Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution.

Koenraad Van Hoorde,Ann Brigitte Cay,Marie-Alice Fraiture,Mathieu Gand,Nancy Roosens,Nadège Balmelle,Philippe Herman,Bavo Verhaegen,Sigrid C J De Keersmaecker,Laura A E Van Poelvoorde

doi:10.3390/cimb43030134

Abstract

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

Highlights

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-stranded
In order to rationalize the monitoring of the virus spread at the level of a country or region regarding the number of samples, the monitoring of wastewater was proposed for the surveillance of SARS-CoV-2 [8,9,10]
It was verified that when an false negatives (FN) result was obtained for a given genome, this was limited to either only the forward or reverse primer or the probe

Summary

Introduction

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-strandedRNA virus. The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-stranded. SARS-CoV-2 can cause severe complications, including death, mostly in the elderly or in people suffering from comorbidities [2,3]. In order to rationalize the monitoring of the virus spread at the level of a country or region regarding the number of samples, the monitoring of wastewater was proposed for the surveillance of SARS-CoV-2 [8,9,10]. There are several limitations associated with wastewater surveillance; generally, a low virus concentration is observed in such samples, which makes detection challenging. The virus detection and quantification can be limited due to the instability of the genome in wastewater, the low efficiency of virus concentration methods and the lack of sensitive detection assays [8]

Methods

Results

Conclusion