Development and optimization of a Muliplex real-time for SARS-CoV-2

Description

The study presented a standardized diagnostic method for SARS-CoV-2 using a multiplex amplification, using different source of primers and probes. This repository contains the raw data of PCR runs during the standardization process and the comparison of results using clinical samples.

Steps to reproduce

Clinical samples and RNA extraction In this study, 155 RNA samples obtained from nasopharyngeal swab samples of respiratory symptomatic patients with clinical suspicion of SARS-CoV-2 infection were selected. These samples were previously evaluated using the commercial GeneFinder COVID-19 Plus RealAmp RT-PCR kit, and 79 negative and 76 positive samples were confirmed. RNA extraction from clinical samples was performed by an in-house method using SpeedBead Magnetic Carboxylate Modified Particles and lysis buffer with guanidine salts developed and standardized at the Virology Laboratory of Universidad El Bosque. Primers and probes, and in silico evaluation Primers and probes for E and N viral genes published by Corman et al. in the Charité University protocol were used. The RNase P gene was used as a human internal control to assess the presence of amplification-susceptible RNA in the samples. The probe for the E gene was labeled with FAM fluorophore, the N probe with Texas Red, and RNase P with HEX, and their respective quenchers. In silico evaluation of primers and probes included in the multiplex systems was performed using 20 Colombian genome sequences reported in the GISAID database. The results showed that all the primers and probes presented a 100% identity with those SARS-CoV-2 lineages. RT-qPCR assays for the detection of SARS-CoV-2 and single vs. multiplex performance comparison Reverse transcription and amplification of the viral genome were performed using the Luna® Universal One-Step RT-qPCR enzyme kit. The E and N viral genes were amplified individually (single reaction) and in combination with the RNase P gene (triplex reaction). Several concentrations of primers and probes were tested and finally selected at 0.2 µM for both reactions (single and multiplex). Samples were considered positive when fluorescence exceeded the detection threshold at Ct less than 37 and a gradual increase during the amplification cycles, generating a typical sigmoidal amplification curve. Samples with Cq values greater than 38 were considered negative. The study also compared the performance of the E and N single reactions with that of the triplex reaction (E, N, and RNAse P) using a commercial SARS-CoV-2 RNA control, diluted to obtain concentrations of 100, 50, and 20 viral copies/µL, and from the latter, serial two-fold dilutions were made to obtain concentrations of 10 and 5 viral copies/µL, using nuclease-free water as the diluent. The single reactions were performed in duplicate and six replicates of the triplex reaction were performed. Finally, to evaluate the performance of triplex RT-qPCR in clinical samples, 155 RNA samples from patients with suspected COVID-19 (79 negative and 76 positive samples for SARS-CoV-2) were selected and analyzed in a CFX-96 thermal cycler.

Institutions

• Universidad El Bosque

Categories

Funders

• Ministerio de Ciencia y Tecnología

  Spain

  Grant ID: Ministerio de Ciencias, Colombia Grant 1308101577416 (Contract 377-2020)

Licence