Alkhatib Mohammad, Carioti Luca, D'Anna Stefano, Ceccherini-Silberstein Francesca, Svicher Valentina, Salpini Romina
Department of Experimental Medicine, University of Rome "Tor Vergata", 00133 Rome, Italy.
Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.
Microorganisms. 2022 Aug 2;10(8):1559. doi: 10.3390/microorganisms10081559.
The performance of diagnostic polymerase chain reaction (PCR) assays can be impacted by SARS-CoV-2 variability as this is dependent on the full complementarity between PCR primers/probes and viral target templates. Here, we investigate the genetic variability of SARS-CoV-2 regions recognized by primers/probes utilized by PCR diagnostic assays based on nucleotide mismatching analysis. We evaluated the genetic variation in the binding regions of 73 primers/probes targeting the Nucleocapsid (N, N = 36), Spike (S, N = 22), and RNA-dependent RNA-polymerase/Helicase (RdRp/Hel, N = 15) of the publicly available PCR-based assays. Over 4.9 million high-quality SARS-CoV-2 genome sequences were retrieved from GISAID and were divided into group-A (all except Omicron, >4.2 million) and group-B (only Omicron, >558 thousand). In group-A sequences, a large range of variability in primers/probes binding regions in most PCR assays was observed. Particularly, 87.7% (64/73) of primers/probes displayed ≥1 mismatch with their viral targets, while 8.2% (6/73) contained ≥2 mismatches and 2.7% (2/73) contained ≥3 mismatches. In group-B sequences, 32.9% (24/73) of primers/probes were characterized by ≥1 mismatch, 13.7% (10/73) by ≥2 mismatches, and 5.5% (4/73) by ≥3 mismatches. The high rate of single and multiple mismatches- found in the target regions of molecular assays used worldwide for SARS-CoV-2 diagnosis reinforces the need to optimize and constantly update these assays according to SARS-CoV-2 genetic evolution and the future emergence of novel variants.
诊断性聚合酶链反应（PCR）检测的性能可能会受到严重急性呼吸综合征冠状病毒2（SARS-CoV-2）变异的影响，因为这取决于PCR引物/探针与病毒靶模板之间的完全互补性。在此，我们基于核苷酸错配分析，研究了PCR诊断检测所使用的引物/探针识别的SARS-CoV-2区域的遗传变异性。我们评估了针对公开可用的基于PCR的检测中核衣壳（N，N = 36）、刺突蛋白（S，N = 22）和RNA依赖性RNA聚合酶/解旋酶（RdRp/Hel，N = 15）的73种引物/探针结合区域的遗传变异。从全球共享流感数据倡议组织（GISAID）检索了超过490万个高质量的SARS-CoV-2基因组序列，并将其分为A组（除奥密克戎毒株外的所有毒株，超过420万个）和B组（仅奥密克戎毒株，超过55.8万个）。在A组序列中，观察到大多数PCR检测中引物/探针结合区域存在很大范围的变异性。特别是，87.7%（64/73）的引物/探针与其病毒靶标显示出≥1个错配，而8.2%（6/73）包含≥2个错配，2.7%（2/73）包含≥3个错配。在B组序列中，32.9%（24/73）的引物/探针具有≥1个错配，13.7%（10/73）具有≥2个错配，5.5%（4/73）具有≥3个错配。在全球用于SARS-CoV-2诊断的分子检测靶区域中发现的高比例单错配和多错配，强化了根据SARS-CoV-2的遗传进化和未来新变种的出现来优化和不断更新这些检测的必要性。
