Identification of vaccine-derived polioviruses using dual-stage real-time RT-PCR.
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ABSTRACT:
SUBMITTER: Kilpatrick DR
PROVIDER: S-EPMC8215842 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polioviruses are members of the Enterovirus C species and asymptomatic fecal shedding allows for their transmission and persistence in a community, as well as the emergence of vaccine-derived polioviruses. Using three serotype-specific real-time RT-PCR (rRT-PCR) assays, the shedding and circulation of oral poliovirus vaccine (OPV) strains was previously investigated in a prospective cohort of Mexican children, their contacts, and nearby sewage. Subsequently, a deep sequencing approach targeting the P1 genomic region was applied to characterize OPV strains previously detected by rRT-PCR. Amplifiable RNA was obtained for sequencing from 40.3% (58/144) of stool samples and 71.4% (15/21) of sewage using nucleic acids extracted directly from primary rRT-PCR-positive specimens. Sequencing detected one or more OPV serotypes in 62.1% (36/58) of stool and 53.3% (8/15) of sewage samples. All stool and sewage samples in which poliovirus was not detected by deep sequencing contained at least one non-polio enterovirus C (NPEV-C) strain. To improve screening specificity, a modified, two-step, OPV serotype-specific multiplex rRT-PCR was evaluated. In stool specimens, the overall agreement between the original assays and the multiplex was 70.3%. By serotype, the overall agreement was 95.7% for OPV serotype-1 (S1), 65.6% for S2, and 96.1% for S3. Furthermore, most original rRT-PCR positive/multiplex rRT-PCR negative results were collected in the summer and fall months, consistent with NPEV-C circulation patterns. In conclusion, this deep sequencing approach allowed for the characterization of OPV sequences directly from clinical samples and facilitated the implementation of a more specific multiplex rRT-PCR for OPV detection and serotyping.
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Project description:Equine coronavirus (ECoV) is a recently identified equine virus, involved mainly in enteric infections. Since the ECoV discovery in 1999, only two real-time RT-PCRs have been developed for viral identification. In this chapter we describe a one-step real-time RT-PCR that has been routinely used in our laboratory for ECoV detection from fecal and respiratory samples.
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Project description:In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.
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Project description:Fragments of four candidate reference genes of Aphelenchoides besseyi, including actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin conjugating-3 enzyme (UBC) and alpha-tubulin (?-tubulin) were cloned from the transcriptome database of A. besseyi. The expression level of these four candidate reference genes and a commonly used reference gene of A. besseyi (18S rRNA) in three experimental conditions, including the four life stages (female, male, juvenile and egg) of two populations and the mixed-stage nematodes of four populations with different origins and hosts were analyzed by RT-qPCR. The expression stability of the five candidate reference genes under the three experimental conditions was analyzed by ?Ct, geNorm, NormFinder and RefFinder respectively. The analysis results of ?Ct, geNorm, NormFinder and RefFinder all indicated that UBC was the gene with the highest average ranking of stability. In conclusion, the expression stability of UBC was optimal under the three experimental conditions, indicating that UBC could be used as a suitable reference gene instead of 18S rRNA in the RT-qPCR analysis for A. besseyi.
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Project description:During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.
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Project description: Not available
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Project description:To validate a panel of selected genes expression in prostate cancer, we performed expression profiling of normal prostate epithelial cells (PrEC) and the prostate cancer cell line LNCaP using qPCR.
2018-09-01 | GSE62409 | GEO
Project description:BackgroundWith the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. Gene expression profiles of a target gene can provide valuable clues towards the understanding of its biological function. Reverse transcription quantitative real-time PCR (qRT-PCR) is the best method for targeted gene expression analysis due to its sensitivity and reproducibility. However, calculating relative expression requires reference genes, which must be stable across various biological conditions. For this purposes, 11 prospective genes namely, 28S RNA, ACT7, CYP, EF1A, EF2, ETIF3E, GAPDH, PP2Ac, PTB, UBC2 and UBI1 were evaluated for their potential use as reference genes in jute.ResultsThe expression stabilities of eleven prospective genes were analyzed in various jute plant tissues, such as the root, stick, bark, leaf, flower, seed and fiber, as well as under abiotic (waterlogged, drought and salinity) and biotic stress (infestation with Macrophomina phaseolina) conditions with different time points. All 11 genes were variably expressed in different tissues and stress conditions. To find suitable reference genes in different sample sets, a comprehensive approach based on four statistical algorithms such as GeNorm, BestKeeper, NormFinder the ?Ct was used. The PP2Ac and EF2 genes were the most stably expressed across the different tissues. ACT7 and UBC2 were suitable reference genes under drought stress, and CYP and PP2Ac were the most appropriate after inoculation with Macrophomina phaseolina. Under salinity stress, PP2Ac and UBC2 were the best genes, and ACT7 and PP2Ac were the most suitable under waterlogged conditions.ConclusionExpression stability of reference genes from jute varied in different tissues and selected experimental conditions. Our results provide a valuable resource for the accurate normalization of gene expression experiments in fiber research for important bast fiber crops.
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Project description:The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.
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Project description:Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.
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