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

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.

Exploring surface water as a transmission medium of avian influenza viruses – systematic infection studies in mallards

“…All samples were analysed by real-time RT–PCR (RT-qPCR). The samples were examined using a generic RT-qPCR targeting a fragment of the NP gene [ 49 ] supplemented with internal control (IC-2 [ 50 ]) to control for PCR inhibitory reactions. Samples with a Cq-value <40 were considered positive.…”

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

“…Chelex 100 resin can be used to extract RNA from pathogens as described above [ 10 , 11 ]. Thus, it is very valuable to establish a method for simultaneously extracting RNA and DNA using Chelex 100 resin.…”

“…We extracted RNA from grass carp reovirus (GCRV) and spring viraemia of carp virus (SVCV) using Chelex 100 resin at the beginning of this study. However, compared with DNA extraction, RNA extraction using Chelex 100 resin was more difficult, despite some successful cases of RNA extraction using Chelex 100 resin [ 10 , 11 ]. We optimized the RNA extraction conditions using Chelex 100 resin, but the extraction effect was still not particularly satisfactory.…”

“…The efficiency and quality of nucleic acid extraction directly affect the accuracy, sensitivity, and overall detection efficiency of PCR diagnosis. At present, different nucleic acid sample preparation techniques and commercial kits are often used to extract nucleic acids in clinical and scientific research applications [ 10 , 11 , 12 , 13 , 14 ]. The separation and purification steps traditionally include utilizing phenol chloroform for extraction and/or ethanol for precipitation [ 15 ], along with a high salt concentration [ 16 ] and excess proteinase K digestion [ 17 ].…”

“…The Chelex 100 resin effectively inhibits nuclease activity in complex samples and stabilizes samples for downstream PCR applications. It has long been used for quick and easy DNA or RNA preparation from many sample types, such as blood [ 21 ], insects [ 22 ], bacteria [ 23 ], and viruses [ 10 , 11 , 24 ]. Carp edema virus (CEV), Cyprinid herpesvirus 2 (CyHV-2), and Chinese giant salamander iridovirus (GSIV) are three common aquatic viral pathogens that cause great losses to aquaculture.…”