Dataset Information

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses.

ABSTRACT: The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B(1), B(2), C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.

SUBMITTER: Lin B

PROVIDER: S-EPMC446281 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses.

Lin Baochuan B Vora Gary J GJ Thach Dzung D Walter Elizabeth E Metzgar David D Tibbetts Clark C Stenger David A DA

Journal of clinical microbiology 20040701 7

The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a lon ...[more]

PMID: 15243087

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Project description:BACKGROUND: The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. RESULTS: We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/. CONCLUSION: We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.

Project description:BackgroundAdenovirus is one of the most common causes of viral acute respiratory infections. To identify the types of human adenoviruses (HAdVs) causing respiratory illness in Beijing, a sentinel surveillance project on the viral aetiology of acute respiratory infection was initiated in 2011.Principal findingsThrough the surveillance project, 4617 cases of respiratory infections were identified during 2011-2013. Throat swabs (pharynx and tonsil secretions) were collected from all the patients, and 15 different respiratory viruses were screened by multiplex one-step PCR method. 45 were identified as adenovirus-positive from sporadic and outbreak cases of respiratory infection by a multiplex one-step RT-PCR method, and a total of 21 adenovirus isolates were obtained. Five HAdV types among three species, including HAdV-3 (species HAdV-B), HAdV-4 (species HAdV-E), HAdV-7 (species HAdV-B), HAdV-55 (species HAdV-B), and an undefined HAdV type (species HAdV-C) were identified. The comparison results of the penton base, hexon, and fiber gene sequences of the Beijing HAdV-3, HAdV-4, HAdV-7, and HAdV-55 strains in this study and those from the GenBank database indicated significant spatial and temporal conservation and stability of sequences within the genome; however, the phylogenetic relationship indicated that both strain BJ04 and strain BJ09 isolated in 2012 and 2013, respectively, may have recombined between HAdV-1 genome and HAdV-2 genome within species HAdV-C, indicating intraspecies recombination.ConclusionsThis study confirmed that at least 5 HAdV types including HAdV-3, HAdV-4, HAdV-7, HAdV-55 and an undefined HAdV type were co-circulating and were the causative agents of respiratory tract infections in recent years in Beijing. HAdV-3, HAdV-4, HAdV-7, and HAdV-55 showed the apparent stability of the genomes, while intraspecies recombination was identified in strain BJ04 and BJ09. The recombinants carrying penton base gene of HAdV-1 as well as hexon and fiber genes of HAdV-2 might be a novel type of HAdV worthy of further study.

Dataset Information

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses.

Publications

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses.

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