Development and validation of a bacterial gastrointestinal multiplex RT-PCR assay for use on a fully automated molecular system.

PCR-based enteric multiplex panels represent a rapid and reliable alternative to conventional "classical" phenotypic stool diagnostics. The aim of this study was to establish a laboratory-developed non-commercial multiplex Real-Time-PCR panel for the detection of the most important bacterial stool pathogens, Salmonella spp., Shigella spp., Yersinia enterocolitica/ pseudotuberculosis and Campylobacter jejuni/coli. on the "open" cobas omni Utility Channel (UC) of the cobas 6800 system (Roche). The aim was to replace the laborious phenotypical stool diagnostics with a high throughput Real-Time PCR method. The respective primers and probes were designed to cover conserved genomic regions of the pathogens and validated using Ultramer oligonucleotides, positive stool material and reference strains. To further validate the multiplex PCR-assay, the following parameters were evaluated: analytical-sensitivity and -specificity, cross-reactivity, linearity and inter- and intra-assay variance as well as limit of detection (LOD). In addition a retrospective analysis of culture positive and negative samples from daily routine was performed using 745 native stool samples. The Gastro assay was linear over a 5-log-unit and within the expected dynamic range with amplification efficiencies ranging from 94.6% to 120%. In addition, all targets showed excellent coefficients of repeatability (≤ 1.11%), intermediate precision (≤ 1.02%) and total variance (≤ 1.39%). In terms of analytical sensitivity the assay demonstrated detection limits ranging from 7.83 copies per reaction to 14.4 copies per reaction. The assay showed excellent agreement with culture methods (>95%) and a 100% sensitivity and specificity after resolution of discrepant results. The multiplex-PCR assay provides a comprehensive, rapid and sensitive alternative to conventional methods for the detection of the major bacterial stool pathogens in diagnostic laboratories.

Post-mortem genetic investigation of cardiac disease-associated genes in sudden infant death syndrome (SIDS) cases.

The sudden infant death syndrome (SIDS) is one of the leading causes of postneonatal infant death. It has been shown that there exists a complex relationship between SIDS and inherited cardiac disease. Next-generation sequencing and surveillance of cardiac channelopathy and cardiomyopathy genes represent an important tool for investigating the cause of death in SIDS cases. In the present study, targeted sequencing of 80 genes associated with genetic heart diseases in a cohort of 31 SIDS cases was performed. To determine the spectrum and prevalence of genetic heart disease associated mutations as a potential monogenic basis for SIDS, a stringent variant classification was applied and the percentage of rare (minor allele frequency ≤ 0.2%) and ultra-rare variants (minor allele frequency ≤ 0.005%) in these genes was assessed. With a minor allele frequency of ≤ 0.005%, about 20% of the SIDS cases exhibited a variant of uncertain significance (VUS), but in only 6% of these cases, gene variants proved to be "potentially informative." The present study shows the importance of careful variant interpretation. Applying stringent criteria misinterpretations are avoided, as the results of genetic analyses may have an important impact of the family members involved.