Multiplexed spa-Typing Protocol for MRSA Identification

A staged spa-typing protocol was developed to enable identification of multiple-strain colonization on a large-scale [27 ]. The polymorphic X region of the protein A gene (spa) was amplified with primers 1095 F: 5′-AGACGATCCTTCGGTGAGC-3′ and 1517R: 5′-GCTTTTGCAATGTCATTTACTG-3′ [28 (link),29 (link)]. PCR reactions consisted of 0.25 mM dNTPs (Qiagen), 0.5 U of GoTaq Flexi DNA Polymerase (Promega), Colorless GoTaq Flexi Buffer, 2.5 mM of magnesium chloride and 0.25 μM of primers in a volume of 10 μl. PCR conditions were 94°C for 2 min; 35 cycles each of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 5 min. PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter).
Samples were sequenced with the same primers as used in PCR. Sequencing reactions used BigDye v3.1 sequencing mix (Applied Biosystems) and were cycled using 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min. Products were purified with Agencourt CleanSEQ beads (Beckman Coulter) and separated on an ABI 3730 DNA Analyzer (Applied Biosystems).
Chromatograms were analyzed using Ridom StaphType v2.0.3 software (Ridom GmbH). The relationships between spa-types were investigated using the BURP clustering algorithm [30 (link)] incorporated into Ridom StaphType.