suspension of bacteria at the time of template adjustment. Therefore, it is estimated that the technique described in Patent Document 1 has insufficient optimization of primer sequences used for PCR and sensitivity is insufficient, and the template DNA concentration is kept within an appropriate range for accurate determination. There is a need.

For these reasons, the technique described in Patent Document 1 requires a certain level of skill to implement.
Therefore, there is still a strong demand for the development of a method that can classify Staphylococcus aureus by genotype more easily and quickly.

JP 2006-141249 A

  In view of the above circumstances, an object of the present invention is to improve the technique described in Patent Document 1 and provide a method for classification of S. aureus genotypes that is more suitable for actual industrial (medical field) use. .

  Another object of the present invention is to provide a primer set for use in the above genotyping method.

  As a result of intensive studies to solve the above problems, the inventor of the present invention has (1) an open reading frame (ORF) of a phage-derived gene, (2) a Staphylococcal cassette chromosome mec on the genome of S. aureus. ; ORFs derived from genomic islands other than SCCmec), (3) ORFs derived from transposons, (4) ORFs derived from SCCmec, and (5) ORFs constituting the genomic islet are detected in any order. It was found that the classification according to the genotype of S. aureus can be performed more effectively by performing the classification according to the genotype by the combination of the presence or absence.

  In the S. aureus genotyping method according to the present invention, the genes in the ORF of (1) are SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998, PV83. , At least one gene selected from the group consisting of SAV0858 and SLTolf257, wherein the genomic island in the ORF of (2) is at least one gene selected from the group consisting of SaGIm, SaPIn1, SaPIn2 and SaPIn3 of the Mu50 strain, The transposon in the ORF (3) is transposon Tn554, and the genes in the ORF (4) are mecA, mec gene complex class A, and mec gene complex class B. It is at least one gene selected from the group consisting of SCCmec kdpC and cassette chromosome recombinase A2, and the genomic islet in the ORF of (5) is MW0919 of the MW2 strain (GenBank Accession Number BA000033), N315 strain (GenBank Accession Number BA000018) SA2259 and SA2124, MRSA-COL strain (GenBank Accession Number CP000046) SACOL1824, and MRSA252 strain (GenBank Accession Number X571856) SAR0228 are preferable.

Further, from the viewpoint of classifying by genotype with higher accuracy, the genes in the ORF of (1) are SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998 and PV83or. It is preferably at least one gene selected from the group consisting of
The genes in the ORF of (1) are at least eight genes selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998 and PV83orf2. More preferably, the genomic islet in the ORF of (5) is the MW0919 and the SA2259,
The gene in the ORF of (1) is at least 11 kinds of genes selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998, and PV83orf2. Further preferred.

Furthermore, it is also preferable that the genomic island in the ORF of (2) is SaGIm of Mu50 strain.
Moreover, the classification method according to the genotype of the present invention includes SEQ ID NOs: 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37. And 38, 39 and 40, 41 and 42, 43 and 44, and 13 sets of primers consisting of combinations of the base sequences shown in 45 and 46, by the PCR (polymerase chain reaction) method (1 The ORF of (2) above is detected by PCR using a pair of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 23 and 24 of the Sequence Listing. The ORF of (3) above was detected by PCR using a pair of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 9 and 10, and SEQ ID NOs: 3, 4, 5, and 6 in the sequence listing. , 7 and 8, 25 and 26, 31 and 32 The ORF of (4) above was detected by PCR using 5 sets of primers consisting of the combined base sequences, and 2 sets of bases shown in SEQ ID NOs: 27 and 28, 29 and 30 in the sequence listing The detection can be carried out by detecting the ORF of (5) above by PCR using two sets of primers comprising a combination of sequences.

  The primer may have additions, substitutions, deletions and insertions of 2 bases or less, but from the viewpoint of classifying by genotype with high accuracy, there are no additions, substitutions, deletions and insertions to the primers. It is preferable.

  Moreover, the classification method according to the genotype of the present invention includes SEQ ID NOs: 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37. 8 or more primers selected from the group consisting of 15 combinations of base sequences shown in 38 and 39, 40, 41 and 42, 43 and 44, 45 and 46, 53 and 54, 55 and 56 Using (1) ORF detection by PCR method, and using a pair of primers consisting of a combination of base sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing, the PCR method (2) The ORF is detected, and the ORF of (3) above is detected by PCR using one set of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing. Numbers 3 and 4, 5 and 6, 7 and 8, 25 and 2 , 31 and 32 are used to detect the ORF of (4) above by the PCR method using 5 sets of primers consisting of a combination of the base sequences shown in SEQ ID NOs: 27 and 28, 29 and 30, 47 And (5) ORF detection by PCR using 5 sets of primers consisting of 5 sets of nucleotide sequences shown in 48, 49 and 50, 51 and 52 Can do.

  From the viewpoint of classifying by genotype with higher accuracy, SEQ ID NOs: 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, PCR using the 11 or more sets of primers selected from the group consisting of 13 sets of base sequences shown in 37 and 38, 39 and 40, 41 and 42, 43 and 44, and 45 and 46. (1) It is preferable to detect ORF.

  The above primers may have additions, substitutions, deletions and insertions of 2 bases or less, but from the viewpoint of genotyping with high accuracy, there are no additions, substitutions, deletions or insertions in the primers. It is preferable.

When a multiplex PCR method is employed as the PCR method, the cost of genotyping can be reduced.
If the detection results of the presence / absence of ORF in (1) to (5) are replaced with 1 (yes) and 0 (no), respectively, and binary coding is performed, the detection results are easy to see.

Furthermore, if the detection results of the presence or absence of ORFs in (4) and (5) are combined into a binary code, MRSA epidemic clones can be easily determined.
By further converting the result of the binary encoding into a decimal encoding, the number of digits of the result is reduced, and the result becomes easier to see.

For carrying out the genotyping method of the present invention,
Sequence numbers 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 And 13 sets of primers consisting of combinations of base sequences shown in 44, 45 and 46, 1 set of primers consisting of combinations of base sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing, and sequences in the sequence listing 1 set of primers consisting of combinations of base sequences shown in Nos. 9 and 10, and base sequences shown in SEQ ID Nos. 3 and 4, 5 and 6, 7 and 8, 25 and 26, and 31 and 32 in the sequence listing Also included within the scope of the present invention is a primer set comprising 5 sets of primers consisting of the above combinations and 2 sets of primers consisting of combinations of the base sequences shown in SEQ ID NOs: 27 and 28, 29 and 30 in the sequence listing.

  The primer may have additions, substitutions, deletions and insertions of 2 bases or less. However, as described above, it is preferable that the primers have no additions, substitutions, deletions and insertions.

  According to the present invention, the classification of Staphylococcus aureus by genotype can be performed simply, quickly, objectively and with high sensitivity without requiring specialized equipment or operator skill. It is possible to promptly determine the route of infection and prevent the spread of nosocomial infections.

FIG. 1 shows an electrophoresis photograph of the example. In FIG. 1, “PC” is an abbreviation for positive control, and numerical values such as “1 2...” Indicate strain numbers. “M” indicates a 50 bp ladder. FIG. 2 shows an electrophoretic photograph of a comparative example. In FIG. 2, the numerical value described as “1 2...” Indicates the strain number. “M” indicates a 100 bp ladder.

Hereinafter, the present invention will be specifically described.
The method for classification of S. aureus genotypes according to the present invention comprises (1) an open reading frame (ORF) of a phage-derived gene on the genome of S. aureus and (2) a genomic other than a staphylococcal cassette chromosome (SCCmec). Island-derived ORFs, (3) transposon-derived ORFs, (4) SCCmec-derived ORFs, and (5) presence or absence of ORFs constituting the genomic islet are detected in an arbitrary order. It is characterized by having a step of classifying by genotype according to the combination of presence or absence of ORF. Hereinafter, these ORFs will be described.

[(1) ORF of phage-derived gene]
In recent years, genome decoding has progressed, and it has been clarified that the S. aureus genome is composed of approximately 2500 to 3000 ORFs.

  Patent Document 1 (Japanese Patent Laid-Open No. 2006-141249) reveals that detection of a combination of several ORFs of phage-derived genes present on the genome is extremely effective for MRSA strain identification. . However, in Patent Document 1, since ORFs of phage-derived genes possessed by various S. aureus have not been studied, the strain discrimination ability of S. aureus other than NewYork / Japan clone tends to be significantly reduced.

  Therefore, the present inventor designed primers for detecting ORFs of many phage-derived genes using GenBank's public information on S. aureus and S. aureus-related phages, and has various genetic backgrounds. Regarding the Staphylococcus aureus, the ORF of the phage-derived gene that they possess was examined.

As a result, SAV0850 (GenBank Accession No. BA000017),
SAV0898 (GenBank Accession No. BA000017),
SAV0866 (GenBank Accession No. BA000017),
SA1774 (GenBank Accession No. BA000018),
SAV1974 (GenBank Accession No. BA000017),
SAV0855 (GenBank Accession No. BA000017),
SAV0881 (GenBank Accession No. BA000017),
SLTolf175 (GenBank Accession No. AB045978),
SA1801 (GenBank Accession No. BA000018),
SAV0913 (GenBank Accession No. BA000017)),
SLTolf182 (GenBank Accession No. AB045978),
The present inventor has found that SAV1998 (GenBank Accession No. BA000017) and PV83orf2 (GenBank Accession No. AB044554) can be classified with high accuracy.

In addition, when these are detected using PCR and electrophoresis described later, DNA is easily amplified and an extra band hardly appears.
SAV indicates the gene of MR50 Mu50 strain, SA indicates the gene of N315 strain, SLTolf indicates the gene of phage SLT, PV83orf2 indicates the gene of phage PV83 Indicates that In addition, the number shown after SAV or the like indicates the gene of the indicated number in the whole genome.

  As other ORFs of phage-derived genes, SAV0858 and SLTolf257, and SAV0890, SAV0904, SAV0910, SAV1985, SA1791, SA1794, SA1806, SLTolf104, SLTor145, and the like listed in Patent Document 1, are classified according to genotype of Staphylococcus aureus. The detection of the presence or absence of these can also classify S. aureus with a certain degree of sensitivity.

The SAV0850 can be detected by PCR using the primers of SEQ ID NO: 11 (reverse) and 12 (forward) in the sequence listing,
SAV0898 can be detected by PCR using the primers of SEQ ID NO: 13 (reverse) and 14 (forward) in the sequence listing,
SAV0866 can be detected by PCR using the primers of SEQ ID NO: 15 (forward) and 16 (reverse) in the sequence listing,
SA1774 can be detected by PCR using primers of SEQ ID NOs: 17 (forward) and 18 (reverse) in the sequence listing,
SAV1974 can be detected by PCR using the primers of SEQ ID NO: 19 (forward) and 20 (reverse) in the sequence listing,
SAV0855 can be detected by PCR using the primers of SEQ ID NO: 21 (forward) and 22 (reverse) in the sequence listing,
SAV0881 can be detected by PCR using the primers of SEQ ID NO: 33 (forward) and 34 (reverse) in the sequence listing,
SLTolf175 can be detected by PCR using primers of SEQ ID NOs: 35 (forward) and 36 (reverse) in the sequence listing,
SA1801 can be detected by PCR using primers of SEQ ID NOs: 37 (reverse) and 38 (forward) in the sequence listing,
SAV0913 can be detected by PCR using the primers of SEQ ID NO: 39 (forward) and 40 (reverse) in the sequence listing,
SLT182 can be detected by PCR using the primers of SEQ ID NO: 41 (forward) and 42 (reverse) in the sequence listing,
SAV1998 can be detected by PCR using the primers of SEQ ID NOs: 43 (reverse) and 44 (forward) in the sequence listing,
PV83orf2 can be detected by PCR using the primers of SEQ ID NO: 45 (forward) and 46 (reverse) in the sequence listing.

Furthermore, by using the primers of SEQ ID NO: 53 (reverse) and 54 (forward) in the sequence listing, it is possible to detect the SAV0858 by PCR,
By using the primers of SEQ ID NO: 55 (forward) and 56 (reverse) in the sequence listing, it is possible to detect the SLTolf257 by PCR. General methods for designing primers for other ORFs will be described later.

  In the above, the detection of the ORF (1) of the phage-derived gene by the PCR method has been mainly described. However, the ORF (1) of the phage-derived gene can also be detected by other methods, for example, by hybridization. Is possible.

[(2) ORFs derived from genomic islands other than SCCmec]
SCCmec is a genomic island encoding methicillin resistance gene (mecA) and the like in the genome of S. aureus. Genomic island is a genome in which a large number of exogenous genes are grouped into the genome. Therefore, in terms of definition of a word, a genomic island includes a gene derived from a phage, but in this specification, a gene derived from a phage is not included in the genomic island. Moreover, what corresponds to the genomic islet mentioned later shall not be included in a genomic island in this specification.

  These genes derived from genomic islands other than SCCmec have a very low possibility of mutation as compared to genes derived from phage. Therefore, by combining the detection results of the presence / absence of possession of ORFs derived from genomic islands other than SCCmec with the detection results of the presence / absence pattern of possession of the phage-derived gene ORF, discrimination of genotype classification of Staphylococcus aureus The ability and the reliability of the determination result can be further improved.

Examples of genomic islands other than SCCmec include SaGIm, SaPIn1, SaPIn2, and SaPIn3.
Any of these can be classified as a target for detection, and S. aureus can be classified with good sensitivity. However, particularly when SaGIm is a target for detection, S. aureus can be classified with particularly high accuracy. SaGIm is a gene with unknown function, and its function has not yet been elucidated.

SaGIm can be detected by PCR using the primers of SEQ ID NO: 23 (reverse) and 24 (forward) in the sequence listing,
SaPIn1 can be detected by PCR using the primers of SEQ ID NO: 59 (forward) and 60 (reverse) in the sequence listing,
SaPIn2 can be detected by PCR using the primers of SEQ ID NO: 61 (forward) and 62 (reverse) in the sequence listing,
SaPIn3 can be detected by PCR using primers of SEQ ID NO: 63 (forward) and 64 (reverse) in the sequence listing. A general method for designing primers for other ORFs will be described later.

  In the above, detection of ORFs (2) derived from genomic islands other than SCCmec has been mainly described by PCR, but ORFs (2) derived from genomic islands other than SCCmec can also be detected by other methods. For example, it can be detected by hybridization.

[(3) ORF derived from transposon]
A transposon is a gene that is said to be involved in gene exchange between strains, has a region encoding an enzyme called transposase, and can move in the genome by the action of this enzyme.

  By determining the detection result of the presence or absence of such a transposon in combination with the detection result of the ORF (1) of the above-mentioned phage-derived gene and the ORF (2) of a genomic island other than SCCmec, the genotype of S. aureus The accuracy of another classification can be increased.

Examples of such transposons include Tn554, Tn552, and Tn916.
In particular, SCCmec Type II possessed MRSA possesses transposon Tn554, while SCCmec Type IV possessed MRSA does not possess much. Therefore, MRSA can be roughly divided into two types by detecting Tn554.

  Tn554 has multiple copies on the S. aureus genome. As described above, since there are a plurality of Tn554s on the genome, even if one Tn554 is mutated by point mutation, PCR amplification is possible with another Tn554, so it is considered that the possibility of false negative is reduced. Therefore, in addition to the detection results of the presence or absence of the ORF (1) of the phage-derived gene and the detection of the presence or absence of the genomic island-derived ORF (2) other than SCCmec, the presence or absence of the transposon Tn554-derived ORF (3) It is expected that determination by combining detection results can prevent false negatives and improve the identification ability of strain identification and the reliability of the results.

  Tn554 can be detected by PCR using the primers of SEQ ID NO: 9 (forward) and 10 (reverse) in the sequence listing. A general method for designing primers for other ORFs will be described later.

  In the above, the detection of the transposon-derived ORF (3) by the PCR method has been mainly described. However, the transposon-derived ORF (3) can also be detected by other methods, for example, by hybridization. Is possible.

[(4) ORC derived from SCCmec]
As described above, SCCmec is a region (genomic island) having a methicillin resistance gene mecA and the like. If SCCmec is present on the genome, the S. aureus is MRSA.

  The types of SCCmec are classified into type I to type V as shown in Table 1 below, depending on the combination of cassette chromosome recombinase type and mec gene complex class. Blanks are combinations that have not been discovered so far.

  As shown in Table 1 below, types of cassette chromosome recombinase include types 1-3 and type C, and classes of mec gene complex include classes A-C. Regarding the type of cassette chromosome recombinase, A is mainly A1 to B3, B is mainly B1 to B3, and C is mainly one type. If the type A is A1, the type B is always B1, and the numbers following A and B are always the same.

The ORF of mec gene complex class A can be detected by PCR using the primers of SEQ ID NO: 31 (reverse) and SEQ ID NO: 32 (forward) in the sequence listing,
The ORF of mec gene complex class B can be detected by PCR using the primers of SEQ ID NO: 5 (forward) and SEQ ID NO: 6 (reverse) in the sequence listing.

If no ORF is detected by any of these, the mec gene complex of MRSA is class C.
If the mec gene complex can be discriminated as described above, the presence or absence of the ORF of the cassette chromosome recombinase type A2 can be detected, and the SCCmec type can be discriminated based on the result.

  The cassette chromosome recombinase type A2 ORF can be detected by PCR using primers of SEQ ID NO: 25 (forward) and SEQ ID NO: 26 (reverse) in the sequence listing.

In addition, among SCCmec type II, it is possible to distinguish between type II a and II b by detecting ORF (kdpC) specific to SCCmec type II a.
When the present inventors collected and analyzed MRSA separated in Japan, the types of SCCmec possessed were mainly IIa, IIb, IV and V.

  Therefore, in SCCmec, not only can the sensitivity of methicillin be detected by detecting the ORF of the methicillin resistance gene mecA, but also the above SCCmec type can be estimated from combinations with other SCCmec-related ORF possession patterns. .

The above SCCmec type II a-specific ORF (kdpC) can be detected by PCR using the primers of SEQ ID NOs: 7 (reverse) and 8 (forward) in the sequence listing,
The ORF of mecA can be detected by PCR using the primers of SEQ ID NO: 3 (forward) and 4 (reverse) in the sequence listing.

  As described above, the type of SCCmec is discriminated, the presence or absence of the ORF of mecA is detected, and the results are combined with the ORF detection results of (1) to (3) above, so that the MRSA strain can be further detailed. It also contributes to ensuring the reliability of the classification obtained from the detection results up to the ORFs (1) to (3). Moreover, it is possible to determine whether Staphylococcus aureus has resistance to methicillin, which is impossible with the technique described in Patent Document 1. Therefore, the genotyping method according to the present invention does not require a test (drug sensitivity test) for determining whether S. aureus has methicillin resistance, which has been performed in the technique described in Patent Document 1. In this regard, the present invention is more economical (lower cost) than the technique described in Patent Document 1.

  In addition, as ORF derived from SCCmec, IS431, ORF constituting pUB110, and the like can be cited. MRSA can also be classified by detecting the presence or absence of these. However, since SCCmec types IIa and IIb can be classified and resistance to methicillin can be determined, in the present invention, ORF of mec gene complex class A, ORF of mec gene complex class B, Cassette is used as ORF derived from SCCmec (4). It is preferable to detect the ORF of chromosome recombinase type A2, the ORF of kdpC and mecA. A general method for designing primers for ORFs other than these ORFs will be described later.

  As described above, the ORF derived from SCCmec (4) includes ORF of mec gene complex class A, ORF of mec gene complex class B, ORF of cassette chromosome recombinase type A2, SCCmec type II a specific ORF (kdpC) and mecA It is desirable from the viewpoint of classifying MRSA with high accuracy by the minimum required ORF.

  In the above, the detection of the SCCmec-derived ORF (4) by the PCR method has been mainly described. However, the SCCmec-derived ORF (4) can also be detected by other methods, for example, by hybridization. Is possible.

[(5) ORF composing genomic islet]
(Genetic characteristics of Staphylococcus aureus isolated in Japan)
Multilocus sequence typing (MLST) analysis is used as a method for systematically classifying the genetic background of S. aureus.

  In MLST analysis, it is necessary to determine the base sequences of seven housekeeping genes, but the combination of detection ORFs required to easily determine the clonal complex (CC) type, which is a set of closely related MLST types Has already been reported. According to the report, it is necessary to detect 16 ORFs to determine the CC type, which is inefficient. Therefore, in order to perform CC type determination more efficiently, it is necessary to determine the minimum ORF sufficient to classify S. aureus isolated in Japan.

  S. aureus has a variety of genetic backgrounds. The genetic background survey results (including detection results of various ORFs) in actual wild strains can be used as fragmentary information in individual research facilities as literature. It is not enough to develop a classification method by genotype that can be classified.

Therefore, the present inventors collected methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolated in Japan, conducted MLST analysis, and investigated their genetic background. As a result, MRSA isolated in Japan was found to have many genotypes obtained by MLST analysis as clonal complex (CC) type 5, CC89 type, CC8 type, CC59 type, CC72 type and CC30 type. It became clear that it was particularly important to distinguish the former three among these genotypes.
Among methicillin-sensitive strains, it was found that there were many CC1, CC5, CC8, CC12, CC15, CC20 and CC45 types.

(ORF that composes genomic islet)
In order to distinguish the CC type, it has been found useful to detect ORFs constituting the genomic islet (see Table 2 below, “0” in Table 2 indicates that the genomic islet does not exist). "1" indicates that the genomic islet exists). Genomic islet refers to the part where the sequence differs by about 5 kbp or less when comparing bacterial genomes, which are scattered throughout the genome and do not affect the survival probability of the individual. It is thought that it was left behind in the process of evolution (reference: Journal of Applied Microbiology 107 (2009) 1367-1374).

As can be seen from Table 2, gene MW0919 of MRSA MW2 strain (GenBank Accession Number BA000033), gene SA2259 and gene SA2124 of N315 strain (GenBank Accession Number BA000018,), gene SACOL1824 of MRSA-COL strain (GenBank Accession Number CP000046) By detecting the gene SAR0228 of MRSA252 strain (GenBank Accession Number X571856), the CC type could be distinguished. It was also found that detection of genes MW0919 and gene SA2259 is effective for classifying CC5, CC8, and CC89 types, which are particularly important.

  In addition, for example, the MW0919 means that it is the 919th gene of the MW2 strain disclosed in GenBank, but this gene does not exist only in the MW2 strain, and strains derived therefrom, It may also exist in a different strain of S. aureus.

The gene MW0919 can be detected by PCR using primers of SEQ ID NOs: 29 (forward) and 30 (reverse) in the sequence listing.
Gene SA2259 can be detected by PCR using primers of SEQ ID NOs: 27 (reverse) and 28 (forward) in the sequence listing,
Gene SA2124 can be detected by PCR using primers of SEQ ID NOs: 51 (forward) and 52 (reverse) in the sequence listing,
The gene SACOL1824 can be detected by PCR using primers of SEQ ID NO: 49 (forward) and 50 (reverse) in the sequence listing,
The gene SAR0228 can be detected by PCR using primers of SEQ ID NOs: 47 (forward) and 48 (reverse) in the sequence listing.

  By detecting the above ORF, it is possible not only to distinguish between CC types but also to determine that MRSA epidemic clones are determined by a combination of clonal complex (CC) type and SCCmec type according to international agreements. Therefore, MRSA epidemic clones can be determined by combining with the detection result of the SCCmec-derived ORF (4).

Furthermore, by combining with other ORF detection results of (1) to (3) above, the strain identification ability in S. aureus in general is improved.
Among the ORFs (1) to (5) described above, Table 3 below shows ORFs that are particularly preferable to be used for classification by genotype and suitable PCR primers for detecting them. In Table 3 below, “bp” represents the size of base pairs amplified by PCR in terms of the number of base pairs. The femA of the amplified ORF shown in SEQ ID NOs: 1 and 2 is a gene encoding factor essential for expression of methicillin resistance, and is commonly found in Staphylococcus aureus, and is used as a positive control.

In addition, as shown in Table 4 below, ORFs that can be preferably used in the present invention as needed and primers useful for detecting them are listed.

[ORF detection means]
ORF of phage-derived genes (1), ORFs derived from genomic islands other than SCCmec (2), ORFs derived from transposon (3), ORFs derived from SCCmec (4) and genomic islet The presence / absence of the ORF (5) constituting the can be detected in any order. Specifically, these ORFs may be detected in any order for each ORF, may be detected collectively in any order for a plurality of ORFs, or all ORFs may be detected simultaneously. Means for detecting the ORF include PCR and hybridization.

<PCR>
When detecting separately for each ORF, for example, using a set of primers (forward primer and reverse primer) corresponding to each ORF to be detected, together with MRSA DNA extraction sample in an independent system, A real time PCR reaction is performed, and the signal fluorescence of the PCR amplification product is detected in real time.

  When detecting multiple ORFs collectively, for example, a primer for detecting femA as a positive control (SEQ ID NOS: 1 and 2 in the sequence listing) and multiple sets of primers corresponding to each of the multiple ORFs. Multiplex PCR can be performed in which (a set of forward primer and reverse primer) is mixed and placed in the same reaction system to perform PCR reaction. In this case, the presence or absence of a PCR amplification product corresponding to each ORF is confirmed by the presence or absence of a band obtained by electrophoresis of the reaction system by electrophoresis, for example, agarose gel electrophoresis or capillary electrophoresis.

When all ORFs are detected simultaneously, for example, a microarray is applied to detect the ORF. In the microarray, probes that hybridize with each ORF to be detected are prepared in each well of the microarray, DNA purified from the culture by column extraction or phenol-chloroform extraction is hybridized, and unreacted DNA is washed. After that, the target ORF can be detected by labeling with a fluorescent dye that binds to double-stranded DNA and measuring with a machine that captures fluorescence.
Among these, multiplex PCR is preferable because a plurality of ORFs can be efficiently and collectively detected with a simple apparatus.

(Primer design)
In addition to ORF (1) of the above phage-derived gene, ORF derived from genomic islands other than SCCmec (2), ORF derived from transposon (3), ORF derived from SCCmec (4) and ORF (5) constituting genomic islet, When designing primers corresponding to each of femA as a positive control, the primer itself bends and the complementary parts are combined to form a duplex, or different primers are complementary to each other. The sequence is such that it does not bind to form a dimer or higher conjugate at the part where it is present.

  Furthermore, in consideration of detection by multiplex PCR, it is preferable that the primer GC ratio is about 50% and that the Tm value is matched. Further, it is desirable to adjust the amplification efficiency so that the PCR amplification product size is about 50 bp to 700 bp, preferably 70 bp to 600 bp so that separation can be performed in a short time during electrophoresis. In addition, in ORF detected by the same reaction system, it is needless to say that primers are designed so that the sizes of PCR amplification products are not the same.

  As for the ORF (1) of the phage-derived gene, ORFs with a base sequence of about 90% are found in clinical isolates and databases. It is preferable to design primers that bind to primers that can be used universally for most S. aureus and are less susceptible to mutations.

  By setting the conditions as described above, it is possible to obtain primers and primer sets that can obtain highly reproducible amplification results in multiplex PCR. Such primer design can be performed by commercially available software.

Specifically, as described in the ORF items (1) to (5) above,
(1) 15 sets of primers (SEQ ID NOS: 11 to 22, 33 to 46, 53 to 56 in the sequence listing) as primers for the ORF of the phage-derived gene,
(2) Four sets of primers (SEQ ID NOs: 23 and 24, 59 to 64 in the sequence listing) as primers for ORFs derived from genomic islands other than SCCmec,
(3) A set of primers (SEQ ID NOs: 9 and 10 in the sequence listing) as primers for ORF derived from transposon,
(4) Five sets of primers (SEQ ID NOs: 3 to 8, 25 and 26, 31 and 32 in the sequence listing) as primers for the ORF derived from SCCmec, and (5) Primers for ORF constituting the genomic islet The present inventor designed a total of 30 primers of 5 sets of primers (SEQ ID NOs: 27 to 30 and 47 to 52 in the sequence listing).

  As will be described later, regarding the ORF (1) of the phage-derived gene, S. aureus can be effectively classified by genotype by using 8 or more primers. Furthermore, when 11 or more sets of primers are used, an accuracy of 95% is guaranteed with respect to the case where all the primers are used.

Among the above primers, considering the efficiency of genotyping and the reliability of the results,
As primers for ORF (1) of a phage-derived gene, SEQ ID NOs: 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36 in the sequence listing , 37 and 38, 39 and 40, 41 and 42, 43 and 44, and 13 sets of primers consisting of combinations of base sequences shown in 45 and 46,
As a primer for ORF (2) derived from genomic islands other than SCCmec, a set of primers comprising a combination of base sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing,
As a primer for transposon-derived ORF (3), a set of primers comprising a combination of base sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing,
As primers for ORF (4) derived from SCCmec, 5 sets consisting of combinations of nucleotide sequences shown in SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 25 and 26, and 31 and 32 in the sequence listing Primers, and
As primers for detection of ORF (5) constituting genomic islet, a total of 22 sets of primers consisting of 2 sets of primers consisting of combinations of nucleotide sequences shown in SEQ ID NOs: 27 and 28, 29 and 30 in the sequence listing A set can be used more suitably.

  When multiplex PCR is performed using the above primer set, femA is detected as a positive control in 11 primer sets, as shown in Table 5-1 and Table 5-2 below as primer mixture 1 and 2. It is desirable to carry out PCR in two reaction systems by mixing and mixing 12 pairs of primers to which primers (SEQ ID NOS: 1 and 2 in the sequence listing) are added.

The specific primers shown above are sequences optimized to amplify the target ORF, and even if these primers are modified such as addition, substitution, deletion, or insertion of about 2 bases, The function of is not lost and the target ORF can be amplified by PCR. Addition means that a base is added to the 5 'or 3' end of the primer shown in the table, and insertion means not between the above ends but between the base and the base inside the primer. Say that a base is added.

  From the viewpoint of exerting high performance as a primer, the change is preferably addition, substitution, deletion, or insertion of 2 bases or less, and addition of 2 bases or less, or 5 ′ side or 3 ′ side of 2 bases or less. Is more preferable, and addition of a base complementary to the target ORF of 1 base or less or deletion of 1 base or less at the 5′-end or 3′-end is particularly preferable. In general, changes on the 5 'side do not easily lose the function as a primer, and changes on the 3' side tend to lose the function of the primer.

<Hybridization>
When detecting ORF by hybridization, DNA purified from a culture (S. aureus) by column extraction or phenol-chloroform extraction is first alkali-denatured and fixed on a nylon membrane, cellulose membrane or microplate. Probes are prepared that hybridize with each ORF detected by the present invention. The probe may be artificially synthesized DNA or may be prepared by PCR using the primers described above. The probe is labeled using biotin, digoxigenin, a fluorescent dye, or the like. The bacteria-extracted DNA is hybridized with the probe, and according to the label of the probe, enzyme addition, color development, light emission, etc. are performed to capture the signal. This is performed for each detected ORF.

[ORF detection genotype classification method implementation method]
Hereinafter, multiplex PCR is performed, and the ORFs described in (1) to (5) above are detected as an example of the ORF that is preferable as the detection target. Will be explained.

(Identification of bacterial species)
From materials such as blood, sputum, pus, throat swabs, nasal swabs, and other patient-derived specimens or medical devices such as catheters and gauze used for patients, mannitol saline medium (for S. aureus isolation), MSO medium Isolate Staphylococcus aureus-like bacteria using a Staphylococcus aureus isolation medium such as (for MRSA isolation). Thereafter, it is identified as Staphylococcus aureus by confirmation of the form by Gram staining and confirmation of biochemical properties.

  A strain identified as Staphylococcus aureus can be added to a liquid medium (Luria-Bertani broth, Brain Heart Infusion medium, Tryptosoy bouillon, etc.) capable of growing S. aureus, or an agar medium (Luria-Bertani agar medium, Brain Heart). Infusion agar, trypsoy agar, normal agar, etc.) are cultured overnight at 37 ° C.

(DNA extraction)
DNA is extracted from the cultured cells by heat extraction, phenol extraction, or using a commercially available DNA extraction kit, and used as a sample.

(PCR reaction)
In multiplex PCR, ORFs (13 ORFs of phage-derived genes, 1 ORF derived from genomic islands other than SCCmec, 1 ORF derived from transposon, 5 ORFs derived from SCCmec, and 2 ORFs constituting genomic islet described in Table 3 above) In addition to 22), femA is detected as a positive control. Specifically, the number of primer pairs (forward primer and reverse primer) corresponding to the ORF group to be detected is divided into two groups of 12 pairs, and each 12 pairs are put in the same reaction tube ((1) (5) Since 22 sets of ORF primers and 1 set of positive control primers per tube, the total number of primers is 24 sets), and PCR reaction is performed.

(Electrophoresis)
The PCR amplification product is electrophoresed using a gel such as an agarose gel in which DNA fragments of approximately 50 bp to 600 bp are sufficiently separated. The migration distance may be about 5 to 6 cm, and in the case of a minigel, it can be sufficiently separated at about 100 V for about 50 minutes. After electrophoresis, photograph by staining with ethidium bromide.

(Result judgment)
A maximum of 12 bands appear per reaction. Unlike PFGE, since the band size is known in advance in the genotyping classification method of the present invention, it is determined whether or not there is a band of the desired size in each strain and reaction system. In some cases, non-specific bands other than the target size may appear, but the non-specific bands are ignored. A binary code is created with a band of the desired size (a band corresponding to each ORF and a band corresponding to a positive control) being 1 if no amplification is seen.

  This code may be created for each ORF of (1) to (5), or may be created as a single code, or a part of the ORFs of (1) to (5) May be created together. When the classification method according to the genotype of the present invention is used in an actual medical field, if all the ORFs (1) to (5) are created as one code, the number of digits becomes large and difficult to see. Since the number of codes increases when each ORF of (1) to (5) is created, it is preferable to create several ORF results together as one code.

  Here, as described above, according to international agreements, MRSA epidemic clones are determined to be determined by a combination of the clonal complex (CC) type and the SCCmec type. As described above, the clonal complex (CC) type can be determined by detecting ORF (5) constituting a genomic islet, and the type of SCCmec can be determined by detecting ORF (4) derived from SCCmec. In addition, the ORFs (1) to (3) do not encode proteins related to drug resistance of S. aureus, and are not important in taxonomics. Useful for enhancing.

  Therefore, it is preferable that the ORF detection results of (4) and (5) are combined into one code, and the ORF detection results of (1) to (3) are combined into one or two codes as appropriate. .

  Then, for example, in order to make the binary code created in this way easier to see by reducing the number of digits, it is converted into, for example, a decimal system to obtain a genotype code. An example of the above code creation is shown in Table 6 below.

First, the presence or absence of each ORF in the S. aureus to be tested is detected by PCR and electrophoresis using appropriate primers, and encoded by the binary method. And this code | cord | chord is converted into a decimal system and a genotype code | cord | chord is obtained (93 (code 1) -147 (code 2) -107 (code 3)).

  The correspondence relationship between the ORF numbers shown in Table 6 and the primers that can detect them is as shown in Table 7 below. In Table 7, “SEQ ID NO” corresponds to the SEQ ID NO in the Sequence Listing.

In the example shown in Table 6, since the code 1 is a summary of the detection results of the ORFs (4) and (5), when genotype codes are generated for a plurality of S. aureus strains, From 1 it can be determined whether the S. aureus is MRSA. Furthermore, by comparing a plurality of MRSA codes 1 determined to be MRSA, it is possible to determine whether or not the plurality of MRSA are the same epidemic clone.

  Code 2 is a summary of a part of the ORF of (1) and the detection results of the ORFs of (2) and (3). Code 3 is a code of the remaining detection results of the ORF of (1). is there. Therefore, for S. aureus, it is determined whether it is MRSA with code 1, and for a plurality of MRSA, it is determined whether the same epidemic clone is obtained by comparing code 1, and then code 2 and code 3 are By comparison, S. aureus strains can be identified in more detail. For example, Staphylococcus aureus collected from patients A and B are both MRSA and the epidemic clone is the same, but the code 2 is different, so the infection route is different, and patients B and C have the same epidemic clone of MRSA, Moreover, since the codes 2 and 3 are the same, it is possible to determine that the infection route is the same. Therefore, the genotyping method of the present invention is considered to be useful as a means for specifying the infection route of S. aureus in the medical field.

  Specifically, for example, in 48 clinical isolates of S. aureus (24 MRSA NewYork / Japan clone strains possessing SCCmec Type II, 13 MRSA strains other than NewYork / Japan clone, 11 methicillin sensitive strains (MSSA)) Table 8 below shows whether or not each ORF (ORF1 to 22) is held in binary coding and decimal coding in the same manner as described above.

In Table 8, the leftmost row is the number of the patient who sampled the strain. Only ATCC33591 is a general name for American type culture collection. In addition, “II b?” Indicates that the strain is a type II b strain but has some mutation.

  By coding the determination results in this way and comparing the obtained genotype codes, it is possible to easily and objectively identify strains separated at different times and facilities. Therefore, according to the classification method of S. aureus genotypes of the present invention, unlike PFGE, it is possible to compare genotypes between classification results of genotypes that are separated in time or distance.

  Further, as is apparent from Table 8 above, MSSA (methicillin-susceptible Staphylococcus aureus) has a code 1 value clearly smaller than that of MRSA (methicillin-resistant Staphylococcus aureus) (64 or more) (0 to 14). Therefore, it can be determined from the code 1 as described above whether the subject S. aureus is methicillin resistant or sensitive.

  Furthermore, the ORFs to be detected in the present invention are ORFs not related to the pathogenicity of Staphylococcus aureus, except for the ORF (4) derived from SCCmec. Genes with specific functions, such as those related to virulence, increase the probability that the Staphylococcus aureus will grow in the host, or the gene product will be the target of the host immune system and conversely reduce the probability of survival. There is. In other words, such genes are thought to be possessed by many of Staphylococcus aureus or vice versa. In the present invention, appropriate ORFs are selected and genotype classification is performed so that such a bias is not applied.

  Further, as will be apparent from Examples described later, when the drug resistance pattern of the clinically isolated Staphylococcus aureus strain and the type of SCCmec are investigated, it is found that there is a correlation between the two. That is, SCCmec type II and I-bearing strains have a strong tendency toward drug resistance, are only sensitive to vancomycin, arbekacin and linezolid, and are almost resistant to other drugs. On the other hand, SCCmec type IV-bearing strains are not prone to drug resistance and are often sensitive to macrolides, tetracyclines, clindamycins and new quinolones. Therefore, by investigating the type of SCCmec (detecting the ORF in (4)), it is possible to capture the drug resistance tendency of Staphylococcus aureus (MRSA).

  After performing genotyping in this way, the present invention may further classify S. aureus by genotype in more detail, for example, by additionally detecting ORFs with ORF numbers 23-30. . Table 9 below shows the status of possession of these ORFs in the 48 strains and the primer SEQ ID NOs for detecting these ORFs.

Furthermore, from Table 8, it can be seen that the above 48 strains can be classified by examining 8 types of phage-derived gene ORF (1). When the arrangement order of the items in Table 8 is changed so that it is easy to understand, Table 10 below is obtained. In Table 10, the shaded portion indicates the detection result of the phage-derived gene ORF (1).

Of the 48 strains in Table 8, 15 combinations in Table 11 (approximate genotype comparison table) shown below show differences of 2 or less phage-derived ORFs. Other combinations have three or more phage-derived ORFs or different ORFs are held in different states, and therefore can be discriminated even if they are typed by any 11 phage-derived ORFs.

However, the 15 pairs indicated by strain numbers 1 and 2 in Table 11 may not be separated when typed with any 11 phage-derived ORFs. Therefore, the ORF effective for discriminating 15 sets of strains is shown in Table 11 above. When two ORFs necessary for identification of two or more combinations of 15 ORFs effective for identification are removed, the strain identification ability may be significantly reduced. Such combinations include the case where ORF 16 and 21 are removed and the case where 5 and 22 are removed.

  When ORF16 and 21 are excluded, strain numbers 2002N192, 2006N438 and 2007N173 cannot be distinguished, and when ORF5 and 22 are removed, strain numbers 2002N035 and 2005N168, and 2002N281 and 2006N512 cannot be distinguished. In both cases, 48 strains are classified into 46 types, and even when any 11 ORFs are selected and detected, 15 sets of primers are detected to detect the ORF shown in (1) above. 95% discriminating ability when using is secured.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.
Forty-seven S. aureus strains clinically isolated at a hospital were classified by genotype by ORF detection according to the following procedure.

<Classification by genotype by ORF detection>
(Cultivation of Staphylococcus aureus)
Forty-seven strains identified as S. aureus were cultured overnight at 37 ° C. using Luria-Bertani agar.

(DNA extraction)
An amount corresponding to approximately 1 colony of the cultured bacteria was suspended in 100 μl of Tris-EDTA buffer containing lysostaphin at a concentration of 1 μg / 100 μl in a 1.5 ml Eppendorf tube, and digestion of the cell wall was performed at 37 ° C. for 10 minutes. Went. Thereafter, the tube was heated at 100 ° C. for 10 minutes. Next, the tube was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was used as a DNA heat extraction sample.

(Preparation of PCR reaction solution)
Roche's FastStart PCR Master was used according to the instructions. At that time, the reaction volume was 20 μl. The composition of 20 μl of the reaction solution is 10 μl of 2 × FastStart PCR Master and 8 μl of primer mixture 1 or 2. 2 μl (1/10 volume of the reaction solution) of the above-mentioned DNA heat extraction sample was added thereto.

  Multiplex PCR was performed on 24 ORFs using 12 pairs of primers (including 1 set of positive control primers) and 2 reaction tubes. The primer combinations (primer mixtures 1 and 2) and their final concentrations are as shown in Tables 12-1 and 12-2 below.

(PCR reaction)
Set the sample in a thermal cycler (Applied Biosytems, GeneAmp PCR System 9700), first treat at 95 ° C for 10 minutes, then 30 cycles of 94 ° C for 30 seconds, 60 ° C for 90 seconds, 72 ° C for 1 minute Repeated. After completion of the cycle reaction, the PCR product was stored at 4 ° C.

(Electrophoresis)
Using an agarose gel (4% NuSieve 3: 1 in TBE buffer), a minigel was prepared, and 5 μl of PCR product was electrophoresed at 100 V for 50 minutes. The migration distance was about 10 cm. After electrophoresis, it was stained with ethidium bromide and photographed.

(Result judgment)
A maximum of 12 bands appeared per reaction. It is determined whether or not there is a band of the target size in each strain and reaction system. If the band of the target size is amplified, 1 is set. A binary code was created. Further, the binary code was converted to the decimal system to obtain a genotype code.

  The results are shown in FIG. 1 and Table 13 below.

In Table 13, “PC” is a positive control and “MSSA” indicates methicillin-sensitive S. aureus.

  In Table 13, code 1 indicates an epidemic clone as described above. For example, all of strains 2 to 8 have the same code 1 of 93, but when codes 2 and 3 are compared, except for strains 2 and 6 Is actually a different strain.

[Comparative example]
<Classification by genotype by ORF detection described in Patent Document 1 (Japanese Patent Laid-Open No. 2006-141249)>
(Cultivation of Staphylococcus aureus)
The same 47 strains used in the examples were cultured overnight at 37 ° C. using Luria-Bertani agar.

(DNA extraction)
An amount corresponding to approximately 1 colony of the cultured bacteria was suspended in 100 μl of Tris-EDTA buffer containing lysostaphin at a concentration of 1 μg / 100 μl in a 1.5 ml Eppendorf tube, and digestion of the cell wall was performed at 37 ° C. for 10 minutes. Went. Thereafter, the tube was heated at 100 ° C. for 10 minutes. Next, the tube was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was used as a DNA heat extraction sample.

(Preparation of PCR reaction solution)
Takara Bio EXTaq was used according to the instructions. At that time, the reaction volume was 20 μl. The composition of 20 μl of the reaction solution is 10 × EXTaq Buffer 2 μl, dNTP mixture 1.6 μl, MgCl _{2 2} mM, the concentration of EXTaq is 0.6 unit / 20 μl, and the volume of the reaction solution excluding 2 μl of DNA heat extraction sample is 18 μl. It was adjusted with purified water (Buffer, dNTP, and MgCl ₂ had concentrations as instructed in the instruction manual, but the EXTaq concentration was set slightly higher than that in the instruction manual (0.25 to 0.50 unit / 20 μl).)

  Four sets of primers were used in combination with four reaction tubes, and multiplex PCR was performed on 16 ORFs. The primer combinations (Mix1 to Mix4) and final concentrations were as shown in Table 2-1 and Table 2-2 in Patent Document 1. However, kdpC detection primer (final concentration 0.15 μM, amplification by-product 123 bp, SEQ ID NOS: 57 and 58 in the sequence listing) was added to Mix2. 2 μl (1/10 volume of the reaction solution) of the above DNA heat extraction sample was added to the PCR reaction solution.

(PCR reaction)
Set the sample on a thermal cycler (GeneAmpTM PCR System 9600, manufactured by PERKIN ELMER), first treat at 94 ° C for 2 minutes, then 30 cycles of 94 ° C for 30 seconds, 53 ° C for 30 seconds, 72 ° C for 1 minute Repeated. After completion of the cycle reaction, the PCR product was stored at 4 ° C.

(Electrophoresis)
Using agarose gel (2.5% Agarose 1000 (Invitrogen) in 0.5X TBE), a minigel was prepared, and 5 μl of PCR product was electrophoresed at 100 V for 25 minutes. The migration distance was about 2 to 3 cm. After electrophoresis, it was stained with ethidium bromide and photographed.

(Result judgment)
A maximum of 4 bands appeared per reaction. It is determined whether or not there is a band of the desired size in each strain and reaction system. If the band of the target size is amplified, 1 is set, and 0 is indicated when amplification is not observed. A binary code was created, and the binary code was converted to a decimal system to obtain a genotype code.

  The results are shown in FIG.

In Table 14, since kdpC is a SCCmec type IIa marker, the strain in which it was detected is methicillin resistant, ie MRSA. However, although kdpC is not detected, for example, strain 1 has been identified as methicillin resistance in advance, it can be seen that it is MRSA, but it cannot be determined only by this genotyping classification.

  Further, as described above, cassette chromosome recombinase A2 and mec gene complex class A are detected from New York / Japan clone, which is SCCmec type II. Among the 47 strains, for example, 33-38 and 41-47. Since at least one of these has not been detected (see Table 13), it is a strain other than New York / Japan clone.

  From Table 13, according to the classification method according to the genotype of the present invention, among strains 33-38 and 41-47, strains 36, 37, 42, 43 and 45 are methicillin-sensitive staphylococci (MSSA), Although it is understood that it is MRSA, in the result obtained by the method of the comparative example, that is, in Table 14, for example, the genotype codes of strains 33 to 38 are all the same, and MRSA and MSSA can be distinguished. Absent.

  Furthermore, from Table 13, according to the classification method according to the genotype of the present invention, the strains 33 to 47 can be classified into 14 ways, but from Table 14, the method of the comparative example can be classified into only 4 ways. Absent. From this, it can be seen that the genotype classification method of the present invention is excellent in strain identification ability.

[Test example]
The drug resistance of Staphylococcus aureus clinical isolates was tested by the disk diffusion method using commercially available antimicrobial drug-containing disks. The results (number of resistant strains and ratio) are shown in Table 15 below.

[The invention's effect]
According to the present invention, the classification of Staphylococcus aureus by genotype can be performed simply, quickly, objectively and with high sensitivity without requiring specialized equipment or operator skill. Decide quickly and prevent the spread of nosocomial infections.

More specifically, the following effects (1) to (5) can be obtained.
(1) By designing a primer that can be used for complex multiplex PCR, it is possible to reduce the number of PCR reaction tubes per sample to two, thereby reducing implementation costs.

(2) It can be applied to a wide range of template concentrations by devising the primer sequence, and the unevenness of the amplification band is less likely to be uneven and easy to judge.
(3) By detecting the SCCmec-related ORF, not only can methicillin-resistant Staphylococcus aureus and sensitive Staphylococcus aureus be distinguished, but the type of SCCmec can be determined, and a pattern of drug resistance can be estimated.

(4) By detecting the ORF of the genomic islet, it is possible to specify the genetic origin of the genome, and the strain identification ability of all S. aureus is improved.
(5) By improving the ORF detection primer sequence of the phage-derived gene, without increasing the number of detection ORFs and without reducing the strain identification ability of NewYork / Japan clone, all S. aureus other than NewYork / Japan clone Strains can be identified.

Claims (17)

On the genome of S. aureus,
(1) Phage-derived gene open reading frame (ORF),
(2) ORFs derived from genomic islands other than Staphylococcal cassette chromosome mec (SCCmec),
(3) ORF derived from transposon,
(4) ORFs derived from SCCmec, and (5) ORFs constituting the genomic islet
A method for classifying Staphylococcus aureus according to genotype, comprising the step of detecting the presence or absence of serotypes in any order and classifying them according to the combination of the presence or absence of these ORFs. The gene in the ORF of (1) is at least one gene selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998, PV83orf2, SAV0858, and STL0257. Yes,
The genomic island in the ORF of (2) is at least one gene selected from the group consisting of SaGIm, SaPIn1, SaPIn2 and SaPIn3 of Mu50 strain,
The transposon in the ORF of (3) is transposon Tn554,
The gene in the ORF of (4) is at least one gene selected from the group consisting of mecA, mec gene complex class A, mec gene complex class B, SCCmec kdpC, and cassette chromosome recombinase A2,
The genomic islet in the ORF of (5) is MW0919 of MW2 strain (GenBank Accession Number BA000033), SA2259 and SA2124 of N315 strain (GenBank Accession Number BA000018), SACOL1824 and MRSA252 strain of MRSA-COL strain (GenBank Accession Number CP000046) The method according to claim 1, which is at least one gene selected from the group consisting of SAR0228 of (GenBank Accession Number X571856).   The gene in the ORF of (1) is at least one gene selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998 and PV83orf2. The method for classification according to genotype of Staphylococcus aureus according to claim 2. The genes in the ORF of (1) are at least eight genes selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998 and PV83orf2.
The method according to claim 2 or 3, wherein the genomic islet in the ORF of (5) is the MW0919 and the SA2259.   The gene in the ORF of (1) is at least 11 genes selected from the group consisting of SAV0850, SAV0898, SAV0866, SA1774, SAV1974, SAV0855, SAV0881, SLTolf175, SA1801, SAV0913, SLTolf182, SAV1998 and PV83orf2. The method according to claim 4, characterized by genotype classification of Staphylococcus aureus.   6. The method according to claim 1, wherein the genomic island in the ORF of (2) is SaGIm of Mu50 strain. Sequence numbers 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 And the detection of ORF of (1) above by PCR (polymerase chain reaction) method using 13 sets of primers consisting of 13 sets of base sequence combinations shown in 44, 45 and 46,
The ORF of (2) above is detected by PCR using a pair of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing,
The ORF of (3) above is detected by PCR using a set of primers consisting of a combination of base sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing,
Using the five primers consisting of combinations of the nucleotide sequences shown in SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 25 and 26, 31 and 32 in the sequence listing, the PCR method (4) Perform ORF detection,
The ORF of (5) above is detected by PCR using two sets of primers consisting of a combination of two sets of base sequences shown in SEQ ID NOs: 27 and 28, 29 and 30 in the sequence listing,
The method according to any one of claims 2 to 6, wherein the primer may have addition, substitution, deletion or insertion of 2 bases or less.   The method according to claim 7, wherein the primer has no addition, substitution, deletion, or insertion. Sequence numbers 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 And 44, 45 and 46, 53 and 54, and 15 or more sets of primers selected from the group consisting of combinations of base sequences shown in 55 and 56, and using the PCR method (1) ORF Detection
The ORF of (2) above is detected by PCR using a pair of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing,
The ORF of (3) above is detected by PCR using a set of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing,
Using the five primers consisting of combinations of the base sequences shown in SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 25 and 26, 31 and 32 in the sequence listing, the PCR method (4) Perform ORF detection,
Using the PCR method, 5 primers consisting of 5 combinations of base sequences shown in SEQ ID Nos. 27 and 28, 29 and 30, 47 and 48, 49 and 50, and 51 and 52 in the sequence listing ( 5) ORF detection
The method according to claim 2, wherein the primer may have addition, substitution, deletion, or insertion of 2 bases or less.   Sequence numbers 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 And (1) the ORF is detected by PCR using 11 or more pairs of primers selected from the group consisting of 13 combinations of nucleotide sequences shown in 44, 45 and 46. The method according to claim 9, wherein the S. aureus genotype is classified.   The method according to claim 9 or 10, wherein the primer has no addition, substitution, deletion, or insertion.   The method for classifying S. aureus according to any one of claims 7 to 11, wherein the PCR method is a multiplex PCR method.   13. The detection result of presence / absence of ORF in (1) to (5) is replaced with 1 (yes) and 0 (no), respectively, and is binary coded. Genotype classification of Staphylococcus aureus in Japan.   14. The method according to claim 13, wherein the detection results of the presence or absence of ORFs in (4) and (5) are combined into a binary code.   15. The method of genotyping S. aureus according to claim 13, wherein the binary encoding result is further decimal encoded. Sequence numbers 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 And 13 sets of primers consisting of combinations of the base sequences shown in 44, 45 and 46,
A set of primers consisting of a combination of the base sequences shown in SEQ ID NOs: 23 and 24 in the sequence listing;
A set of primers consisting of a combination of base sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing;
5 sets of primers comprising combinations of base sequences shown in SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 25 and 26, 31 and 32 in the sequence listing;
A primer set for classification according to genotype of Staphylococcus aureus, characterized in that it comprises two sets of primers consisting of combinations of base sequences shown in SEQ ID NOs: 27 and 28, 29 and 30 in the sequence listing,
A primer set for classification according to genotype of Staphylococcus aureus, which may have addition, substitution, deletion, or insertion of 2 bases or less.   The primer set according to claim 16, wherein the primer has no addition, substitution, deletion, or insertion.