Elaswad Ahmed, Fawzy Mohamed
Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.
Department of Virology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.
Pathogens. 2021 Mar 19;10(3):371. doi: 10.3390/pathogens10030371.
Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3' end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.
最近，在几种动物物种中检测到了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）。病毒传播到动物体内后，随着对新动物宿主适应过程的推进，其基因组会积累突变。因此，我们研究了这些突变是否会导致与诊断性PCR检测方法不匹配，并相应地对寡核苷酸序列提出了适当的修改建议。使用28种诊断性PCR检测方法和从动物中分离出的793个公开可用的SARS-CoV-2基因组进行了全面的生物信息学分析。在所研究的28种PCR检测方法中，有16种在0.5%的阈值下与其靶标至少存在一个错配。分别在针对开放阅读框1ab（ORF1ab）、刺突、包膜和核衣壳基因的7种、2种、2种和6种检测方法中检测到错配。其中一些错配，如引物或探针3'端的缺失和错配，预计会对诊断性PCR检测产生负面影响，导致假阴性结果。对寡核苷酸序列的修改应能使寡核苷酸与模板的结合更强，检测方法的灵敏度更高，结果的可信度更高。随着病毒在动物体内继续进化，有必要监测诊断性PCR检测的靶标是否会出现任何未来的突变。
