Department of Biosciences, Faculty of Science, COMSATS University Islamabad, Islamabad, Pakistan.
Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Food Environ Virol. 2022 Dec;14(4):364-373. doi: 10.1007/s12560-022-09522-3. Epub 2022 May 4.
Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RT‑LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.
基于逆转录环介导等温扩增 (RT-LAMP) 和微流控技术集成的微流控芯片 (LOC) 系统的开发有望加速 SARS-CoV-2 的诊断，以便进行早期干预。在目前的工作中，对来自 COVID-19 热点的七个废水样本的提取 RNA 进行了逆转录定量聚合酶链反应 (RT-qPCR) 和 RT-LAMP 检测。还在没有 RNA 提取的废水样本上进行了 RT-LAMP 检测。目前对 SARS-CoV-2 的检测主要是通过 ORF (ORF1ab) 和 N 基因的 RT-qPCR，因此我们针对这两个基因来寻找检测 SARS-CoV-2 的最佳靶基因。我们还在微流控装置内进行了有/无 RNA 提取的 RT-LAMP 实验，以针对这两个基因。提取 RNA 进行的 RT-qPCR 和 RT-LAMP 的阳性率分别为 100.0%（7/7）和 85.7%（6/7）。RT-qPCR 结果显示，所有 7 个废水样本的 N 基因均为阳性（Ct 范围为 37-39），而 ORF1ab 为阴性，这表明 N 基因可能是检测 SARS-CoV-2 的最佳靶基因。对未提取 RNA 的废水样本进行 N 基因和 ORF（ORF1a）基因的 RT-LAMP 检测表明，所有 7 个样本均呈粉红色（阴性）。除了 7 个样本中的 6 个样本在 RNA 提取后针对 N 区进行 RT-LAMP 时，所有微通道的颜色均保持粉红色（黄色）。本研究表明，成功地使用 RT-LAMP 在微流控芯片中从废水样本中检测到 SARS-CoV-2。本研究的新颖之处在于，在没有先前病毒浓缩和有/无 RNA 提取的情况下，使用废水样本进行 SARS-CoV-2 的检测。
