Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia.

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3' end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3' end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Álvarez-Díaz DA ，Franco-Muñoz C ，Laiton-Donato K ，Usme-Ciro JA ，Franco-Sierra ND ，Flórez-Sánchez AC ，Gómez-Rangel S ，Rodríguez-Calderon LD ，Barbosa-Ramirez J ，Ospitia-Baez E ，Walteros DM ，Ospina-Martinez ML ，Mercado-Reyes M ... -  《-》

Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline.

During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.

Kuchinski KS ，Jassem AN ，Prystajecky NA 《-》

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Moore SC ，Penrice-Randal R ，Alruwaili M ，Randle N ，Armstrong S ，Hartley C ，Haldenby S ，Dong X ，Alrezaihi A ，Almsaud M ，Bentley E ，Clark J ，García-Dorival I ，Gilmore P ，Han X ，Jones B ，Luu L ，Sharma P ，Shawli G ，Sun Y ，Zhao Q ，Pullan ST ，Carter DP ，Bewley K ，Dunning J ，Zhou EM ，Solomon T ，Beadsworth M ，Cruise J ，Crook DW ，Matthews DA ，Davidson AD ，Mahmood Z ，Aljabr W ，Druce J ，Vipond R ，Ng L ，Renia L ，Openshaw PJM ，Baillie JK ，Carroll MW ，Stewart J ，Darby A ，Semple M ，Turtle L ，Hiscox JA ... -  《Viruses-Basel》

Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection.

Gand M ，Vanneste K ，Thomas I ，Van Gucht S ，Capron A ，Herman P ，Roosens NHC ，De Keersmaecker SCJ ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

The establishment of reference sequence for SARS-CoV-2 and variation analysis.

Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.

Wang C ，Liu Z ，Chen Z ，Huang X ，Xu M ，He T ，Zhang Z ... -  《-》