A Fast, Visual, and Instrument-free Platform Involving Rapid DNA Extraction, Chemical Heating, and Recombinase Aided Amplification for On-Site Nucleic Acid Detection.

Xiao Fu Wang,Xiao Yun Chen,Lei Yang,Jun Feng Xu,Jian Li Guo,Jian Ca,Wen Qiang Chen,Wei Wei,Xiao Li Xu,Cheng Peng

doi:10.3389/fbioe.2021.764306

Abstract

The nucleic acid-based technique has been widely utilized in many fields including for on-site detection. However, traditional molecular detection techniques encounter limitations like relying on instruments, time consuming or complex operation, and cannot meet the demands of on-site testing. In this study, a rapid DNA extraction method (RDEM), recombinase aided amplification (RAA), and chemical heating packet (CHP) are integrated and termed as RRC platform for on-site detection of nucleic acid. For demonstration purposes, SHZD32-1 (a new transgenic soybean line from China) was detected using the novel platform to demonstrate its feasibility and capability for on-site detection. Using the RDEM, high-quality DNA appropriate for molecular detection was quickly extracted in 3–5 min. The heat energy generated by CHP was met the temperature requirements of RAA. Using the RRC platform, the whole detection process can be accomplished within only 30 min, and the results can be visually detected with glasses under blue light. No special or expensive instrument was needed for the detection process. This study provides a novel approach for on-site detection of nucleic acids besides providing valuable insight on related future research.

Highlights

At present, numerous nucleic acid-based analytical techniques were developed
In this study, putting into account the challenges encountered in the three steps of nucleic acid-based detection method, we developed a simple, fast, visual, as well as instrument-free platform for on-site detection of nucleic acid
The expected size of the 18S rRNA gene was amplified and observed for all the samples in the polymerase chain reaction (PCR) and recombinase aided amplification (RAA) assays (Figures 1E,F). These findings suggested that, the yield and purity of the DNA extracted using rapid DNA extraction method (RDEM) were not as high as those of the QIAGEN kit, they were sufficient for the subsequent molecular tests like PCR and RAA

Summary

Introduction

Numerous nucleic acid-based analytical techniques were developed. It has been extensively applied in many fields including disease detection, food safety, and environmental monitoring (Van Nguyen et al, 2019; Zhang et al, 2020). The nucleic acid-based method generally comprises three steps including nucleic acid extraction, amplification, as well as product detection. Nucleic acid extraction is the most critical step in the on-site detection of nucleic acid. The most commonly used method is the DNA extraction method, which entails the traditional reagent extraction and commercial kit extraction techniques. These DNA extraction techniques are time-consuming (1–2 h), and require auxiliary equipment like

Methods

Results

Conclusion