Biomedical subjects

Matthew P Stevens

Publications and source records attributed to Matthew P Stevens.

4 recordsLinked to original sources

PubMed2006

Human papillomavirus genotyping using a modified linear array detection protocol.

A standardized PCR-based linear array genotyping assay has been developed (Roche Molecular Systems) allowing the concurrent typing of 37 human papillomavirus (HPV) types. A component of this assay, the PCR amplicon detection protocol, requires the use of a shaking water-bath incubator for amplicon/probe hybridization and subsequent stringent washing, which many laboratories may find cumbersome or simply not possess. In this study, the utility of a dry-air incubator in substitution of the recommended shaking water-bath for use in this assay was confirmed. DNA was extracted by AmpliLute method from 150 PreservCyt specimens collected from patients undergoing ablation treatment for histologically confirmed cervical abnormality. The DNA was amplified by PCR and amplicons detected by both standard and modified protocols, varying only by the method of incubation for DNA hybridization and stringent washing. Identical HPV-type profiles were generated in 149/150 (99.3%) of the specimens tested by both protocols, representing a near perfect level of concordance (kappa = 0.98). It was proposed that this modification would markedly simplify the detection process, reduce setup time and eliminate the potential for water spillover, thereby allowing more laboratories to adopt this method subsequent to in-house validation.

Cervix Uteri↗

PubMed2006

Sequence variation in the Newcastle disease virus genome.

Full-length genome sequences of five virulent and five avirulent strains of Newcastle disease virus isolated between 1998 and 2002 in Victoria and New South Wales, Australia were determined. Comparisons between these strains revealed that coding sequence variability in the haemagglutinin-neuraminidase (HN), matrix (M) and phosphoprotein (P) gene sequences appeared to be more variable than in the fusion (F), nucleocapsid (N) and RNA dependent-RNA replicase (L) genes. Sequence analysis of a number of other isolates made during the recent virulent NDV outbreaks, also identified the presence of a number of variants with altered F gene cleavage sites, which resulted in altered biological properties of those viruses. Quasispecies analysis of a number of field isolates indicated the presence of virulent virus in one particular isolate. Gene sequence analysis of the progenitor virus isolated in 1998 showed very little sequence variation when compared to that of a progenitor-like virus isolated in 2001, demonstrating that in the field, viral genome sequence variation appears to be biologically restricted to that of a consensus sequence.

Amino Acid Sequence↗

PubMed2006

Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests.

Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (kappa=0.93) and 95.1% (135 of 142) (kappa=0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P=0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated.

Cervix Uteri↗

PubMed2004

Characterization of Chlamydia trachomatis omp1 genotypes detected in eye swab samples from remote Australian communities.

Chlamydia trachomatis conjunctival samples collected over a 6-month period from individuals with clinical signs of trachoma and located in remote communities in the Australian Northern Territory were differentially characterized according to serovar and variants. The rationale was to gain an understanding of the epidemiology of an apparent increased prevalence of acute trachoma in areas thought to be less conducive to this disease. Characterization was performed through sequencing of a region of the omp1 gene spanning the four variable domains and encoding the major outer membrane protein. Nucleotide and deduced amino acid sequences were genotyped by using a BLAST similarity search and were examined by phylogenetic analyses to illustrate evolutionary relationships between the clinical and GenBank reference strains. The predominant genotype identified corresponded to that of serovar C (87.1%), followed by the genotype corresponding to serovar Ba (12.9%). All nucleotide and amino acid sequences exhibited minor levels of variation with respect to GenBank reference sequences. The omp1 nucleotide sequences of the clinical samples best aligned with those of the conjunctival C. trachomatis reference strains C/TW-3/OT and Ba/Apache-2. All clinical samples (of serovar C) exhibited four or five nucleotide changes compared with C/TW-3/OT, while all serovar Ba samples had one or two nucleotide differences from Ba/Apache-2. Phylogenetic analyses revealed close relationships between these Northern Territory chlamydial samples and the respective reference strains, although the high proportion of sequence variants suggests an evolutionarily distinct C. trachomatis population causing eye infections in Australia. Given that such genotypic information has gone unreported, these findings provide knowledge and a foundation for trachoma-associated C. trachomatis variants circulating in the Northern Territory.

Adolescent↗