We describe and validate a novel PCR assay to detect the pandemic hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) lineage ST 239. Results based on previously uncharacterized isolates from a hospital in northeast Thailand support the view that at least 90% of HA-MRSA isolates in mainland Asia correspond to ST 239 or close relatives.Methicillin-resistant Staphylococcus aureus (MRSA) strains constitute a considerable health and resource burden in the hospital environment. Although there is currently a paucity of data (17), the evidence available suggests a high frequency of methicillin resistance throughout mainland Asia (9,14). Two recent papers have used multilocus sequence typing (MLST) to characterize hospital-acquired MRSA (HA-MRSA) isolates from nine Asian countries from Saudi Arabia to the Philippines (4, 15). These data suggest that at least 90% of the cases of HA-MRSA within a region accounting for Ͼ60% of the world's population can be accounted for by a single clonal subgroup, ST 239. This genotype has also been detected in 26 countries outside of Asia, corresponding to the EMRSA-1, -4, -7, -9, and -11, Brazilian (1), Portuguese, Hungarian (8), and Viennese clones, although in many countries, it has recently been replaced by other clones (1,5,7,8,13,16,(18)(19)(20).The global dissemination of this clone is consistent with heightened transmissibility, and two recent reports noted an association between ST 239 and increased virulence (2, 10). ST 239 has evolved through a large-scale recombination event involving the import of ϳ20% of the genome from CC 30 (ST 30-like), while the remaining 80% (genomic backbone) corresponds to CC 8 (ST 8-like) (21). As these two lineages are unrelated (6), this mosaic structure provides the means to develop a rapid, accurate, and sensitive PCR-based assay. We designed two pairs of discriminatory nonredundant primers based on two variable genes, one of which (SA2003) lies within the ST 8-like backbone, while the other (SA0317) lies within the ST 30-like imported region (21) (Fig. 1). As the primers were designed to amplify products of different sizes (220 bp for SA2003 and 484 bp for SA0317), it is possible to identify cases where both products are amplified from a single PCR. The presence of both amplicons of the predicted sizes indicates the presence of both the ST 30-and ST 8-like sequences in the hybrid genome of ST 239.The primer sequences used were ST 239/ST 30-like specific primers 5Ј-TCGCACTCTCGTTGAACA-3Ј (SA0317Forward) and 5Ј-AAATCCGCTTCGACAAACATT (SA0317Reverse) (Fig. 1) and ST 239/ST 8-like specific primers 5Ј-CACTTTAAATA CTGACGAAAAT-3Ј (SA2003Forward) and 5Ј-TTGAAAAT TGATCATTCAGCAA-3Ј (SA2003Reverse).Heteroduplex PCR was carried in a 25-l mixture consisting of 1ϫ PCR buffer, 1.5 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphate, 0.7 M of each of the four primers, 1.25 U of HotStart Taq DNA polymerase, and 2.5 l of DNA. PCR conditions were 95°C for 15 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. Figu...

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