Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

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).

Microfluidics Technology in SARS-CoV-2 Diagnosis and Beyond: A Systematic Review

“…The whole assay was completed within 70 min with an LOD of two copies/reaction [ 102 ]. Xiong H et al developed a rotating microfluidic fluorescence system detection based on RT-LAMP, Ramachandran A et al developed isotachophoresis coupled RT-LAMP based amplification and clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12 based detection, Huang Q et al developed a portable microfluidic chip-based system with two-stage isothermal amplification, Soares et al developed the modular centrifugal microfluidic platform to perform RT-LAMP, all detect nucleic acids of SARS-CoV-2 by fluorescence-based detection [ 103 , 104 , 105 , 106 ]. The limit of detection of rotating microfluidic fluorescence systems detection based on RT-LAMP and isotachophoresis coupled RT-LAMP based amplification and CRISPR–Cas12 based detection is the same (10 copies/μL).…”

“…This assay demonstrated 94.12% specificity and 95.83% sensitivity for SARS-CoV-2 with a very low detection limit of about 10 copies. So, it is ideal for resource-constrained areas and point-of-care testing (POCT) ( Table 3 ) [ 105 ].…”

“…As for the material, PMMA was frequently used, followed by PDMS, polycarbonate, 192.24 IFC, nylon, glass, clear resin, and clear methacrylate-based resin [ 81 , 84 , 85 , 94 , 102 , 104 , 108 , 109 , 110 ]. Among fluid manipulation techniques, centrifugal force was the most commonly used strategy, followed by electrochemical pumping, capillary action, manual pipetting, hydrostatic pressure, isotachophoresis, and syringe pump [ 82 , 84 , 85 , 94 , 102 , 104 , 105 , 109 ]. Yang et al used manual pipetting followed by capillary action for the fluid manipulation strategy [ 107 ].…”

Advances in Biosensing Technologies for Diagnosis of COVID-19

“…In another set of studies, the microfluidic chamber is integrated with LAMP and RPA for the detection of SARS-CoV-2 and measles virus (MV) in parallel simultaneously by the fluorescence signal change. This point-of-care testing method showed 100% clinical specificity and sensitivity for MV and 94.12% specificity and 95.83% sensitivity for SARS-CoV-2 with the LOD of ten copies within one hour [ 46 ].…”

Editorial for the Special Issue on MEMS and Microfluidic Devices for Analytical Chemistry and Biosensing

“…To reach the goal of developing lower-cost and easy to use point-of-care analysis systems, Qin Huang et al [3] propose a novel microfluidic lab-on-chip for isothermal parallel detection of multiple targets. The approach is based on a two-stage recombinase polymerase amplification and subsequent loop-mediated isothermal amplification (RPA + LAMP) microfluidic platform, which is demonstrated in a disc-shaped milled PMMA lab-on-chip.…”