JP4379308B2 - Genotyping method of methicillin-resistant Staphylococcus aureus and primer set used therefor - Google Patents

Description

  The present invention relates to a methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) genotype classification method (strain identification method) and a primer set used therefor.

MRSA is a pathogen that is isolated in many hospitals. A hospital in Tokyo (925 beds) reported that MRSA was detected in 72 inpatients in one month, and another hospital in Kumamoto (550 beds) also reported 35 in one month. Reported that MRSA was detected in hospitalized patients. According to the Ministry of Health, Labor and Welfare's survey of infectious diseases, more than 20,000 cases are reported annually from only about 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 bacterium causing nosocomial infection, and it is necessary to specify the infection route in order to reduce nosocomial infection.

  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. In addition to the fact that the spread of infection has already been completed when the test results are obtained, it has a complicated band pattern. In order to make a determination, subjective factors tend to be included, and it is difficult to determine whether or not the genotype is the same except for strains that migrated simultaneously. Therefore, it was virtually impossible to compare genotypes between PFGE analyzes separated by time or distance.

  In addition, since the meaning of each band obtained by PFGE is unknown, it is difficult to objectively evaluate the subtle difference in size of the bands, and there is a difference in determining whether the genotype is the same by the judge May come out.

  Specifically, since genotyping by PFGE depends on the position of the restriction enzyme recognition site on the genome, even if one base of the restriction enzyme recognition site is mutated, three band differences may appear. is there. Therefore, it is said that in order to prove that two strains are different, at least four bands, preferably seven, are necessary (see Non-Patent Document 1).

On the other hand, even if there are many mutations other than the restriction enzyme recognition site, if there is no change in the length of the cleaved DNA fragment, it cannot be recognized as a band difference. For this reason, the same band pattern may be exhibited despite different strains. Therefore, the change in the band pattern due to PFGE does not necessarily match the number of gene mutations, making it difficult to classify bacteria by genotype based on the band pattern due to PFGE, especially MRSA genotype classification with many similar strains. Yes.

Furthermore, PFGE is complicated and requires skill of the operator, and the work time is long, and special equipment such as a pulse field gel electrophoresis apparatus is required, and high cost and time are required to perform genotyping. For this reason, even when mass infection is suspected, it is often difficult to perform PFGE on all strains.

For these reasons, it is difficult to use PFGE as a routine test, and there are almost no places where PFGE is performed in hospital laboratories.
In contrast, several methods using PCR as a rapid genotyping method to replace PFGE have been studied.

A typical example is the random amplification of polymorphic DNA (RAPD) method. The RAPD method is a method for detecting PCR amplification products by non-specific annealing of primers. However, since a large number of bands can be obtained, it is necessary to judge with a complex band pattern like PFGE, and furthermore, it depends on non-specific annealing of the primer, so identification for judging whether the strains are the same or not In addition to inferior ability, there is a problem that reproducibility is low, and it has not been put into practical use.

In addition, 16S-23S rDNA spacer amplification method to detect the difference in length of spacer DNA sandwiched between 16S rDNA-23S rDNA, protein A-gene using polymorphism of A protein specific to Staphylococcus aureus PCR, coagulase gene-PCR using the coagulase polymorphism specific to S. aureus, PCR characterisation of the hypervariable using the polymorphism near the methicillin resistance gene (mecA) Region (HVR) adjacent to mecA method has been tried. However, since these mainly focus on toxin-related genes and resistance genes as detection targets, sufficient discrimination ability is obtained from the viewpoint of genotyping of MRSA, which has many genetically closely related strains. It has not been put into practical use in clinical settings. Thus, the development of alternative methods for PFGE, especially the development of MRSA genotyping methods using PCR, has been extremely difficult (see Non-Patent Document 2).

Therefore, the development of MRSA genotyping methods that can be more easily, quickly and objectively determined is still strongly desired.
Tenover et al., J Clin Microbiol 1995,33: 2233-9 Stranden et al., J Clin Microbiol 2003, 41: 3181-6.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel MRSA genotype classification method that can be an alternative method of PFGE.
Another object of the present invention is to provide a primer set for use in the above genotyping method.

MRSA genotyping methods according to the present invention include (1) phage-derived open reading frame (ORF) and (2) Staphylococcal cassette chromosome on the genome of methicillin-resistant Staphylococcus aureus (MRSA). It is characterized by detecting the presence or absence of ORFs derived from genomic islands other than mec; SCCmec), and (3) ORFs derived from transposon in any order, and classifying by genotype according to the combination of the presence or absence of these ORFs.

  In the MRSA genotyping method, it is preferable that the genomic island other than the SCCmec is SaGIm of the Mu50 strain, and the transposon is transposon Tn554.

Furthermore, in the MRSA genotyping method,
Sequence number 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 And 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52 , 53 and 54, 55 and 56, 57 and 58, and 59 and 60 by PCR (polymerase chain reaction) method using 13 or more primers selected from the group consisting of combinations of base sequences The (1) phage-derived ORF is detected, and a PCR method is used to detect the (2) genomic island-derived ORF by using a set of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 in the Sequence Listing. ORF detection and sequence Wherein the of SEQ ID NO: 1 and PCR using a pair of primers comprising a combination of 2 to the indicated nucleotide sequence (3) mentioned as it is a preferred embodiment to detect the ORF from transposon,
Sequence number 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 And (1) the phage-derived ORF is detected by PCR (polymerase chain reaction) using 13 sets of primers consisting of combinations of the nucleotide sequences shown in Nos. 28, 29 and 30, and SEQ ID NO: (2) Genomic island-derived ORF was detected by PCR using a pair of primers consisting of combinations of base sequences shown in 3 and 4, and shown in SEQ ID NOs: 1 and 2 in the sequence listing. A more preferable embodiment is to detect the ORF derived from the (3) transposon by PCR using a pair of primers comprising a combination of base sequences.

In the MRSA genotyping method of the present invention, the PCR method is preferably a multiplex PCR method.
In the MRSA genotyping method according to the present invention, binary detection is performed by replacing the detection results of ORF presence / absence (1) to (3) with 1 (yes) and 0 (no), respectively. Preferably, the binary encoding result is further converted to a decimal encoding.

  The primer sets for classification according to genotype of MRSA according to the present invention are SEQ ID NOS: 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 13, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, and 13 pairs of primers, and bases shown in SEQ ID NOs: 3 and 4 in the sequence listing It is characterized by comprising one set of primers consisting of a combination of sequences and one set of primers consisting of a combination of base sequences shown in SEQ ID Nos. 1 and 2 in the sequence listing.

  According to the present invention, MRSA genotyping can be carried out simply, quickly, objectively and with high sensitivity without requiring specialized equipment or operator skill, so that the path of nosocomial infection can be quickly established. Can determine and prevent the spread of nosocomial infections.

More specifically, the following effects (1) to (5) can be obtained.
(1) By using a PCR method in which the result is found in a short time, it is possible to shorten the time until the result is obtained. Since the PCR reaction itself is completed in about 2 hours, the genotype is found within 4 hours from the sample preparation, and the infection source can be investigated quickly, which helps to prevent the spread of infection.
(2) Since a PCR reaction that can be performed only by a simple pipetting operation is used, anyone can perform it with a little training. Moreover, since the time required for the work can be greatly shortened, the labor cost for performing genotyping can be reduced.
(3) Since the ORF to be detected is known in advance, the result determination is clear and the reliability is improved.
(4) Since the genotype can be determined by the ORF possession pattern with different possession states for each strain, it is only necessary to determine whether or not the ORF is possessed, and the objectivity of the determination result can be improved. Furthermore, by coding the determination results, it is possible to easily and objectively compare the determination results of genotype classification performed at different times and facilities. Therefore, unlike PFGE, genotypes can be compared between judgment results that are separated in time or distance.
(5) Since all operations are performed in a liquid phase system and the operation is simple, automation by a machine is also possible. Therefore, genotype classification can also be carried out in the laboratory in the hospital for all MRSA detected in the routine examination.

Hereinafter, the present invention will be specifically described.
The MRSA genotyping method according to the present invention comprises (1) an open reading frame (ORF) derived from a phage, (2) an ORF derived from a genomic island other than a staphylococcal cassette chromosome (SCCmec), on the MRSA genome, And (3) The presence or absence of possession of ORFs derived from transposon is detected in an arbitrary order, and the classification according to the genotype is performed by the combination of presence or absence of different ORFs derived from these three types.

<(1) ORF derived from phage>
In recent years, genome decoding has progressed, and it has become clear that the S. aureus genome is composed of approximately 2500 to 3000 ORFs. Genomic differences in MRSA isolated in Japan are very small, and the main difference is known to be only in exogenous genomic islands and phages, but phage-derived genes are frequently mutated (acquired, dropped out). Because there is no data on the combination of multiple ORFs derived from phages that are actually possessed by individual MRSA isolates, to the best of our knowledge, we use ORFs derived from phages for genotyping. No attempt has been made to do so.

The present inventors paid attention to the phage lysogenized in this MRSA, and devised and examined the use of the presence / absence of the phage-derived ORF in genotype classification.
Each of the phages is composed of about 65 ORFs, and the sequence of ORFs and the combinations thereof are different for each phage. Therefore, the PubMed DNA database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) was searched, and from the search results, seven types of S. aureus-derived phages (phi11, phi12, phi13, phiETA, phiPVL, phiPV83, phiSLT) and five types of phage lysed to MRSA3 strain (N315 strain, Mu50 strain, MW2 strain) (N315 strain: 1 species (φN315), Mu50 strain: 2 species (φMu50A, φMu50B) ), MW2 strain: 2 types (φSa2mw, φSa3mw)) information is obtained, and the ORFs possessed by these 12 types of phages are used as ORFs to investigate whether MRSA isolates actually possess It was decided.

For the purpose of selecting those useful for genotyping from the approximately 780 (12 × 65) ORFs possessed by these 12 types of phages, the following examination was added.
As a selection method, among 780 ORFs, those that are common among multiple phages are compared on a computer, then 12 ORFs that are frequently shared among multiple phages are selected as the first candidate, and MRSA Among the isolates, 32 strains with large variations in the PFGE band pattern (29 SCCmec Type II strains, which account for over 90% of MRSA detected in domestic hospitals, and about several percent of MRSA isolated in domestic hospitals) SCCmec Type IV possessed strains 3) were used as representative strains, and it was confirmed by PCR whether or not these ORFs were retained. The confirmation work by PCR is to design the forward primer and reverse primer according to the conventional method so as to correspond to the base sequence contained in the specific ORF to be checked for possession, and to connect these primers and each DNA of MRSA. A PCR reaction was performed by a conventional method, and it was confirmed by confirming whether or not amplification of a DNA fragment corresponding to the above base sequence occurred.

In addition, by repeating the process of checking the presence of ORFs in the vicinity of ORFs that have been confirmed to be retained in the same way, the number of ORFs to be examined will be increased, and eventually a total of 60 ORFs will be obtained. Individual ORFs were examined. And about all these 60 ORFs, the holding situation in the above 32 representative stocks among MRSA isolates was investigated.

As a result, 19 ORFs were either possessed or not possessed by almost all of the above 32 strains.
Regarding the remaining 41 ORFs, it became clear that there was a difference in the state of possession by strains, and it was found that the presence or absence of these phage-derived ORFs and their combinations are useful for strain identification. Table 1 shows the relationship between the origin of these 41 ORFs and 41 sets of primers designed to be annealed with the nucleotide sequences contained in each of these 41 ORFs (Table 1; ORFs 3 to 43, SEQ ID NO: 5 to 86 in the sequence listing).

Furthermore, 28 ORFs (Table 1; ORFs 3 to 30) of them are possessed by about 20 to 80% of the 32 MRSA isolates, and the strains can be identified efficiently and accurately. It has been found. In particular, the possession pattern of 13 of these ORFs (Table 1; ORF 3-15) was specific to each strain, so using these 13 ORFs would allow sufficient identification of strains even with the smallest number of ORFs. It turns out that ability is demonstrated.

As mentioned above, there is a concern that the phage-derived genes are frequently mutated (acquired and dropped out), but with regard to the 13 ORFs (Table 1; ORFs 3 to 15), MRSA strains generated within the same population For example (for example, strains isolated in the same nosocomial infection case or at different times from the same person), it has been confirmed that even isolates with a temporal gap have the same possession pattern. This is presumed to be because MRSA strains isolated in special environments such as hospitals have few opportunities for contact with other strains and are less likely to mutate than previously thought. . Therefore, even when only the detection of the 13 phage-derived ORFs is used for classification according to the genotype of MRSA separated in a hospital or the like, it can be said that the reliability of the determination result is secured to some extent.

More specifically, 41 sets of primers (SEQ ID NOs: 5 to 86 in the sequence listing) designed to anneal with the base sequences contained in each of these 41 ORFs (ORFs 3 to 43 in Table 1).
Preferably, using 28 sets of primers (SEQ ID NOs: 5 to 60 in the sequence listing) corresponding to the base sequences respectively contained in 28 ORFs (ORF 3 to 30 in Table 1), Preferably more than 13 of these primers, particularly preferably 13 ORFs (ORF 3-15 in Table 1)
13 sets of primers (SEQ ID NOs: 5 to 30 in the sequence listing) corresponding to the nucleotide sequences contained in each
) To detect the presence or absence of phage-derived ORF on the MRSA genome by confirming whether or not amplification of the DNA fragment corresponding to the above base sequence has occurred. Can do.

Thereby, the identification information of the MRSA strain regarding the presence or absence of the phage-derived ORF can be obtained.
<(2) ORFs derived from genomic islands other than SCCmec>
In addition to SCCmec, 3 genomic islands, 5 Mu50 strains, and 4 MW2 strains have been inserted into the MRSA genome in addition to SCCmec. Are known. In the present specification, a genomic island is different from the above-described phage.

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 pattern of presence / absence of ORF derived from genomic islands other than SCCmec with the pattern of presence / absence of possession of phage-derived ORF, the discrimination ability of MRSA genotype classification and the reliability of judgment results are further improved. Can be improved.

Since the three genomic islands derived from the N315 strain are the same as those inserted into the Mu50 strain, the primers are among the ORFs specific to nine types (3 + 5 + 4-3 = 9) of genomic islands excluding duplicates. 19 easy-to-design items were selected and 32 of MRSA isolates were selected as representative strains in the same manner as the above-mentioned selection of phage-derived ORFs, and it was confirmed whether these 19 ORFs were possessed. . For confirmation, the forward primer and reverse primer are designed in a conventional manner so as to correspond to the base sequence contained in the specific ORF to be examined for possession, and these primers and each MRSA DNA are used for routine confirmation. PCR was carried out by this method, and it was confirmed by confirming whether or not amplification of a DNA fragment corresponding to the above base sequence occurred.

As a result, 32 representative MRSA isolates (29 SCCmec Type II strains, which account for over 90% of MRSA detected in domestic hospitals, and SCCmec, which accounts for several percent of MRSA isolated in domestic hospitals) 3 type IV possession strains) were divided into two strains of the OR50 derived from the Mu50 strain genomic island: the retained strain (10 strains) and the non-retained strain (22 strains). On the other hand, all the strains possessed the genomic island-derived ORF common to the N315 strain and the Mu50 strain, but none of the strains except the ORF derived from the MW2 strain genomic island possessed. This one ORF (derived from MW2 strain genomic island) is the same as the ORF derived from Mu50 strain genomic island, and the above 32 strains were divided into two strains (10 strains) and a strain not possessed (22 strains). Therefore, it is considered that the detection result of the phage-derived ORF can be complemented by detecting the presence or absence of the ORF and combining the detection result with the detection result of the phage-derived ORF described above.

Therefore, as the marker for classification by genotype, we adopted this ORF, which is the only ORF derived from the Mu50 strain genomic island, which also possesses the MW2 strain. Table 1 shows the relationship between the ORF and a set of primers designed by a conventional method so that they can be annealed with the nucleotide sequence contained in the ORF (Table 1; ORF2, SEQ ID NOs: 3 and 4 in the Sequence Listing). .

In addition, SCCmec Type IV possessing shares that account for about several percent of MRSA separated in hospitals in Japan for the sake of completeness, in addition to the above 3 shares, 7 shares were added, and a total of 10 MRSAs were also subject to the above ORF (Table 1; ORF2 )), It was found that these stocks were also divided into two, one with the ORF (8 shares) and one with no ORF (2 shares).

Therefore, in addition to the pattern of presence / absence of possession of phage-derived ORFs, the determination of the presence / absence pattern of possession of ORFs derived from genomic islands other than SCCmec will lead to improved strain identification ability and reliability of results. There is expected.

More specifically, a set of primers (SEQ ID NOs: 3 and 4 in the sequence listing) is designed by a conventional method so as to anneal with the base sequence contained in this ORF (Table 1; ORF2). Detect the presence or absence of ORFs derived from genomic islands on the MRSA genome by performing PCR reaction using the pair and confirming whether or not amplification of the DNA fragment corresponding to the above base sequence has occurred. be able to.

Thereby, the identification information of the MRSA strain regarding the presence or absence of ORF derived from genomic islands other than SCCmec can be obtained.
<(3) ORF derived from transposon>
MRSA possessing SCCmec Type II possesses Tn554, which is a kind of gene structure called transposon, which is said to be involved in gene exchange between strains, but does not possess most of MRSA possessing SCCmec Type IV. Therefore, MRSA can be roughly divided into two types by detecting Tn554.

Tn554 also has multiple copies on the MRSA genome. Since there are multiple Tn554s in this way, even if one Tn554 is mutated by point mutation, PCR is performed with another Tn554.
Since amplification is possible, the possibility of false negatives is thought to be low. Therefore, in addition to the detection result of the presence or absence of the phage-derived ORF and the detection result of the presence or absence of the genomic island-derived ORF other than the SCCmec, the detection result of the presence or absence of the possession of the transposon-derived ORF is also determined in combination. It is expected to be able to prevent negatives and improve strain specific identification ability and reliability of results.

Comparison of Tn554 sequences in the PubMed DNA database revealed that the transposase B subunit had few mutations, so the ORF of the transposase B subunit was adopted as a marker for MRSA genotyping. Table 1 shows the relationship between the ORF and a set of primers designed by a conventional method so that they can be annealed with the nucleotide sequence contained in the ORF (Table 1).
ORF1, SEQ ID NO: 1 and 2 in the sequence listing).

More specifically, a set of primers (SEQ ID NOs: 1 and 2 in the sequence listing) is designed by a conventional method so as to be annealed with the base sequence contained in this ORF (Table 1; ORF1). Detect the presence or absence of transposon-derived ORFs on the MRSA genome by performing a PCR reaction using the pair and confirming whether amplification of the DNA fragment corresponding to the above base sequence has occurred. Can do.

Thereby, the identification information of the MRSA strain regarding the presence or absence of transposon-derived ORF can be obtained.
Thus, in the present invention, in addition to the pattern of the presence or absence of possession of the phage-derived ORF, and the presence or absence of possession of the ORF derived from genomic islands other than SCCmec, the pattern of presence or absence of possession of the transposon-derived ORF is also detected, By determining, the MRSA strain specific identification ability and the reliability of the result can be further improved.

<ORF detection means>
Detection of the presence or absence of (1) phage-derived ORF, (2) genomic island-derived ORF other than SCCmec, and (3) transposon-derived ORF on the MRSA genome described above can be performed in any order. Specifically, any of a means for separately detecting these ORFs for each ORF, a means for collectively detecting each ORF, and a means for simultaneously detecting all ORFs may be employed as the detection means.

As a means for detecting each ORF separately, for example, using a set of primers (forward primer and reverse primer) corresponding to each ORF to be detected, in an independent system, together with an MRSA DNA extraction sample, A means for performing real time PCR reaction and detecting the generated signal fluorescence of the PCR amplification product in real time can be mentioned. In addition, as a means for collectively detecting each of a plurality of ORFs, for example, 3 to 4 sets of primers (a set of forward primer and reverse primer) corresponding to 3 to 4 ORFs are mixed to make the same reaction system. Multiplex PCR can be performed in which the PCR reaction is performed. In this case, each ORF
The presence or absence of a PCR amplification product corresponding to 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. Examples of means for simultaneously detecting all ORFs include detection of genes using a microarray.

Among these, multiplex PCR is preferable because a plurality of ORFs can be efficiently and collectively detected with a simple apparatus.
<Primer design>
In designing primers corresponding to the above (1) phage-derived ORF, (2) genomic island-derived ORF other than SCCmec, and (3) transposon-derived ORF,
Considering that the primers themselves do not form a double strand or bind to each other to form a dimer, the GC ratio is set to approximately 50% in consideration of detection by multiplex PCR. It is preferable to devise to match the Tm value. Further, it is desirable to adjust the amplification efficiency so that the PCR amplification product size is about 100 bp to 700 bp, preferably 150 bp to 500 bp so that separation can be performed in a short time during electrophoresis.

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.
Specifically, as described in (1) Phage-derived ORF, (2) Genomic island-derived ORF other than SCCmec, and (3) Transposon-derived ORF, (1) Primer for phage-derived ORF, respectively. 41 sets of primers (SEQ ID NO: 5 to 86 in the sequence listing), (2) 1 set of primers (SEQ ID NO: 3 and 4 in the sequence listing) as primers for genomic island-derived ORF other than SCCmec, and (3 ) A total of 43 sets of primers (SEQ ID NOs: 1 and 2 in the sequence listing) as primers for the transposon-derived ORF can be used in the genotyping method of the present invention.

Among these, considering the efficiency of genotyping and the reliability of the results, (1) as primers for phage-derived ORF, SEQ ID NOs: 5 and 6, 7 and 8, 9 and 10
11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 And 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60 13 or more primers selected from the group consisting of 28 base sequence combinations shown in (2), and (2) primers for genomic island-derived ORFs other than SCCmec are shown in SEQ ID NOs: 3 and 4 in the Sequence Listing. As a primer for a transposon-derived ORF, a set of primers consisting of a combination of base sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing Reimer, primer set consisting of can be suitably used, further, as a primer for the among at even (1) phage-derived ORF, and SEQ ID NO: 5 6,7 and 8
, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30 (2) As a primer for a genomic island-derived ORF other than SCCmec, a set of primers consisting of a combination of the base sequences shown in SEQ ID NOs: 3 and 4 in the sequence listing, and ( 3) As a primer for transposon-derived ORF, a total of 15 primer sets consisting of a set of primers consisting of a combination of base sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing can be used more suitably. .

When performing multiplex PCR using the above primer set, Table 2-1
And as shown as Mix 1-4 in Table 2-2, it is desirable to combine and use 3-4 sets of primers among these primer sets.

<Method to implement ORF detection genotype classification method>
Hereinafter, the procedure of the classification method according to the genotype of MRSA of the present invention will be described by taking the case of performing multiplex PCR as an example.

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). Subsequently, MRSA is confirmed by confirming the morphology by Gram staining and biochemical properties by confirming the susceptibility to Staphylococcus aureus by drug susceptibility testing by the diffusion disk method or MIC method, or mecA gene detection by PCR.

  For strains identified as MRSA, liquid media that can grow S. aureus (Luria-Bertani broth, Brain Heart Infusion media, tryptic soy bouillon, etc.) or agar media (Luria-Bertani agar, Brain Heart Infusion agar) Medium) at 37 ° C. overnight using a medium, tryptosoy agar medium, ordinary agar medium, etc.

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:
ORF (preferably, a total of 15 ORFs derived from 13 phages, 1 ORF derived from genomic islands and 1 ORF derived from transposon) is detected by multiplex PCR. Specifically, the number of primer pairs (forward primer and reverse primer) corresponding to the ORF group to be detected is divided into 3 to 4 groups, and every 3 to 4 groups are put in the same reaction tube, and the PCR reaction is performed. Do.

Electrophoresis:
The PCR amplification product is electrophoresed using an agarose gel in which DNA fragments of approximately 100 bp to 700 bp are sufficiently separated. The migration distance may be about 2 to 3 cm, and in the case of a minigel, it can be sufficiently separated at 100 V for about 25 minutes. After electrophoresis, take a photograph after staining with ethylene bromide.

Result judgment:
Up to 4 bands appear per reaction. Unlike PFGE, since the band size is known in advance, 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 1 if the band of the desired size is amplified and 0 if no amplification is seen. A binary code is converted to a decimal system to obtain a genotype code.

Specifically, for example, 32 MRSA isolates (29 SCCmec Type II holdings and SCCmec Type IV)
Table 3 shows whether or not 15 ORFs (Table 1; ORF 1 to 15) are held in binary code and further decimal code.

  Thus, by encoding the determination result and comparing the obtained genotype codes, it is possible to easily and objectively specify the strains separated at different times and facilities. Therefore, according to the genotype classification method of MRSA of the present invention, genotypes can be compared between genotype classification results that are separated in time or distance, unlike PFGE.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.
[Examples and Comparative Examples]
For MRSA 165 strains isolated at 4 hospitals in Aichi, Mie and Ishikawa prefectures, genotype classification by ORF detection (Example) and genotype classification by PFGE (comparative example) according to the following procedure.
Went.

<Classification by genotype by ORF detection (Example)>
MRSA culture:
A strain identified as MRSA was cultured overnight at 37 ° C. using Luria-Bertani agar.

DNA extraction:
Suspend the amount corresponding to approximately 1 colony of the cultured bacteria in 100 μl of distilled water containing 1 μg / 100 ml of lysostaphin in a 1.5 ml Eppendorf tube, digest the cell wall at 37 ° C. for 5 minutes, and then heat at 100 ° C. for 10 minutes did. Next, the mixture 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 was 10 × EXTaq Buffer 2 μl, dNTP mixture 1.6 μl, MgCl _{2 2} mM, and the concentration of EXTaq was 0.6 unit / 20 μl. (Buffer, dNTPs, but the MgCl ₂ is the concentration of the instruction as the instruction manual, ExTaq concentration was set slightly higher than the mark's instructions (0.25~0.50 unit / 20μl).)
Multiplex PCR was performed on 15 ORFs in 4 reaction tubes by combining 3-4 sets of primers. Table 2 shows primer combinations (Mix1 to Mix4) and final concentrations.
-1 and Table 2-2. 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 in a thermal cycler (GeneAmp ^™ 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 times. After completion of the cycle reaction, it was stored at 4 ° C.

Electrophoresis:
Using an agarose gel (2% DNA Agar (Marine BioProducts, BC Canada) in 0.5X TBE), a minigel was prepared, and 5 μl of the 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 ethylene bromide and photographed.

Result judgment:
A maximum of 4 bands appeared per reaction. Determine whether there is a band of the desired size in each strain and reaction system, and create a binary code with 1 if the band of the desired size is amplified and 0 if no amplification is seen did. Further, the binary code was converted to the decimal system to obtain a genotype code.

The results are shown in Tables 4-1 to 4-3.
<Classification by genotype by PFGE (comparative example)>
According to the conventional method, classification according to genotype by PFGE was performed by the following method.
MRSA culture:
The culture was shaken overnight at 37 ° C. using Luria-Bertani broth medium.
Creating a fungus block:
Place 150 μl of the culture in a 1.5 ml Eppendorf tube and centrifuge at 8,000 rpm for 3 minutes.
The supernatant was discarded and collected. The collected strain was suspended in 200 μl of 0.8% agarose gel containing 1 μg / 100 μl of lysostaphin, and then agarose was coagulated to prepare a bacterial block.
Lysis treatment:
The fungus block was then immersed in 0.5M EDTA containing lysostaphin at a ratio of 0.2 μg / 100 μl.
Digested at 4 ° C for 4 hours. Subsequently, the solution was replaced with 0.5 M EDTA containing 1% / ml of Proteinase K and N-Lauroylsarcosine, and digested overnight at 50 ° C.
Inactivation of Proteinase K:
Then, change the solution to Tris EDTA (TE) buffer containing Pefabloc SC at a rate of 1 mg / ml, wash twice for 30 minutes with gentle shaking at 50 ° C, inactivate Proteinase K, and then use TE buffer. Washed for 1 hour.
Restriction enzyme treatment:
The bacterial block was cut into about one third of the original size, washed with a restriction enzyme buffer for 30 minutes, and treated with restriction enzyme (SmaI, 30 unit / sample) at 30 ° C. for 4 hours.
Electrophoresis:
The bacterial block that has been treated with restriction enzymes is further cut into thin sections, applied to a 1% agarose gel (BIO RAD, Pulsed Field Certified Agarose), and switching time 1 using a PFGE electrophoresis apparatus (BIO RAD, CHEF-DR III). Electrophoresis was performed for 22 hours under conditions of ˜50 seconds, inner angle of 120 °, and 14 ° C.

The gel after electrophoresis was stained with ethylene bromide and photographed.
Image analysis and result determination:
The obtained band pattern was analyzed by image analysis software (BIO RAD, FingerPrinting II) to give the name PFGE. Cluster names were added alphabetically to a set of strains with relative homology of 80% or more in the analysis results. Subsequently, sub-cluster names are assigned with Roman numerals to a set showing a relative value of homology between strains classified into the same cluster of 80 to 90%, and at least one band is different within the same sub-cluster. Added pattern names with Arabic numerals. Therefore, the genotype name represented by alphabet-Roman numeral-Arabic numeral was used.

The results are shown in Tables 4-1 to 4-3.
As a result, it was classified into 82 types by PFGE, and 86 types by genotyping by ORF detection. In addition, strains derived from the same nosocomial infection case were the same type for both ORF and PFGE detection.

More specifically, from Table 4-1, it can be seen that there is a one-to-one correlation between the determination results of the genotype classification method of the present invention and the conventional method PFGE for 52 MRSA isolates.
From Table 4-2, it can be seen that 42 MRSA isolates can be classified into more genotypes according to the genotype classification method of the present invention even if the PFGE determines a single genotype. In the classification method according to the genotype of the present invention, genomic DNA varies depending on the strain depending on the combination of (1) ORF derived from phage, (2) ORF derived from genomic island other than SCCmec, and (3) ORF derived from transposon. This difference is presumed to be because the ability to discriminate strains is improved because false negatives can be prevented and detected efficiently.

Further, from Table 4-3, 71 strains of MRSA isolates were determined to be a single genotype according to the genotype classification method of the present invention, even though the strain was determined to be a plurality of genotypes by PFGE. I understand. This is presumably because the genotyping classification method of the present invention can objectively determine the result based on whether or not the ORF is held, so that there is no variation in determination result reading as in PFGE.

Forward primer for methicillin-resistant Staphylococcus aureus ORF.
Reverse primer for methicillin-resistant Staphylococcus aureus ORF.

Claims (5)

On the genome of methicillin-resistant Staphylococcus aureus (MRSA), (1) phage-derived open reading frame (ORF), (2) MuF strain SaGIm- derived ORF, and (3) transposon Tn554- derived MRSA genotype classification method that detects the presence or absence of ORF in any order, and classifies by genotype according to the combination of the presence or absence of these ORFs ,
Sequence number 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 And (1) detection of the ORF derived from the phage by PCR (polymerase chain reaction) method using 13 sets of primers consisting of a combination of the nucleotide sequences shown in FIGS.
(2) Detection of ORF derived from SaGIm of the Mu50 strain by PCR using a set of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 in the sequence listing, and
The detection of ORF derived from the above-mentioned (3) transposon Tn554 is carried out by PCR using a set of primers consisting of a combination of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing.
MRSA genotype classification method. 2. The method according to claim 1 , wherein the PCR method is a multiplex PCR method. The MRSA gene according to claim 1 or 2 , wherein the detection results of the presence or absence of ORF in (1) to (3) are replaced with 1 (yes) and 0 (no), respectively, and binary coded. Classification by type. 4. The MRSA genotyping method according to claim 3 , wherein the binary encoding result is further decimal encoded. Sequence number 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 And 13 sets of primers consisting of combinations of base sequences shown in 28, 29 and 30;
A set of primers consisting of a combination of base sequences shown in SEQ ID NOs: 3 and 4 in the sequence listing;
A primer set for classification according to genotype of MRSA, comprising a set of primers comprising a combination of base sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing.