Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis.

There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.

Approximating the Set of Local Minima in Partial RNA Folding Landscapes

Motivation: We study a stochastic method for approximating the set of local minima in partial RNA folding landscapes associated with a bounded-distance neighbourhood of folding conformations. The conformations are limited to RNA secondary structures without pseudoknots. The method aims at exploring partial energy landscapes pL induced by folding simulations and their underlying neighbourhood relations. It combines an approximation of the number of local optima devised by Garnier and Kallel (2002) with a run-time estimation for identifying sets of local optima established by Reeves and Eremeev (2004).

Results: The method is tested on nine sequences of length between 50 nt and 400 nt, which allows us to compare the results with data generated by RNAsubopt and subsequent barrier tree calculations. On the nine sequences, the method captures on average 92% of local minima with settings designed for a target of 95%. The run-time of the heuristic can be estimated by O(n2D?ln?), where n is the sequence length, ? is the number of local minima in the partial landscape pL under consideration and D is the maximum number of steepest descent steps in attraction basins associated with pL.

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16s rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16s rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

A nine-base nucleotide sequence in the porcine circovirus type 2 (PCV2) nucleocapsid gene determines viral replication and virulence

Porcine circovirus type 2 (PCV2) was retrospectively identified by serology in swine populations as an asymptomatic infection at least 25 years prior to the first reported case of PCV2-associated postweaning multisystemit wasting syndrome (PMWS). To investigate the sudden emergence of PMWS, viral sequences were amplified from frozen archived (1970-1971) porcine tissues and the complete genome of archival PCV2 was determined. The ORF1 gene product (viral DNA replicase) was homologous to contemporary PCV2 ORF1. In ORF2 (viral nucleocapsid gene) archival PCV2, a consistent linear nine-base sequence difference at base positions 1331 through 1339 was observed.The deduced amino acid sequence from these base changes alters the nucleocapsid conformation within the second immunogenic epitope from a hydrophobic (contemporary PCV2) to a hydrophilic (archival PCV2) configuration.

Fast RNA conjugations on solid phase by strain-promoted cycloadditions

Strain promoted cycloaddition is presented as a tool for RNA conjugation on the solid phase; RNA-cyclooctyne conjugates are prepared by cycloaddition to both azide (strain-promoted azide-alkyne cycloaddition, SPAAC) and nitrile oxide dipoles (strain-promoted nitrile oxide-alkyne cycloaddition, SPNOAC). The conjugation is compatible with 2'-OMe blocks and with 2'-O-TBDMS protection on the ribose moieties of the sugar. Nitrile oxide dipoles are found to be more reactive click partners than azides. The conjugation proceeds within 10 min in aqueous solvents, at room temperature without any metal catalyst and tolerates dipoles of varying steric bulk and electronic demands, including pyrenyl, coumarin and dabcyl derivatives. © 2012 The Royal Society of Chemistry.