Nested VP1 PCR and Nanopore Sequencing from Stool and ES Samples v1.11 v2

Abstract

This protocol is updated from the protocol described in the paper "Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing" by Shaw et al in the Journal of Clinical Microbiology (2020), DOI: 10.1128/JCM.00920-20. The protocol aims to amplify the VP1 region of poliovirus through a nested PCR using panEV primers followed by amplification of the VP1 sequence using the Q8/Y7 primer set. We advise the use of barcoded primers where possible as this greatly simplifies the subsequent library preparation process. Additional steps have however been included in the case that the second PCR step is performed either with standard Q8/Y7 primers or using Q8/Y7 with the barcode adaptors (BCA) attached. Primer sequences for the panEV primers, Q8/Y7 primers and modified Q8/Y7 primers are found in Dataset S1 of the publication. Sequencing of the panEV product is also possible through the removal of the VP1 nested PCR steps and the preparation of the panEV product for sequencing. Version updates: v1.1 – Updated to LSK-109 v1.2 – Minor formatting edits v1.3 – Streamlining and minor alterations v1.4 – Written inclusion of negative control in PCR v1.5 – Updated to dreamtaq and Y7R primers v1.6 – Updated MinKNOW instructions & minor edits v1.7 – Changed back to Y7 primers v1.10 – Updated to LSK-110, removed cleaning and quantification prior to pooling, updated taq volume in rtPCR, altered nested VP1 to use neat panEV product. V1.11 – Updated RNA extraction to include Proteinase K, removed DNA CS addition, added pooling of Y7/Q8 primers.

Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients

“…The entire-capsid-coding region (approximately 3.9 kbp) was amplified by RT-PCR with 5′NTR-529-552+ (5′NTR) and Cre-4485-4460- (Cre) primers [ 5 ] (EC RT-PCR) using two commercial kits. The RT-PCR with a SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (named kit A hereafter, Invitrogen, Carlsbad, CA, USA) was performed according to the procedure developed by Shaw et al [ 6 , 15 ]. In this procedure, the 5′NTR primer was added after the RT reaction.…”

“…Non-barcoded Y7/Q8 primers were also used to evaluate the effect of the attached barcodes on the sensitivity of the detection. For the PCR reaction with barcoded primers, a DreamTaq DNA polymerase kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the previous report [ 6 , 15 ]. With non-barcoded primers, an EmeraldAmp PCR Master Mix kit (Takara Bio, Shiga, Japan) was used.…”

“…Both PCR reactions were performed with 2 µL of the first PCR products in a 25 µL of reaction mixture. The PCR conditions consist of a denaturation step at 95 °C for 2 min; 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min; and a hold step at 72 °C for 10 min [ 6 , 15 ]. For the PCR with the type-specific primers, a One-Step RT-PCR Kit (Qiagen, Venlo, The Netherlands) was employed, which is usually used for RT-PCR with viral RNA according to a manual by the WHO [ 3 ].…”

“…Nanopore sequencing. Nanopore sequencing was performed as previously reported [ 6 , 15 ]. The sequence library was prepared with an NEB Next Companion Module for Oxford Nanopore Technologies Ligation Sequencing and a SQK-LSK110 kit (Oxford Nanopore Technologies, Oxford, UK).…”