SCCmec typing

SCCmec types can be determined by dentifying specific genes or gene alleles that are required for classification by PCR. We recommend to determine the type of ccr and class of mec first, then to investigate differences in J regions. The following PCR methods have been reported.

I. SCCmec type assignment

1.  PCRs for assigning ccr types

The ccr type could be determined by identifying ccr genes, which are located in the midst of the ccr gene complex.

(1) Identification of ccr1, ccr2, ccr3（Ito et al. 2001, Okuma et al. 2002）

　　A multiplex PCR to identify three gene alleles in a reaction with a primer common to three ccrB genes (ccrB1, ccrB2, and ccrB3), and three primers that are specific to each ccrA1, ccrA2, ccrA3 gene, respectively.

(2) Identification of ccr1, ccr2, ccr3, ccr5（ccrC） (Zhang et al. 2005)。

　　A multiplex PCR with a primer pair to identify ccrC and primers used in (1).

(3) Identification of ccr1, ccr2, ccr3, ccr4, and ccr5 (ccrC) (Kondo et al. in press).

　A multiplex PCR with two primer pairs to identify ccr4 and ccrC, and primers used in (1).

2．PCRs for assigning mec classes

The mec classes can be determined by identifying genes or gene alleles comprising a mec gene complex, e.g., mecA, mecR1, mecI, IS431, and IS1272. (1) Traditional PCR to identify each gene or gene allele (Okuma et al.2002).

(1) Traditional PCR to identify each gene or gene allele (Okuma et al.2002. Hisata et al. 2005. Chongtracool et al. 2006)

(2) A multiplex PCR to identify mec classes A and B（Zhang et al. 2005）

(3) A multiplex PCR to identify mec classes A, B, and C（Kondo et al. in press）

3. Decision of SCCmec type

The type of SCCmec is decided upon the type of ccr and class of mec, that are obtained by PCR.

II. Subtyping of SCCmec elements based on the structural differences in J regions

 PCRs to identify specific ORFs in J1 regions or to identify integrated copy of drug resistance plasmids or transposons, that are mostly integrated at J2 and J3 regions of SCCmec elements, have been reported.

1. PCRs for assigning J region differences

(1) Multiplex PCR （Oliveira et al. 2001）

The first multiplex PCR for assignment of SCCmec elements. The multiplex PCR can identify ORFs locate at J1 region of type-I.1 SCCmec and type-II.1.

(2) PCRs with pairs of primers specific to ORFs in J1 regions（Okuma et al.2002. Ma et al. 2005. Hisata et al. 2005）

Traditional PCR to identify specific regions locate at J1 region of SCCmec elements, e.g., type-II.1, type-II.2, type-IV.1, type-IV.2, type-IV.3, and type-IV.4.

(3) Multiplex PCR （Zhang et al. 2005）

A multiplex PCR to identify ORFs locate at J1 region of type-I.1, type-II.1, type-IV.1, type-IV.2, and type-IV.4 SCCmec elements. Note that a primer pair to identify type-III.1 SCCmec is designed on SCCmercury, and a primer pair to identify type-IV.3 SCCmec is designed on the outside of SCCmec designated IE25923.

(4) Multiplex PCR #3  (Kondo et al. in press)

A multiplex PCR to identify ORFs locate at J1 region of type I.1, type IV.1, type IV.2, type IV.3, type IV.4 SCCmec elements.

(5) Muliplex PCR #4 （Kondo et al. in press）

A multiplex PCR to identify ORFs locate at J1 region of type II.1, type II.2, type II.3, type II.4, type III.1, type V.1 SCCmec elements.

(6) Multiplex PCR#5 （Kondo et al. in press）

A multiplex PCR to identify transposons Tn554 and ΨTn554 at the J2 region of type II and type-III SCCmec elements.

(7) Multiplex PCR#6 （Kondo et al. in press）

A multiplex PCR to identify plasmids pUB110 and pT181, which integrated downstream of mecA.

2. Description of SCCmec subtypes

II. Primers used for PCR

Primers and reactions used for each PCR are listed

1. PCRs to identify ccr

2．PCRs to identify mec classes

3. Subtyping of SCCmec elements based on the structural differences in J regions

PCR

A.The method at the early stage

All PCR reactions were carried out in a GeneAmp PCR system 9600 (Perkin Elmer, U. S. A.). The reactions were performed in a final volume of 100 ml and contained 10 ng of template DNA, two oligonucleotide primers (0.1 mM), 200 mM of each dNTP, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl_2, 0.1% (w/v) gelatin and 2U of Taq DNA polymerase (Perkin Elmer, U. S. A). The PCR reaction consisted of 30 cycles of denaturation (94°C, 1 min), annealing (50°C, 1 min), and extension (72°C, 2 min). 

The method now used at Department of Bacteriology, Juntendo University.

1. Prepare the pre-mixture and aliquot to each tube. Pre-mixture should contain the following components in a volume of 49 mL per aliquot: 5 mL 10 x commercial reaction buffer containing MgCl₂, 250 mM of each dNTP, 2-4 oligonucleotide primers (0.1 mM), Taq DNA polymerase (1 unit), and DEPC-H₂O to adjust the final volume to 49 mL.

2. Add 1 mL of template DNA to each tube and subject to PCR.

3. The PCR conditions are: denaturation (94 °C, 1 min), 30 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 30 sec or 1 min), and extension (72 °C, 2 min). Keep samples at 4 °C following PCR.

4. Mount the agarose gel in the electrophoresis tank, and add enough 1x TAE buffer to cover the gel.

5. Mix 4 mL sample from each PCR reaction with 1 mL loading buffer and load samples into the wells of the gel. The gel is usually run at high voltage (100V). Stop running at appropriate time. Usually, we stop the procedure when the bromophenol blue has run 2/3 of the gel length.

6. DNA fragments in the agarose gel are stained by soaking in 0.01% Ethidium Bromide solution for 20 min.

7. Take a photograph using transmitted UV light with Fas II (UV sample camera, Toyobo, Tokyo, Japan).

B. Multiplex PCR (Oliveira et al. 2001)

The multiplex PCR was performed in a 50-µl volume with the GeneAmp PCR kit (Applied Biosystems, Foster City, Calif.) containing the following: 1x PCR buffer II; 200 µM (each) deoxynucleoside triphosphate; 400 nM concentrations of primers CIF2 F2, CIF2 R2, MECI P2, MECI P3, RIF5 F10, RIF5 R13, pUB110 R1, and pT181 R1; 800 nM concentrations of primers DCS F2, DCS R2, MECA P4, MECA P7, and IS431 P4; 200 nM concentrations of primers KDP F1, KDP R1, RIF4 F3, and RIF4 R9; 1.25 U of AmpliTaq; and approximately 5 ng of template DNA. PCR amplifications were performed in a DNA Thermal Cycler 480 (Applied Biosystems) with the following parameters: predenaturation for 4 min at 94°C; 30 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min; postextension for 4 min at 72°C; and soaking at 4°C. PCR products (10 µl) were resolved in a 2% SeaKem LE (BioWhittaker Molecular Applications, Rockland, Maine) agarose gel in 0.5x Tris-borate-EDTA buffer (Bio-Rad, Hercules, Calif.) at 100 V and visualized with ethidium bromide.

C. Multiplex PCR (Zhang et al. 2005)

All PCR assays were performed directly from bacterial suspensions obtained after the rapid DNA extraction method. An aliquot of 2 µl of this suspension was added to 23 µl of PCR mixture containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate (dATP, dUTP, dGTP, and dCTP) (Invitrogen Inc., Carlsbad, CA), various concentrations of the respective primers (Table 2), and 1.0 unit of Platinum Taq DNA polymerase (Invitrogen Inc., Carlsbad, CA).

The amplification was performed in a GeneAmp PCR system 9700 or 9600 Thermal Cycler (Applied Biosystems, Foster City, CA) beginning with an initial denaturation step at 94°C for 5 min followed by 10 cycles of 94°C for 45 seconds, 65°C for 45 seconds, and 72°C for 1.5 min and another 25 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 1.5 min, ending with a final extension step at 72°C for 10 min and followed by a hold at 4°C. For the single target amplification, PCR was run in 23 µl of PCR mixture but containing 0.2 µM of each primer, with cycling parameters beginning with an initial denaturation step at 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, ending with a final extension step at 72°C for 10 min. All PCR assay runs incorporated a reagent control (without template DNA). The PCR amplicons were visualized using a UV light box after electrophoresis on a 2% agarose gel containing 0.5 µg/ml ethidium bromide.

D. M-PCR#1 (M-PCR for identification of ccr) (Kondo et al. in press)

 Reaction mixtures contained 10 ng chromosomal DNA, oligonucleotide primers (0.1 mM), 200 mM each dNTPs, Ex Taq buffer, and 2.5 U Ex Taq polymerase (Takara Bio Inc., Kyoto, Japan) in a final volume of 50 ml. The concentration of MgCl₂ was 3.2 mM. A Takara PCR Thermal Cycler was used for amplification with an initial denaturation step (94°C, 2 min); 30 cycles of denaturation (94°C, 2 min), annealing (57°C, 1min), extension (72°C, 2 min); and a final elongation at 72°C for 2 min. PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.

E. For M-PCR#2-5 (M-PCR for identification of mec and J regions) (Kondo et al. in press)

Reaction mixtures contained 10 ng chromosomal DNA, oligonucleotide primers (0.1 mM), 200 mM each dNTPs, Ex Taq buffer, and 2.5 U Ex Taq polymerase (Takara Bio Inc., Kyoto, Japan) in a final volume of 50 ml. The concentration of MgCl₂ was 2.0 mM. A Takara PCR Thermal Cycler was used for amplification with an initial denaturation step (94°C, 2 min); 30 cycles of denaturation (94°C, 2 min), annealing (60°C, 1min), extension (72°C, 2 min); and a final elongation at 72°C for 2 min. PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.

F. M-PCR#6 (Kondo et al. in press)

M-PCR#6, we performed long-range PCR using the Expand High Fidelity PCR system following the manufacturer’s recommendations (Roche Diagnostics Co. Indianapolis, IN, USA). Briefly, reactions were performed in a final volume of 50 ml and contained 10 ng template DNA, oligonucleotide primers (0.3 mM), 200 ml of each dNTP, 1x Expand High Fidelity buffer, 1.5 mM MgCl₂ , and 2.6 U Expand High Fidelity PCR system enzyme mix. The PCR consisted of denaturation (94°C, 2 min); 10 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 8 min); 20 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 12 min); and final  elongation (72°C, 7 min).

 PCR products were electrophoresed in a 1% agarose-gel with 1 X TAE buffer at 100V for approximately 30 min. The DNA fragments were stained by soaking the agarose gel in 0.01% ethidium bromide solution for 20 min and were visualized using a UV transilluminator.