Iqbal Bushran N, Arunasalam Shiyamalee, Divarathna Maduja V M, Jabeer Aaom, Sirisena Pdnn, Senaratne Thamarasi, Muthugala Rohitha, Noordeen Faseeha
Diagnostic and Research Virology Laboratory, Department of Microbiology, Faculty of Medicine, University of Peradeniya, Peradeniya 20400, Sri Lanka.
ImmunifyMe Health Tech Pvt Ltd, New Delhi, India.
J Clin Virol Plus. 2022 Aug;2(3):100081. doi: 10.1016/j.jcvp.2022.100081. Epub 2022 May 5.
Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.
Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 °C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay.
A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%.
The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.
在疫情期间，使用简单的实时分子检测方法检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）将有助于中低收入国家的疫情缓解工作。我们开发并验证了一种快速简单的实时环介导等温扩增检测法（LAMP），用于筛查已知感染和未感染个体中的SARS-CoV-2感染。
设计了六组针对SARS-CoV-2 N基因（登录号MN994468）的引物。使用热启动2X预混液和实时PCR仪在65℃下进行LAMP反应60个循环，每个循环15秒。通过在紫外线下观察浊度和通过实时PCR仪的FAM通道进行实时荧光检测来读取结果。我们使用新开发的LAMP检测法对总共320个样本进行了检测，其中包括240个经实时逆转录PCR检测为SARS-CoV-2阳性（Ct值<40）的样本和80个SARS-CoV-2阴性样本。
240个SARS-CoV-2阳性样本中共有206个通过新开发的LAMP检测法检测为阳性，灵敏度为86%。所有80个SARS-CoV-2阴性样本通过新开发的LAMP检测法检测为阴性，特异性为100%。
与用于检测SARS-CoV-2的实时逆转录PCR相比，新开发的实时LAMP检测法的灵敏度为86%，特异性为100%。如果采用这种新检测法来尽量减少时间和成本，将有助于筛查大量样本。
