Six multiplex PCRs for SCCmec type assignment. (A) M-PCR 1 for identification of ccr genes for the assignment of the type of ccr gene complex. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, N315; lane 3, 85/2082; lane 4, CA05; lane 5, WIS; lane 6, HDE288. DNA fragments of the expected sizes for each ccr gene were amplified. The DNA fragment corresponding to mecA served as an internal control in each lane. (B) M-PCR 2 for identification of three gene alleles for assignment of the mec gene complex. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, N315; lane 3, 85/2082; lane 4, CA05; lane 5, WIS. DNA fragments of the expected sizes for class A mec (lanes 2 and 3), class B mec (lanes 1 and 4), and class C mec (lane 5) were amplified. (C) M-PCR 3 for J1 region difference-based subtyping of type I and type IV SCCmec elements, which carry the class B mec. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, CA05; lane 3, 8/6-3P; lane 4, 81/108; lane 5, JCSC4469. DNA fragments of the expected sizes for each J1 region were amplified. (D) M-PCR 4 for J1 region-based subtyping of type II and type III SCCmec elements, which carry the class A mec, and the type V SCCmec, which carries the class C mec. Chromosomal DNAs from standard strains were used as templates. Lane 1, N315; lane 2, JCSC3063; lane 3, BK351; lane 4, RN7170; lane 5, 85/2082; lane 6, WIS. (E) Identification of resistance determinants. Transposons (Tn554 and ΨTn554) were assigned by M-PCR 5 (lanes 1 to 3), and plasmids (pUB110 and pT181) were assigned by M-PCR 6 (lanes 4 and 5). SCCmercury was assigned with an M-PCR with the four sets of primers listed in Table 2 (lane 6). Chromosomal DNAs from standard strains were used as templates. Lanes 1 and 4, N315; lane 2, BK645; lanes 3, 5, 6, and 85/2082. MWM, molecular weight marker.