Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Moore, Matthew D.; Jaykus, Lee-Ann

iously described 37 . Briefly, extracted GII.4 New Orleans RNA (Accession Number: JN595867) was first amplified with T7GII.4F and GII.4R primers (Table 1) using the Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) and 5 μ l template. A 15 min reverse transcription cycle at 50 °C was then followed by enzyme inactivation at 95 °C for 2 min. Amplification was performed for 30 cycles of 95 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 30 sec. The 460 nucleotide amplicon was gel purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and subjected to in vitro transcription using the MEGAshortscript T7 kit (Thermo Fisher, Waltham, MA) according to manufacturer's instructions. The transcribed RNA was purified and quantified (NanoPhotometer Pearl, Denville Scientific, South Plainfield, NJ). It was then serially diluted and used to construct a standard curve with RT-qPCR using the JJV2F-COG2R-Ring2-TP primer probe set (Table 1 ) and the SuperScript One-Step RT-PCR kit with 45 cycles and a 54 °C annealing temperature. The RNA standard curve was used to estimate RNA copy number from extracted patient stool samples. Cq was evaluated with a threshold of 30 flourescence units using Bio-Rad CFX Manager software. All reactions were performed in triplicate.