Guidelines for in-house verification of the DDNS method for poliovirus direct detection v2

This document describes a pragmatic approach to conducting in-house verification of the DDNS method for detection of polioviruses from stool samples, evaluating and verifying technical performance of each process. Laboratories should conduct in-house verification of the complete DDNS method to confirm that the required performance characteristics can be met within the settings. Verification of the DDNS method will be assessed based on the following parameters: accuracy and sensitivity.To support DDNS method performance, the laboratory should ensure that lab personnel are adequately trained, competent, and qualified to perform the protocol. Note: We recommend working closely with members of the Polio Sequencing Consortium (PSC) before and during in-house verification of the DDNS method to ensure that the laboratory has all the required components and competency to perform the full protocol. Purpose To outline the key stages in conducting in-house verification of the DDNS method for poliovirus detection from stool suspensions proving technical abilities and performance. These will include proving sufficient recovery of viral nucleic acid during RNA extraction and achieving efficient PCR amplification during STAGE 1 and STAGE 2. STAGES 3 and 4 further target sample handling, accuracy and adherence to protocol QC recommendations. Library preparation, sequencing, data analysis and interpretation are also assessed during verification. Performance characteristics are evaluated based on results from spiking experiments using preparations of the assay positive control (Coxsackievirus A20 material), testing known positive and negatives samples, and testing a set of blinded samples. The completed in-house verification will demonstrate that assay results are in line with how the DDNS method has been designed to perform. This will allow obtaining reliable data and establishing a QA process to ensure data integrity. Results from the consecutive stages of the in-house verification should be recorded in the verification report document, discussed with the Polio Sequencing Consortium and the reports become part of the laboratory’s QA records. Overview of the procedure is shown in figure below. Detailed protocols Viral RNA extraction: dx.doi.org/10.17504/protocols.io.81wgbzpyogpk/v2 DDNS: dx.doi.org/10.17504/protocols.io.rm7vzbyyxvx1/v6