A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on RT-RPA with dipstick assay at point-of-care testing.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on reverse transcriptase isothermal recombinase polymerase amplification (RT-RPA) with dipstick assay was developed. The integrated handheld device, consisting of amplification, detection and sealing modules, was developed to perform rapid nucleic acid amplification and detection under a fully enclosed condition. After RT-RPA amplification with a metal bath or a normal PCR instrument, the amplicons were mixed with dilution buffer prior to being detected on a lateral flow strip. To avoid aerosol contamination causing false-positive, from amplification to final detection, the detection 'pen' had been enclosed to isolate from the environment. With colloidal gold strip-based detection, the detection results could be directly observed by eyes. By cooperating with other inexpensive and rapid methods for POC nucleic acid extraction, the developed 'pen' could detect COVID-19 or other infectious diseases in a convenient, simple and reliable way.

[Isothermal amplification technology based on microfluidic chip].

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic.

PCR has been widely used in different fields including molecular biology, pathogen detection, medical diagnosis, food detection and etc. However, the difficulty of promoting PCR in on-site point-of-care testing reflects on challenges relative to its speed, convenience, complexity, and even cost. With the emerging state-of-art of microfluidics, rapid PCR can be achieved with more flexible ways in micro-reactors. PCR plays a critical role in the detection of SARS-CoV-2. Under this special background of COVID-19 pandemic, this review focuses on the latest rapid microfluidic PCR. Rapid PCR is concluded in two main features, including the reactor (type, size, material) and the implementation of thermal cycling. Especially, the compromise between speed and sensitivity with microfluidic PCR is explored based on the system ratio of (thermal cycling time)/(reactor size). Representative applications about the detection of pathogens and SARS-CoV-2 viruses based on rapid PCR or other isothermal amplification are discussed as well.