Direct Detection of poliovirus and Nanopore Sequencing (DDNS) - Environmental Samples v2

Le résumé fourni par la source

This protocol covers amplification of poliovirus nucleic acid from concentrated sewage samples following enrichment by sewage concentration and RNA purification with the subsequent sequencing of barcoded VP1 amplicons. An overview of the full procedure if shown in Figure 1. The protocol was successfully tested on concentrates prepared with two-phase separation method, spin-column concentration and bag-mediated filtration system. Poliovirus molecular detection sensitivity will inherently depend on what viral content had been successfully captured in the starting wastewater concentrate sample. Viral RNA should be purified in duplicates from each concentrate following the method for MagMAX Viral RNA Isolation Large volume extraction protocol using 1.2 mL of concentrate for each extraction. It is recommended to perform the extraction fresh so the RNA can be used immediately after purification. Each RNA sample is then subjected to two step amplification in triplicates using a semi-nested PCR approach. A 1:10 dilution using nuclease free water of the first round PCR is included to increase assay sensitivity and reduce chimera formation. Once controls are confirmed by gel electrophoresis, the PCR products are carried forward for nanopore sequencing. The PCR amplicons are pooled and purified before end-repair and dA-tailing. Following sequencing adapter ligation, the sequencing library is prepared and loaded onto the flow cell. This protocol is for use with Oxford Nanopore kit14 chemistry ligation sequencing reagents and the prepared sequencing mix can be run on MinION Mk1B or GridION sequencer.

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