Research Assistant Center, Tainan Municipal Hospital (Managed by Show Chwan Medical Care Corporation), Tainan 701033, Taiwan.
Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan.
Viruses. 2023 Jul 16;15(7):1562. doi: 10.3390/v15071562.
We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma ( < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.
我们开发了一种简便的方法，无需病毒培养即可使用内部 RT-PCR 扩增完整的 SARS-CoV-2 序列。从 8 例 SARS-CoV-2 感染的多个时间点收集了 41 份储存的咽喉拭子和血液标本。使用 QIAamp 病毒 RNA 迷你试剂盒提取总 RNA，并混合以提高 RNA 水平。仅选择那些经商业实时 RT-PCR（RT-qPCR）阳性的标本，并用内部 RT-PCR 进行完整序列扩增，然后进行测序。使用 MEGA 11 和 Simplot 3.5.1 软件进行系统发育树和探索性分析。拭子样本的总 RNA 浓度明显高于血浆（<0.01）。阳性结果主要在拭子中发现，但在一份血浆中也发现了阳性结果。成功的基因扩增取决于 Ct 值（Ct<38）。在 ORF1ab/Nsp3 中发现了一个非同义突变（在 NC045512.2 位置 6312，C 突变为 A），大多数刺突蛋白突变发生在 S1 亚基（残基 14-685）。该方法省时可靠，可用于快速基因组分析。增加样本量并将其混合提取 RNA 可以在不培养的情况下提高 RNA 浓度。结合基因组特定可变区的核苷酸序列比常规方法更有效。
