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

Abstract

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.

Keywords: COVID-19 diagnostic testing; Genetic diversity; Next-generation sequencing; RT-PCR; SARS-CoV-2.

Fig. 1

Target hybridization regions of the primers/probes employed by the principal in-house protocols for molecular detection of SARS-CoV-2. The protocols targeted 13 different genome regions inside the Orf1ab (Nsp9, Nsp10, Nsp11, RdRp, ExoN), E (Envelope) y N (Nucleocapsid) genes. Target hybridization regions of the primers / probes employed by the principal in-house protocols for molecular detection of SARS-CoV-2. The different genes and protein products, as well as the coordinates in kilobases (Kb) of the genes and protein products to which they are directed were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. 2

Genetic diversity at the target region of the Hong_Kong (2020)_HKU-NP assay. Hong_Kong (2020) _HKU-NP probe displayed mismatches at positions 29,187–88 and 29,197–98 (highlighted in red), with all the Colombian sequences and the RefSeq displaying CA and CC, respectively. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Genetic diversity at the target region of the Corman_Berlin (2020) _RdRP assay. The Colombian genome hCoV/Colombia/Valle_del_Cauca/79943/2020 displayed a substitution (G to A) at position 15,451 (highlighted in red), affecting the primer Corman_Berlin (2020) _RdRP_SARSr-F2 hybridization region. The substitution is located in the penultimate position at the 3 ‘end of the promer, generating a mismatch that could interfere with the 3′ pentamer stability. A degenerate base (S = C or G) in the primer Corman_Berlin (2020) _RdRP_SARSr-R1 at position 15,519 (highlighted in red) produces a mismatch with all the Colombian genomes and the RefSeq as they have T in the sense sequence, so the degenerate base should be one that includes an A among degenerate alternatives. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Genetic diversity at the target region of the Zhu_2020_CDC-Set I assay. The Colombian genome hCoV/Colombia/Antioquia/79253/2020 displayed a substitution (C to T) at position 13,384 (highlighted in red) where the probe Zhu_2020_CDC-Set I Probe (ORF1ab) hybridizes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Genetic diversity at the target region of the Zhu_2020_CDC-China_Set II assay. Three Colombian genomes, hCoV/Colombia/Bogota/78390/2020, hCoV/Colombia/ Valle_del_Cauca/81279/2020 and hCoV/Colombia/Valle_del_Cauca/81251/2020 have the same pattern of substitutions (GGG to AAC) in primer Zhu_2020_CDC-China_Set II Fw (N) in the first three nucleotides at the 5′ end (highlighted in red). Furthermore, the hCoV/Colombia/Quindio/79911/2020 and hCoV/Colombia/Quindio/80663/2020 genomes displayed a substitution (G to T) at position 28,899 where this oligonucleotide hybridizes (highlighted in red). Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. S1

Alignment of Pasteur 2020 (France)nCoV IP2 primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. S2

Alignment of Hong Kong (2020) HKU-ORF1b-nsp1 primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. S3

Alignment of Corman-Berlin(2020) E Sarbeco primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. S4

Alignment of CDC 2020(USA) 2019-nCoV N1 (Fig. S4) and DMC-MH 2020(Thailand)WH-NIC N primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. S5

Alignment of CDC 2020(USA) 2019-nCoV N3 and Corman-Berlin(2020) N Sarbeco primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).

Fig. S6

Alignment of Nao 2020(Japan)NIID 2019-nCOV N and CDC 2020(USA) 2019-nCoV N2 primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were estimated according to the SARS-CoV-2 reference genome available at GenBank (NC_045512.2).