A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.
EBioMedicine
; 61: 103036, 2020 Nov.
Article
in English
| MEDLINE | ID: covidwho-844322
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
ABSTRACT
BACKGROUND:
Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis.METHODS:
A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios.FINDINGS:
The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative.INTERPRETATION:
SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic.FUNDING:
Detailed funding information is available at the end of the manuscript.Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Bacterial Proteins
/
RNA, Viral
/
Endodeoxyribonucleases
/
Real-Time Polymerase Chain Reaction
/
CRISPR-Associated Proteins
/
Clustered Regularly Interspaced Short Palindromic Repeats
/
Betacoronavirus
Type of study:
Diagnostic study
/
Prognostic study
/
Systematic review/Meta Analysis
Limits:
Humans
Language:
English
Journal:
EBioMedicine
Year:
2020
Document Type:
Article
Affiliation country:
J.ebiom.2020.103036
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