Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA.
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ABSTRACT:
SUBMITTER: Hepp C
PROVIDER: S-EPMC8486776 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (<20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against extracted RNA from 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 91% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 ± 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n = 52), showing average detection times of 12.68 ± 2.56 min for positive samples (n = 34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geolocalization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.
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Project description:BACKGROUND: Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. METHODS: Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. RESULTS: The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. CONCLUSION: Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.
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Project description:RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols.
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Project description:The variability of a small supernumerary marker chromosome (sSMC)-related phenotype is determined by the molecular component, the size, and shape of the marker chromosome. As fluorescence in situ hybridization has limitations regarding the resolution, efficiency, and accuracy. Recently, array comparative genomic hybridization (aCGH) was used for sSMC characterization. In this study, twenty cases with sSMCs were characterized by aCGH and FISH. Chromosomal origin of the marker chromosomes were successfully identified in seventeen of them. For the three cases with negative aCGH results, two of them were more likely due to that the sSMCs only contained centromere heterochromatin, whereas the reason for the remaining case with negative aCGH finding was uncertain. In order to establish a stronger genotype-phenotype correlation for clinical service in the future and avoid miss characterization, more sSMC cases were needed to be detailed characterized. This will help to clarify the variable clinical characteristics of sSMCs and provide additional information to aid clinical service and future research.
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Project description:Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.
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