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In many families with histories suggestive of BRCA1-or BRCA2-related disease, the proband is deceased. Reliable assessment of archived tissue blocks not amenable to full gene sequencing would be helpful. In this study, a polymerase chain reaction (PCR) assay using primers that bracket the BRCAmutation site and microfluidics-based detection of heteroduplex/amplicon size differences was developed to circumvent artifacts associated with low quality DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Genomic DNA was extracted from 100 FFPE specimens from patients that had previously undergone BRCAgene sequence analysis on blood specimens. Conventional PCR amplification products were differentiated using the Agilent 2100 Bioanalyzer. One FFPE specimen failed to amplify the wild-type alleles for all three sites and was therefore called indeterminate. All 62 FFPE specimens with known Ashkenazi Jewish founder mutations had both the wild-type and the correct mutated allele amplified, including one specimen that failed to amplify the mutant allele in other real-time PCR assays. Appropriately, 21 FFPE specimens known to have other BRCA1/2mutations and 16 without any mutation had only the wild-type allele correctly amplified for each target. Therefore, by changing the primer location and detecting amplicons via heteroduplexes formed by size differences, we identified mutations from FFPE tissues missed using real-time methods.

Nasal carriage of Staphylococcus aureusis considered a source of subsequent infection in health care settings. Utilization of real-time polymerase chain reaction (PCR) for detection of S. aureushas the potential to dramatically affect infection control practice by rapidly identifying S. aureus-colonized patients. We developed and validated the use of real-time PCR for detection of S. aureuscolonization in two patient populations. Paired nasal swabs were collected from 299 neonates and from 151 adult patients at Evanston Hospital. One swab was used for culture and the other placed into a bacterial lysis solution containing achromopeptidase. The DNA liberated was used as the template for real-time PCR with primers for the femAgene. SYBR Green was used for amplicon detection. In the neonatal population the sensitivity, specificity, predictive value positive and predictive value negative for culture and PCR was 92%versus96%, 100%versus100%, 100%versus100%, and 98%versus99%, respectively. In the adults the results were 90%versus100%, 100%versus98%, 100%versus96%, and 95%versus100%, respectively. Real-time PCR was able to detect S. aureusin 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

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