Multiplexed detection and identification of respiratory pathogens using the NxTAG® respiratory pathogen panel.

The Luminex® NxTAG® Respiratory Pathogen Panel (NxTAG RPP) is an IVD-cleared assay for the simultaneous detection and identification of nucleic acids from 18 respiratory viruses and 2 (or 3 outside of the U.S.) atypical bacterial pathogens in nasopharyngeal swabs. Its scalability allows concurrent testing of up to 96 samples in a single batch. Nucleic acid extracted from 200 µL of raw specimen using the easyMAG® extractor is added directly to pre-plated, lyophilized bead reagents (LBRs), where multiplexed RT-PCR and hybridization to MagPlex-TAG™ microspheres occurs within a sealed reaction well using a single cycling program. Data acquisition is done on the MAGPIX® instrument which reads and sorts the reaction products directly from the sealed well following transfer of the assay plate from the thermal cycler. NxTAG is the newest innovation in bead-based nucleic acid chemistry developed by Luminex. Here we provide the detailed assay protocol and present data which describe the clinical and analytical performance characteristics of NxTAG RPP.

Clinical Evaluation of the Luminex NxTAG Respiratory Pathogen Panel.

An evaluation of the Luminex NxTAG Respiratory Pathogen Panel was performed on 404 clinical respiratory specimens. Clinical sensitivities and specificities of the assay compared to those of the reference methods were 80.0% to 100.0% and 98.9% to 100.0%, respectively. Correct genotyping information was provided for 95.5% of influenza virus A specimens. The closed-tube format of the assay simplified the workflow and minimized carryover contamination.

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster.

BACKGROUND: The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in Drosophila different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in Drosophila Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response. RESULTS: Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3rd instar larvae and we show that 20E-induced levels of EcR isoforms EcR-RA, ER-RC, and EcR-RD/E differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types. CONCLUSIONS: We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.

Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster.

During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions.