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.

Keywords: Coronavirus disease 2019; Isothermal amplification; Microfluidics; Nucleic acid testing; Severe acute respiratory syndrome coronavirus 2.

Fig. 1

Design of the disc-shaped microfluidic chip and workflow for the detection of respiratory viruses. (a) Schematic diagram of the microfluidic chip; (b) layout of the designated reaction chambers for each targeted virus and control on the chip; (c) mechanism of NASBA; in the non-cyclic phase, primer 1 anneals to the target sequence and forms an RNA:DNA hybrid. RNase H hydrolyzes the RNA from this RNA:DNA hybrid; next, primer 2 anneals to the single-stranded DNA (ssDNA) and synthesizes a double-stranded DNA (dsDNA) by reverse transcriptase (RT); in the cyclic phase, the reaction repeats the cycles of transcription of RNA from dsDNA templates, the synthesis of cDNA, the hydrolysis of the RNA chain in the RNA:DNA hybrid, and the formation of dsDNA; (d) workflow of virus detection. Left to right: receiving the inactivated clinical samples, extracting RNA from samples, loading reagents into the chip, sealing the chip with tape, loading the chip into the instrument, distributing reagents into the reaction chambers on the chip, and performing amplification and detection in the RTisochip™-W system.

Fig. 2

Design of the RTisochip^TM-W system. (a) Photograph of the 16-channel RTisochip™-W with four piled analyzers, each of which contains four sub-modules. (b) Exploded view of the analyzer containing four sub-modules. 1: sheet metal frame; 2: right side panel; 3: upper panel; 4: sub-module; 5: front panel; 6: rear panel; 7: fixed upper cover panel of switch; 8: switch; 9: fixed panel of switch; 10: network port mounting panel; 11: partition panel; 12: power socket mounting panel; 13: socket-type power filter; 14: power supply for the system; 15: power supply for the microchip heater; 16: left side panel; 17: rubber foot. (c) Schematic of the structure of the sub-module inside the instrument. Up to 16 identical sub-modules can be connected to the computer via a router. PMT: photomultiplier tube; LED: light emitting diode; PID: proportional-integral-derivative.

Fig. 3

Comparison of different RNA extraction methods for on-chip virus detection. (a) Comparison of the RNA extraction efficiencies of the QIAamp® and the TRIzol™ kits by detecting four different virus targets (n = 3); (b) comparison of the manual TRIzol™ kit and the automated KingFisher™ Flex Purification System in the detection of HCoV-229E/NL63 and H1N1 2009 (n = 3). All error bars represent one standard deviation. TCID₅₀: median tissue culture infectious dose.

Fig. 4

Sensitivity and repeatability tests of the RTisochip™-W system. (a) LOD of 19 respiratory viruses tested by the on-chip assays; (b) typical amplification curves and linear fitting between the Tp and the template concentration of H1N1 2009 (n = 3); (c) typical amplification curves and linear fitting of SARS-CoV-2 S gene (n = 3); (d) repeatability tests of three microfluidic chips with synthesized RNA templates of H1N1 2009 and SARS-CoV-2 S gene (n = 10); (e) repeatability of the detection of SARS-CoV-2 S and N genes with extracted RNA samples (n = 10). All error bars represent one standard deviation.

Fig. 5

Anti-interference, cross-reaction, and competitive effect tests of the RTisochip™-W. (a) Similar Tp values and amplification curves of seven selected targets in reactions with or without mucin. (b) Final concentrations of the interfering substances added to the reaction buffer. (c) Similar Tp values of seven selected targets in reactions with or without mixed interfering substances. (d) Cross-reaction tests of the platform. Positive results are shown as orange squares, while azure squares represent negative results. P1–P19: virus templates; N1–N6: NCs. (e) Competitive effect tests of mixed target samples. Two different concentrations of single- or mixed-target samples were tested and influenza B was missed at the low concentration of 500 copies·μL⁻¹. ns: not significant.

Fig. 6

Clinical identification of respiratory viruses using the RTisochip™-W. (a) Positive rates of each virus target in clinical samples collected in 2019–2020. Samples from the Beijing Tsinghua Changgung Hospital were collected in the flu season from 2019 to 2020 (n = 101), and samples from the Capital Institute of Pediatrics were collected in the non-flu season of 2019 (n = 100). (b) Comparison of the SARS-CoV-2 detection results obtained by the RTisochip™-W and the conventional RT-PCR. “+” represents a positive result and “–” is negative. (c) Repeatability tests of the RTisochip™-W using clinical samples. Positive and negative results are shown as orange and azure squares, respectively. (d) Positive rates of a total of 614 clinical throat swab samples from suspected or confirmed COVID-19 patients. The detailed number was indicated.

Fig. 7

COVID-19 testing guideline. NGS: next generation sequencing; dPCR: digital PCR; IgM: immunoglobin M; IgG: immunoglobin G.