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PROVIDER: PRJNA896155 | ENA |
REPOSITORIES: ENA
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Project description:Gene expression profiling of very few or even single cells is of particular interest in many applications. However, detection of a large number of mRNA sequences from a small number of cells is limited by the sensitivity of available methods. High-throughput multiplex reverse transcription followed by PCR amplification (RT-PCR) has much to offer to these studies due to its inherent sensitivity, efficiency and cost-effectiveness. A multiplex RT-PCR based high-throughput gene profiling system is described in this communication. With this system >1000 different mRNA species can be amplified in a single tube to a detectable amount. By using specially designed PCR primers, the long-standing low specificity problem associated with high-throughput gene expression profiling has been solved. Amplified sequences are then resolved by microarray with probes that only hybridize to sequences amplified from mRNA. The method is so sensitive that mRNA in single cells can be reliably detected. Differentially expressed genes identified with the high-throughput approach in the breast cancer cell line, MCF-7, and its drug resistant variant, MCF-7/AdrR, could be validated by a different method. The approach may greatly facilitate the analysis of combinatorial expression of known genes in any cells in many important applications with a limited amount of RNA. Keywords: drug resistence
2007-06-01 | GSE5920 | GEO
Project description:Multiplex PCR amplicon-based sequencing of sewage SARS-CoV-2
| PRJNA1057461 | ENA
Project description:This series includes 278 microarrays used to detect respiratory viruses in a set of nasopharyngeal lavage specimens from children with respiratory tract infections Objective: To assess the utility of a pan-viral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections. Study Design: The Virochip was compared to conventional clinical direct fluorescent antibody (DFA) and PCR-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children. Results: The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity 85-90%, specificity 99%, PPV 94-96%, NPV 97-98%) in the detection of respiratory syncytial virus, influenza A, and rhino-/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect. Conclusions: Given its favorable sensitivity and specificity profile and greatly expanded spectrum of detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric respiratory tract infections. Keywords: viral detection
2008-07-09 | GSE10294 | GEO
Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as RT-PCR are the gold standard, during the current pandemic the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers, and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis of active infection and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in-vitro de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples
2022-01-12 | PXD021080 | Pride
Project description:This series includes 278 microarrays used to detect respiratory viruses in a set of nasopharyngeal lavage specimens from children with respiratory tract infections Objective: To assess the utility of a pan-viral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections. Study Design: The Virochip was compared to conventional clinical direct fluorescent antibody (DFA) and PCR-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children. Results: The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity 85-90%, specificity 99%, PPV 94-96%, NPV 97-98%) in the detection of respiratory syncytial virus, influenza A, and rhino-/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect. Conclusions: Given its favorable sensitivity and specificity profile and greatly expanded spectrum of detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric respiratory tract infections. Keywords: viral detection The series includes 278 clinical specimens
2010-05-26 | E-GEOD-10294 | biostudies-arrayexpress
Project description:Fruit trees, as apricots, can be infected by and are constantly exposed to the attack of viruses. As they are propagated on a vegetative way, this risk is present not only at the field, where they exists for decades, but also during propagation. Metagenomic diagnostic methods, based on next generation sequencing, offer unique possibilities to reveal all the presenting pathogens in the investigated sample. Using small RNA NGS, a special fields of this technique, we tested leaf samples of different varieties of apricot in isolator house and at open air stock nursery. As a result, we identified Cherry Virus A (CVA) and Little Cherry Virus1 (LChV1) first time in Hungary. Gained results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral PCR products enabled us to investigate their phylogenetic relationships. Our results demonstrate, that small RNA NGS can offer a sensitive virus diagnostics method, moreover beside obligatory tested viruses we could detect CVA and LChV1. However as these pathogens haven’t been described in our country before, their role in symptom development and modification during coinfection with other viruses requires further investigations.
2018-06-20 | GSE114251 | GEO
Project description:BACKGROUND: 50% to 80% of asthma exacerbations are precipitated by viral upper respiratory tract infections (RTI), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood due to the limited scope and throughput of conventional viral detection methods. METHODS: We investigated the capability of the Virochip, a DNA microarray-based viral detection platform, to characterize the viral diversity in RTIs in asthmatic and non-asthmatic adults. RESULTS: The Virochip detected viruses in a higher proportion of samples (65%) than culture isolation (17%), while exhibiting high concordance (98%), sensitivity (97%) and specificity (98%) with pathogen-specific PCR. A similar spectrum of viruses was identified in the RTIs from each patient subgroup; however, unexpected diversity among the coronaviruses (HCoVs) and HRVs was revealed. All but one of the HCoVs corresponded to the newly-recognized HCoV-NL63 and HCoV-HKU1 viruses, and over 20 different serotypes of HRVs were detected, including a set of 5 divergent isolates that form a distinct genetic subgroup. CONCLUSIONS: The Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger scale studies will be necessary to determine if particular substrains of viruses confer an elevated risk of asthma exacerbation Keywords: Virus detection
2007-11-07 | GSE8029 | GEO