Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry.
Chembiochem
; 21(15): 2214-2218, 2020 08 03.
Article
in English
| MEDLINE | ID: covidwho-186217
ABSTRACT
The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the inâ
situ detection of viral transcripts as early as 4 hours after infection.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA, Messenger
/
RNA, Viral
/
Oligonucleotide Probes
/
In Situ Hybridization, Fluorescence
/
Early Diagnosis
/
Click Chemistry
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
Chembiochem
Journal subject:
Biochemistry
Year:
2020
Document Type:
Article
Affiliation country:
Cbic.202000081
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