Rapid Detection of SARS-CoV-2 Virus Using Dual Reverse Transcriptional Colorimetric Loop-Mediated Isothermal Amplification

Abstract

The outbreak and pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a public health emergency of international concern. The rapid and accurate detection of the virus is a critical means to prevent and control the disease. Herein, we provide a novel, rapid, and simple approach, named dual reverse transcriptional colorimetric loop-mediated isothermal amplification (dRT-cLAMP) assay, to accelerate the detection of the SARS-CoV-2 virus without using expensive equipment. The result of this assay is shown by color change and is easily detected by the naked eye. To improve the detection accuracy, we included two primer sets that specifically target the viral orf1ab and N genes in the same reaction mixture. Our assay can detect the synthesized SARS-CoV-2 N and orf1ab genes at a low level of 100 copies/μL. Sequence alignment analysis of the two synthesized genes and those of 9968 published SARS-CoV-2 genomes and 17 genomes of other pathogens from the same infection site or similar symptoms as COVID-19 revealed that the primers for the dRT-cLAMP assay are highly specific. Our assay of 27 clinical samples of SARS-CoV-2 virus and 27 standard-added environmental simulation samples demonstrated that compared to the commercial kits, the consistency of the positive, negative, and probable clinical samples was 100, 92.31, and 44.44%, respectively. Moreover, our results showed that the positive, but not negative, standard-added samples displayed a naked-eye-detectable color change. Together, our results demonstrate that the dRT-cLAMP assay is a feasible detection assay for SARS-CoV-2 virus and is of great significance since rapid onsite detection of the virus is urgently needed at the ports of entry, health care centers, and for internationally traded goods.

Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method

“…3B ). All results indicated that the triplex primer combination of the set3, set1, and set5 (S3+S1+S5) exhibited the best performance and was therefore selected as the optimal LAMP primer combination for further studies ( Ji et al, 2021 ).…”

“…SARS-CoV-2 constantly changes through genetic mutations, with novel variants of concern (VOC) occurring over time, such as Alpha, Beta, Gamma, Delta and Omicron, which are more transmissible, more pathogenic, or have better capability for immune escape ( Hacisuleyman et al, 2021 ; Hoffmann et al, 2021 ; Wang et al, 2021 ). All of these VOCs and some other genomic mutations make it difficult to detect SARS-CoV-2 with only one single target ( Ji et al, 2021 ; Mohon et al, 2020 ). Therefore, LAMP primer sets targeting different SARS-CoV-2 regions may provide more accurate diagnosis results.…”

“…Furthermore, all of these VOCs usually mutated in the Spike(S) gene, and in our studies, we chose ORF1ab, E and N genes of SARS-CoV-2, which were the most conserved regions within the SARS-CoV-2 genome. Moreover, compared to performing multiple singleplex RT-LAMP reactions, multiplex RT-LAMP assays reduce the cost and time for two or more targets being simultaneously amplified in one reaction ( Ji et al, 2021 ; Kim et al, 2019 ; Mohon et al, 2020 ).…”

Recent Advances of Representative Optical Biosensors for Rapid and Sensitive Diagnostics of SARS-CoV-2

“…The color change can be observed with the naked eye, even at low concentrations of the target analyte. Simultaneously, colorimetric biosensors have been used in POC for their advantages of direct readout, convenient operation, low instruments requirements, and portability [ 61 , 62 , 63 ]. In recent years, a series of smart materials that can induce color changes have been developed using noble metals, metal oxides, and carbon materials [ 64 , 65 ].…”

Research Progress on Nano Photonics Technology-based SARS-CoV-2 Detection※

“…比色法(Colorimetry)是指利用检测设备中可视化的 颜色变化, 反映相关生物化学信号的一种方法, 可以借 助荧光分子、纳米金颗粒等标记探针, 直观地检测病 毒 [70][71] 、蛋白质 [72] 等生物标志物. 比色法的方式有很多, RT-PCR 作为目前 SARS-CoV-2 的核心检测方法, 正是 比色法的一种.…”