Fig 1.
After selective pre-enrichment in overnight broth, sample cultures are screened for nuc presence in the primary detection step (Day 1). Negative results are released the same day and sample cultures positive for nuc are subcultivated on selective agar plates. The following day (Day 2), phenotypical MRSA colonies from subcultivated material are by PCR analyzed for presence of both nuc and mecA genes in a secondary detection step. MRSA positive as well as negative results from this PCR are released the same day. On the following day (Day 3), negative results from non-MRSA phenotypical samples are released.
Table 1.
nuc targeted oligonucleotides.
Fig 2.
Hydrolysis probe PCR oligonucleotides complementary to nuc gene.
Alignments were performed on the nuc gene in S. aureus EF529590.1 and in CC75 lineage/S. argenteus MSHR1132. Separate reverse primers were designed to enable complement to both aligned nuc sequences.
Fig 3.
Nuc amplification of CCUG31966 and CC75-08 achieved in hydrolysis probe PCR.
(a) S. aureus (CCUG31966) amplified with nuc-F+nuc-R1+nuc-R2 and nuc-F+nuc-R2. (b) CC75 lineage strain/S. argenteus (CC75-08) amplified with nuc-F+nuc-R1+nuc-R2 and nuc-F+nuc-R1.
Fig 4.
Nuc amplification of CC75-08 detected by degenerate probe in hydrolysis probe PCR.
Nuc amplification detection of CC75 lineage strain/S. argenteus (CC75-08) by degenerate probe and probe mismatching in one position. Amplification was performed using primers nuc-F, nuc-R1 and nuc-R2.
Fig 5.
Increased PCR sensitivity for CC75-08 in the hydrolysis probe PCR.
Analytical sensitivity equal for S. aureus (CCUG31966) in hydrolysis probe PCR and SybrGreen PCR. Analytical sensitivity increased for CC75 lineage/S. argenteus (CC75-08) strains in hydrolysis probe PCR.
Table 2.
Calculated PCR efficiency and lowest CFU detected/PCR reaction.
Table 3.
Results for primary PCR detection and culture for clinical samples.
Fig 6.
Increased stability using hydrolysis probe PCR.
Cq values from PCR control collected during one year from hydrolysis probe PCR (a) and SybrGreen PCR (b) show increased stability in hydrolysis probe PCR.