A High-Throughput, Multi-Index Isothermal Amplification Platform for Rapid Detection of 19 Types of Common Respiratory Viruses Including SARS-CoV-2

Abstract

Fast and accurate diagnosis and the immediate isolation of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are regarded as the most effective measures to restrain the coronavirus disease 2019 (COVID-19) pandemic. Here, we present a high-throughput, multi-index nucleic acid isothermal amplification analyzer (RTisochip™-W) employing a centrifugal microfluidic chip to detect 19 common respiratory viruses, including SARS-CoV-2, from 16 samples in a single run within 90 min. The limits of detection of all the viruses analyzed by the RTisochip™-W system were equal to or less than 50 copies·μL −1 , which is comparable to those of conventional reverse transcription polymerase chain reaction. We also demonstrate that the RTisochip™-W system possesses the advantages of good repeatability, strong robustness, and high specificity. Finally, we analyzed 201 cases of preclinical samples, 14 cases of COVID-19-positive samples, 25 cases of clinically diagnosed samples, and 614 cases of clinical samples from patients or suspected patients with respiratory tract infections using the RTisochip™-W system. The test results matched the referenced results well and reflected the epidemic characteristics of the respiratory infectious diseases. The coincidence rate of the RTisochip™-W with the referenced kits was 98.15% for the detection of SARS-CoV-2. Based on these extensive trials, we believe that the RTisochip™-W system provides a powerful platform for fighting the COVID-19 pandemic.

Rapid electrochemical detection of coronavirus SARS-CoV-2

“…Examples of commercially available qRT-PCR test kits are the Xpert Xpress SARS-CoV-2 test 9 , CDC 2019-novel coronavirus Real-Time RT-PCR Diagnostic Panel 10 , ExProbeTM SARS-CoV-2 Testing Kit 11 , Abbott RealTime SARS-CoV-2 RT-PCR Kit 12 , PerkinElmer® New Coronavirus Nucleic Acid Detection Kit 13 , and TaqPath COVID-19 Combo Kit 14 . However, there are disadvantages with these methods as they are time-consuming, requiring a specialized laboratory setting with expensive instruments and trained personnel 15 .To overcome these drawbacks, various isothermal nucleic acid amplification assays such as recombinase polymerase amplification (RPA) 16 and loop-mediated isothermal amplification (LAMP) [17][18][19][20] , deoxyribonucleic acid (DNA) nanoscaffoldbased hybrid chain reaction 21 , and nucleic acid sequence-based amplification 22 , have been developed to overcome the need for sophisticated thermal cycling equipment associated with qRT-PCR.…”

Rapid and sensitive triple-mode detection of causative SARS-CoV-2 virus specific genes through interaction between genes and nanoparticles

“…But for patients with mild infection, the performance of our triple-mode biosensor needs to be improved by incorporating gene amplification processing. Recently, Yan [ 49 ] and Xing[ 50 ] reported the signal amplification strategy by using fluorescence molecular amplification technology, resulting in the ultrasensitive gene detection at the level of ∼aM. We believe that the proposed triple-mode biosensor could reach much lower detection limit by incorporating the fluorescence signal amplification strategy.…”

Nucleic Acid Testing of SARS-CoV-2

“…Therefore, unlike RT-qPCR, isothermal amplification methods do not require thermal cycling instruments or specialized technicians for disease diagnosis [ 132 ]. Current isothermal nucleic acid amplification methods used for SARS-CoV-2 detection include, but are not limited to, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), strand-displacement amplification (SDA), and rolling circle amplification (RCA) [ 133 , 134 , 135 , 136 , 137 ] ( Figure 1 C). Herein, we describe the general procedures and components of isothermal amplification methods commonly used for diagnosis of SARS-CoV-2.…”

“…First described by Notomi et al [ 138 ], this method uses strand displacement activity of DNA polymerase and a set of inner and outer primers (four or six specific primer sequences) to amplify the target nucleic acids. LAMP is carried out at a single temperature between 60 and 65 °C, and generates up to 10 9 copies of DNA in less than an hour [ 133 , 137 , 139 ]. The LAMP procedure is initiated by hybridization of the forward inner primer (FIP) toward the target DNA template, which synthesizes the complementary strand.…”

“…Other than LAMP and RPA, isothermal amplification methods such as NASBA, SDA, and RCA have been used for the detection of SARS-CoV-2 [ 136 , 137 , 158 ]. Although we will not describe each technique in detail here, Table 3 presents the general features and components of each isothermal amplification method.…”