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.

中文翻译：

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在哥伦比亚病毒遗传变异的背景下，对几种内部rRT-PCR方案进行SARS-CoV-2检测的分子分析。

SARS-CoV-2引起的COVID-19大流行是最近人类历史上前所未有的公共卫生问题。已经报道了用于SARS-CoV-2诊断的不同的内部实时RT-PCR（rRT-PCR）方法以及引物区域中具有突变的基因组的出现。因此，来自局部传播的SARS-CoV-2株的全基因组数据有助于了解其全局变异性以及诊断方案的开发和微调。为了描述哥伦比亚SARS-CoV-2基因组在SARS-CoV-2检测的主要内部方法的寡核苷酸杂交区域中的遗传变异性，通过下一代处理已确认SARS-CoV-2分子诊断的RNA样品排序。在大流行早期，由7个机构建议并由WHO整合的13个靶区的SARS-CoV-2检测引物/探针序列与Muscle工具进行了比对，以评估可能影响其性能的遗传变异性。最后，在每个引物的3'端的相应密码子位置，鉴定每个基因/蛋白质产物的开放阅读框检查。从30例COVID-19病例中获得了完整的SARS-CoV-2基因组，代表了该国当前的流行病学。观察到至少一个哥伦比亚序列与靶向RdRP和N基因的五个寡核苷酸不匹配。与第三个密码子位置对齐的4个引物的3'端显示出高风险的核苷酸替换和在此关键位置的潜在错配。在一些引物/探针区域的哥伦比亚SARS-CoV-2序列中检测到遗传变异，可用于WHO COVID-19技术指南中的内部rRT-PCR诊断测试；应通过实验评估其对性能和假阴性率的影响。强烈建议对SARS-CoV-2进行基因组监测，以及早发现关键区域的突变，并就特定的诊断测试提出建议，以确保覆盖局部循环的遗传变异。