key: cord-0859777-tbvqkuum
authors: Chen, Hong; Wang, Yunxiang; Wei, Hongjuan; Rong, Zhen; Wang, Shengqi
title: A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus
date: 2022-01-25
journal: RSC advances
DOI: 10.1039/d1ra07756b
sha: 19c41c23b88ff514389700fbb1badf18875157bf
doc_id: 859777
cord_uid: tbvqkuum

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per μL and 14.23 copies per μL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

1. The assembly of DNA fragments in vitro. Figure S1 . The image of agarose gel electrophoresis indicated that DNA fragments with length of 1 kb could be assembled in vitro by water bath PCR. Electrophoretogram of 1-5: DNA fragments with length of 1 kb.

2. Determination of the shortest incubation time in each water bath. Figure S2 . Temperature profiles of the PCR mixture and water bath in an entire PCR protocol. Temperatures were recorded by two thermocouples fixed inside and outside the reaction tube.

3. Optimization of the concentration of the used SA-QBs. Figure S4 . The signal-to-noise ratio of positive results (10 4 copies/μL of SARS-CoV-2 and IBV) to negative control at different concentrations of digoxin antibody and TAMRA antibody.

5. Agarose gel electrophoresis of PCR products. Figure S5 . Images of agarose gel electrophoresis at different concentrations of (A) SARS-CoV-2 and (B) IBV in the range of 10 2 -10 5 copies/μL. In each electrophoretogram, 1: negative control, 2-5: 10 5 -10 2 copies/μL.

The nucleic acid template of the influenza B virus and SARS-CoV-2

 TTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAG  TGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTG  CGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCC  CTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAA  ATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAA  AATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGC  CAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTT  GCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAA  TCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATT  GCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCT  TCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGC  AGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGC  TGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAAT  GTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTG  CTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCA  TACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGG  AAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATT  GGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGT  CGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACA  GGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCAT  TTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGC  CTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCA  GAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGG  ATGATTTCTCCAAACAA  TTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAA