SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires bot...
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doaj-30798df488df4e9bbce770cad00a1a912021-02-15T00:01:47ZengMDPI AGMicromachines2072-666X2021-02-011219719710.3390/mi12020197SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal AmplificationMeiqing Liu0Haoran Li1Yanwei Jia2Pui-In Mak3Rui P. Martins4State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, ChinaState-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, ChinaState-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, ChinaState-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, ChinaState-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau 999078, ChinaThe emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.https://www.mdpi.com/2072-666X/12/2/197SARS-CoV-2DNA probeduplex-specific nucleasesignal amplificationRNA detection |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Meiqing Liu Haoran Li Yanwei Jia Pui-In Mak Rui P. Martins |
spellingShingle |
Meiqing Liu Haoran Li Yanwei Jia Pui-In Mak Rui P. Martins SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification Micromachines SARS-CoV-2 DNA probe duplex-specific nuclease signal amplification RNA detection |
author_facet |
Meiqing Liu Haoran Li Yanwei Jia Pui-In Mak Rui P. Martins |
author_sort |
Meiqing Liu |
title |
SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification |
title_short |
SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification |
title_full |
SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification |
title_fullStr |
SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification |
title_full_unstemmed |
SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification |
title_sort |
sars-cov-2 rna detection with duplex-specific nuclease signal amplification |
publisher |
MDPI AG |
series |
Micromachines |
issn |
2072-666X |
publishDate |
2021-02-01 |
description |
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection. |
topic |
SARS-CoV-2 DNA probe duplex-specific nuclease signal amplification RNA detection |
url |
https://www.mdpi.com/2072-666X/12/2/197 |
work_keys_str_mv |
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