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.

Single, short primers of arbitrary nucleotide sequence were used in polymerase chain reactions to amplify regions of DNA isolated from several melanopline and oedipodine grasshoppers collected from local Saskatchewan populations. This represents one of the first applications of the method, called randomly amplified polymorphic DNA (or RAPD), to natural populations. Twenty-four different oligonucleotide primers, nine nucleotides in length, yielded clear and reproducible bands corresponding to amplified products and separable by agarose gel electrophoresis. On average, about 8.1 bands (range 0-17) were obtained per primer per individual. The mean percent similarity between band profiles of conspecific individuals was 51.2%, whereas the mean value for individuals representing different species or genera was 35.0%. Clearly, greater numbers of insects and primers will be required to achieve a satisfactory level of phylogenetic resolution. Given RAPDs technical advantages and ease of execution, however, this should not be problematic to the molecular systematist.

A reassortant influenza A(H1N1) virus of swine origin distinct from the pandemic H1N1 2009 strain was isolated from 3 patients, all of whom worked at the same large hog operation in Saskatchewan, Canada. The genomic composition of the isolates has not been previously reported, to our knowledge, and was the product of a genetic reassortment between seasonal H1N1 and triple-reassortant influenza virus that emerged in North American swine during the late 1990s. The neuraminidase and hemagglutinin genes of A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, and A/Saskatchewan/5131/2009 were derived from human H1N1 virus and were closely related to those of A/Brisbane/59/2007.

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 mixedgenotype 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.Infection by hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide (19). The overall prevalence of HCV infection in the United States is 1.8%, with most of the patients unaware of their infection and at risk for developing cirrhosis and hepatocellular carcinoma (15,21). HCV is a positive-sense, single-stranded RNA virus that displays extensive genetic heterogeneity (1). At least six major HCV genotypes comprising numerous, more closely related subtypes have been identified (26). HCV genotypes display significant differences in their global distribution and prevalence, making genotyping a useful method for determining the source of HCV transmission in an infected localized population (11). Furthermore, in addition to viral load and liver histology, the genotype of the infecting HCV strain appears to be an important determinant of the severity and aggressiveness of liver infection, as well as patient response to antiviral therapy (26). Consequently, several methods for genotyping HCV have been developed, including direct DNA sequencing (2, 5, 6), typespecific PCR (17), restriction fragment length polymorphism (16), line probe assays (22, 23), primer-specific and mispair extension analysis (7,8), heteroduplex mobility analysis by temperature gradient capillary electrophoresis (14)...

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Molecular diagnostic tools capable of identifyingShiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes , and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe , TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n ‫؍‬ 36) and a panel of O157 and non-O157 strains (n ‫؍‬ 64). PCR assays targeting stx1 and stx2 had variable specificity and sensitivity values with enriched stool samples. Molecular assays using DNA from pure cultures revealed that some primers were not sensitive to all stx2 variants. This evaluation concluded that the TaqMan-based probes were most appropriate in high throughput clinical diagnostic laboratories in consideration of cost , turn around time , and assay performance. (J Mol

Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation. Neonatal blood spots collected from all newborns from the high risk area were genotyped for the F188Δ mutation for seven consecutive years. Using DNA analysis, we estimated that the heterozygote frequency for the mutant allele for HHH syndrome to be about 1 in 19 individuals, predicting one affected child with HHH syndrome for approximately every 1,500 individuals (1 in 1,550 live births; 1 child every 12 years) in this isolated population. The frequency for the mutant allele for HHH syndrome in this isolated community is probably the highest in the world for this rare disorder. We determined that ornithine levels, by tandem mass spectrometry, were not abnormal in newborns with F188Δ mutation, carriers and normals. Ornithine rises to abnormally high levels at some time after birth well past the time that the newborn screening blood spot is collected. The timing or the reasons for the delayed rise of ornithine in affected children with HHH syndrome have not been determined. Newborn screening for HHH Syndrome in this high risk population is only possible by detection of the mutant allele using DNA analysis.

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the “gold standard” for molecular typing in epidemiology.