ABSTRACT
A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.
Subject(s)
COVID-19 , SARS-CoV-2 , Binding Sites , Humans , Mutation , RNA, Viral/genetics , Sensitivity and SpecificityABSTRACT
We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occurring in the Wuhan region of China in December 2019. From China, the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on March 2, 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequencing data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates, from which 21 whole genome sequences were generated. Our analysis shows that both the early A (19B) and the later observed B (20A/C) clade are present in Mali, indicating multiple and independent introductions of SARS-CoV-2 to the Sahel region.
Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Genome, Viral/genetics , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Betacoronavirus/isolation & purification , COVID-19 , Child , Child, Preschool , Female , Genetic Variation/genetics , Genomics , Humans , Male , Mali/epidemiology , Middle Aged , Pandemics , Phylogeny , SARS-CoV-2 , Whole Genome Sequencing , Young AdultABSTRACT
We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occurring in the Wuhan region of China in December 2019. From China, the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on March 2, 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequencing data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates, from which 21 whole genome sequences were generated. Our analysis shows that both the early A (19B) and the later observed B (20A/C) clade are present in Mali, indicating multiple and independent introductions of SARS-CoV-2 to the Sahel region.