Magmax 96 viral rna isolation kit

RNA was extracted from samples using the MagMAX-96 viral RNA isolation kit (Thermo Fisher Scientific) on the KingFisher Flex automated extraction platform (Thermo Fisher Scientific). Sample (50 μl) was added to 130 μl of lysis buffer (MagMAX-96 viral RNA isolation kit, Thermo Fisher Scientific) and then the manufacturer’s protocol for extraction was followed. Final elution volume for RNA was 90 μl. RNA was analyzed by reverse transcriptase PCR (rRT-PCR) on the ABI 7500 (Applied Biosystems) using the previously described Callahan protocol (35 (link)). No concentration or pooling of samples was used. A standard curve of the challenge virus was used to produce equivalent TCID₅₀/ml titers for each sample.
Plaque assays were carried out using a fetal goat tongue cell line (ZZ-R 127) (36 (link)). Freshly prepared monolayers of cells in 6-well plates were infected with 200 μl of sample, overlaid with indubiose and incubated for 48 h. Serial dilutions of samples were used where necessary and each sample was tested in duplicate. No concentration or pooling of samples was carried out. Virus was inactivated using citric acid, then overlay removed and cells stained using naphthol blue. Plaque counts were made and recorded by visual inspection of plates. Challenge virus was included as a positive control.

RNA extractions were performed on MJ, serum, oral swabs, and tissue suspensions using the Applied Biosystems MagMAX-96 Viral RNA Isolation Kit (AMB1836-5, Life Technologies, Burlington, ON, USA) together with a MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies) as described previously [28 (link)].
The commercial Tetracore VetAlert™ FMDV RNA Test Kit (Tetracore Inc., Rockville, MD, USA) real-time reverse transcription polymerase chain reaction (rRT-PCR) was used to test the extracted RNA from various samples for the presence of FMDV genome. The assay was performed according to the manufacturer’s instructions using the Applied Biosystems 7500 Real-Time PCR System (4351106, Life Technologies). Crossing threshold (Ct) values less than 40 were considered positive.

Total RNA was extracted from 50-µl plasma aliquots obtained from each viraemic monkey using the MagMAX-96 Viral RNA Isolation kit (Life Technologies) in conjunction with the Thermo Scientific KingFisher Flex automated extraction platform and were eluted in 60 μl AVE buffer. The portion of gag encoding capsid in each sample was then individually amplified by RT–PCR using a SuperScript IV One-Step RT–PCR System (Life Technologies) according to the manufacturer’s recommended protocol. Amplification of the SHIV capsid-encoding region in each sample was performed using primers SIV-CA-F (5′-CCAAAAACAAGTAGACCAACAG-3′) and SIV-CA-R (5′-TGCAAAAGGGATTGGCAC-3′), and the products were subjected to population-level bulk sequencing at Elim Biopharmaceuticals using the same primer set. To identify codon changes, Sequencher version 4.9 (Gene Codes) was used to align DNA encoding sequences of the SHIV capsid for each sample with that of the parent virus obtained from each infected placebo-treated control animal. This provided a control for potential genetic drift that might have occurred during the 24-week efficacy study.