Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription.
Anal Chim Acta
; 1212: 339909, 2022 Jun 15.
Article
in English
| MEDLINE | ID: covidwho-1821092
ABSTRACT
Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2â¯fM and 200â¯fM within 1â¯h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Reverse Transcription
/
COVID-19
Type of study:
Diagnostic study
Topics:
Long Covid
Limits:
Humans
Language:
English
Journal:
Anal Chim Acta
Year:
2022
Document Type:
Article
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