ABSTRACT
Sequencing technology is the most commonly used technology in molecular biology research and an essential pillar for the development and applications of molecular biology. Since 1977, when the first generation of sequencing technology opened the door to interpreting the genetic code, sequencing technology has been developing for three generations. It has applications in all aspects of life and scientific research, such as disease diagnosis, drug target discovery, pathological research, species protection, and SARS-CoV-2 detection. However, the first- and second-generation sequencing technology relied on fluorescence detection systems and DNA polymerization enzyme systems, which increased the cost of sequencing technology and limited its scope of applications. The third-generation sequencing technology performs PCR-free and single-molecule sequencing, but it still depends on the fluorescence detection device. To break through these limitations, researchers have made arduous efforts to develop a new advanced portable sequencing technology represented by nanopore sequencing. Nanopore technology has the advantages of small size and convenient portability, independent of biochemical reagents, and direct reading using physical methods. This paper reviews the research and development process of nanopore sequencing technology (NST) from the laboratory to commercially viable tools; discusses the main types of nanopore sequencing technologies and their various applications in solving a wide range of real-world problems. In addition, the paper collates the analysis tools necessary for performing different processing tasks in nanopore sequencing. Finally, we highlight the challenges of NST and its future research and application directions.
ABSTRACT
The COVID-19 pandemic spreads rapidly and globally. To quell the pandemic propagation, rapid and accurate detection of SARS-CoV-2 is urgently needed. Here, we present a nanopore coupled RT-LAMP method for SARSCoV-2 detection. After comparing all information from the nanopore experiment, we develop a method to use the event rate change of the amplicons translocation event to measure the amplification. As a result, our platform can distinguish positive from negative samples in 15 min with around 65 copies/reaction limit of detection and 100% specificity. We believe that the nanopore coupled RT-LAMP platform would provide a sensitive and specific detection for SARS-COV-2. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.