Biomedical subjects

Ryan R McDonald

Publications and source records attributed to Ryan R McDonald.

4 recordsLinked to original sources

PubMed2006

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR.

BACKGROUND: Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization. OBJECTIVES: To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection. STUDY DESIGN: Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases. RESULTS: Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR. CONCLUSIONS: A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Caliciviridae Infections↗

PubMed2005

High-throughput qualitative multiplex 5' nuclease assay using post-only PCR analysis.

Homogenous fluorescence PCR assays offer distinct advantages for qualitative testing and are gaining immense popularity in fields like diagnostic microbiology. To meet the demand of high-volume laboratories, we developed a protocol for qualitative multiplex 5' nuclease assays using post-only PCR analysis. This novel approach overcomes throughput problems encountered with the established methods for TaqMan detection on the ABI PRISM 7700 Sequence Detection system and permits off-site TaqMan PCR, which can be analyzed several days after the reactions are completed. We have validated this novel protocol using an assay for the identification of Bordetella pertussis against real-time and plate-read analysis methods and have shown that our proposed protocol produces no difference in qualitative calls.

Bordetella pertussis↗

PubMed2005

Rapid genotyping of hepatitis C virus by primer-specific extension analysis.

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5' untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n=312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n=190), 12.8% type 2 (n=40), 20.2% type 3 (n=63), 0.3% type 4 (n=1), and 0.3% other (n=1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.

5' Untranslated Regions↗

PubMed2005

Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates. This assay facilitates the rapid identification of PVL-positive isolates of MRSA.

Bacterial Toxins↗