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Keywords = TRIzol 6

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20 pages, 1316 KiB  
Article
Immunocapture RT-qPCR Method for DWV-A Surveillance: Eliminating Hazardous Extraction for Screening Applications
by Krisztina Christmon, Eugene V. Ryabov, James Tauber and Jay D. Evans
Appl. Biosci. 2025, 4(3), 40; https://doi.org/10.3390/applbiosci4030040 - 6 Aug 2025
Abstract
Deformed wing virus (DWV) is a major contributor to honey bee colony losses, making effective monitoring essential for apiary management. Traditional DWV detection relies on hazardous RNA extraction followed by RT-qPCR, creating barriers for widespread surveillance. We developed an immunocapture RT-qPCR (IC-RT-PCR) method [...] Read more.
Deformed wing virus (DWV) is a major contributor to honey bee colony losses, making effective monitoring essential for apiary management. Traditional DWV detection relies on hazardous RNA extraction followed by RT-qPCR, creating barriers for widespread surveillance. We developed an immunocapture RT-qPCR (IC-RT-PCR) method for screening DWV-A infections by capturing intact virus particles from bee homogenates using immobilized antibodies. Validation demonstrated strong correlation with TRIzol®-based extraction (r = 0.821), with approximately 6 Ct reduced sensitivity, consistent with other published immunocapture methods. Performance was adequate for moderate–high viral loads, while TRIzol® showed superior detection for low-dose infections. Laboratory-produced reverse transcriptase showed equivalent performance to commercial enzymes, providing cost savings. IC-RT-PCR eliminates hazardous chemicals and offers a streamlined workflow for surveillance screening where the safety and cost benefits outweigh the sensitivity reduction. This method provides a practical alternative for large-scale DWV-A surveillance programs, while TRIzol® remains preferable for low-level detection and diagnostic confirmation. Full article
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9 pages, 1467 KiB  
Article
The Effect of Common Viral Inactivation Techniques on 16S rRNA Amplicon-Based Analysis of the Gut Microbiota
by Zachary McAdams, Kevin Gustafson and Aaron Ericsson
Microorganisms 2021, 9(8), 1755; https://doi.org/10.3390/microorganisms9081755 - 17 Aug 2021
Cited by 3 | Viewed by 2852
Abstract
Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched [...] Read more.
Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched fecal samples from sixteen female CD-1 mice of the same GM background prior to fecal DNA extraction. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA. Treatment-dependent effects on DNA yield, genus-level taxonomic abundance, and alpha and beta diversity metrics were assessed. A sodium dodecyl sulfate (SDS)-based inactivation method and Holder pasteurization had no effect on measures of microbial richness, while two Buffer AVL-based inactivation methods resulted in a decrease in detected richness. SDS inactivation, Holder pasteurization, and the AVL-based inactivation methods had no effect on measures of alpha diversity within samples or beta diversity between samples. Fecal DNA extracted with TRIzol-treated samples failed to amplify and sequence, making it unsuitable for microbiome analysis. These results provide guidance in the 16S rRNA microbiome analysis of fecal samples requiring viral inactivation. Full article
(This article belongs to the Section Gut Microbiota)
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