Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards.

Ahmed Elnagar,Martin Beer,Timm C Harder,Sandra Blome,Bernd Hoffmann

doi:10.3390/ijms222312915

Ahmed Elnagar, Martin Beer + Show 3 more

Open Access

https://doi.org/10.3390/ijms222312915

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Abstract

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Highlights

The Flinders Technology Associates (FTA®) Whatman filter paper cards are based on a chemically-treated cellulose membrane, which lyses cells, their nuclei, and organelles from a variety of sources
The African swine fever virus (ASFV) EDTA blood sample has been diluted with ASFV-negative EDTA blood, and the Influenza A virus (IAV)-positive sample has been diluted with negative allantoid fluid
Some FTA cards are designed for the isolation and purification of nucleic acids, while other cards are consisting of filter papers that are specialized for the collection, transport, and storage of biological samples

Summary

Introduction

The Flinders Technology Associates (FTA®) Whatman filter paper cards are based on a chemically-treated cellulose membrane, which lyses cells, their nuclei, and organelles from a variety of sources (e.g., blood, saliva, plant tissue). The matrix protects the nucleic acids from damaging agents (e.g., nucleases, oxidative agents, and bacterial growth) which serves to reduce degradation [1] They are commercially available in a variety of configurations to meet application requirements and custom configurations. FTA cards are impregnated with chaotropic agents that inactivate infectious agents and reduce the biohazard potential of the sample, thereby minimizing risks of exposure to the technical staff during sample processing. This enables the storage of biological material on FTA cards at room temperature for extended periods. Different procedures for the release of the nucleic acids from the FTA cards were described by the commercial suppliers or in context with specific viruses and cards [12]

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