Beer, Martin

Ebinger, Arnt*

Höper, Dirk

Rubbenstroth, Dennis

English

OA OpenAgrar repository

Poster Presentations Diagnostics   167 D05  Impact of RNA quality on diagnostic sensitivity for BoDV-1 in FFPE-tissues  Arnt Ebinger, Dennis Rubbenstroth, Martin Beer, Dirk Höper  Institute for Diagnostic Virology, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health   Keywords: Borna disease virus 1, formalin-fixed paraffin-embedded, diagnostic sensitivity, RNA-extraction   Archived formalin-fixed paraffin-embedded (FFPE) brain tissues of presumptive Borna disease virus 1 (BoDV-1) cases of dead-end hosts (incl. humans, horses, sheep) are from particular interest to study its molecular epidemiology and geographical distribution. However, molecular techniques (RT-qPCR, NGS) targeting the extracted RNA are affected by the low RNA quality due to fixation and embedding. Therefore, we analyzed the impact of a longer and a shorter BoDV-1-specific RT-qPCR amplicon assay (P gene: 162 bp, M gene: 75bp) on the diagnostic sensitivity for 88 FFPE brain samples originating from BoDV-1-positive animals and humans from 2004-2018 and compared in detail the RNA-quality level (DV200) with the Cq values for 48 samples. While the 75bp-amplicon detected 96.5% BoDV-1 cases, the diagnostic sensitivity was reduced for the 162bp-amplicon (79.5%). In “low” and “too degraded” RNA-quality levels the Cq values for 162bp-amplicon was significantly higher, whereas there were no differences for “medium” and “high” levels. Based on those results, we evaluated three RNA extraction protocols, in terms of total RNA-yield, DNA content, ß-actin, DV200, and BoDV-1 RNA and developed a superior semi-automated protocol in combination with Covaris truXTRAC FFPE tNA, Agentcourt RNAdvance Tissue Kit, and the KingFisher Flex. This study highlights the significance of selecting an appropriate extraction method and PCR assay for a reliable diagnostic in low-quality samples.   

Impact of RNA quality on diagnostic sensitivity for BoDV-1 in FFPE-tissues

https://www.openagrar.de/servlets/MCRFileNodeServlet/openagrar_derivate_00026188/SD201954796.pdf

Impact of RNA quality on diagnostic sensitivity for BoDV-1 in FFPE-tissues

Abstract

Similar works

Full text

Available Versions

OA OpenAgrar repository