Abstract
A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).
Highlights
The recognition of infection pathogens is crucial for the assessment of disease state and treatment efficacy; diagnosing infectious diseases accurately and in a timely manner is important, especially for diseases caused by multiple pathogens [1,2,3]
To enhance the limits of detection, improve specificity, and enable parallel detection of multiple nucleic acid targets, we proposed a two-stage isothermal amplification, with an integrated microfluidic chip for the detection of different types of pathogens in a closed system, with a volume of 1 μL recombinase polymerase amplification (RPA), plus 10 μL loop-mediated isothermal amplification (LAMP) for each target, during about one hour process; this consists of first-stage basic RPA and second-stage real-time LAMP, while products of RPA serve as templates for LAMP
Two viruses were targeted in this study: (1) SARS-CoV-2, which is a pathogen that caused the outbreak of coronavirus disease 2019 (COVID-19) worldwide, and (2) Measles Virus (MV), which is an enveloped negative-strand
Summary
The recognition of infection pathogens is crucial for the assessment of disease state and treatment efficacy; diagnosing infectious diseases accurately and in a timely manner is important, especially for diseases caused by multiple pathogens [1,2,3]. El-Tholoth M. used a two-stage isothermal amplification (Penn-RAMP), which was developed by Song J. in 2017 [34] for the detection of SARS-CoV-2 [35] These methods were performed in tubes, which means large volume (25 to 50 μL) and the detection of only one target in a single tube. The results led us to believe that the closed microfluidic chip system with the two-stage isothermal amplification method has high sensitivity, specificity and reproducibility, and consumes small volume reaction components with a fast reaction speed, which is suitable for real-time multiple pathogen diagnosis in a single detection run
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