Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2.
Mol Diagn Ther
; 25(5): 617-628, 2021 09.
Article
in English
| MEDLINE | ID: covidwho-1328680
ABSTRACT
BACKGROUND AND OBJECTIVE:
Since the initial coronavirus disease outbreak in late 2019 (COVID-19), reverse-transcription real-time polymerase chain reaction (RT-qPCR) has become the gold standard test to detect severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, a more sensitive and accurate diagnostic tool was required. Therefore, droplet digital polymerase chain reaction (ddPCR) was suggested as an alternative method. Here, we evaluated the performance of ddPCR to detect SARS-CoV-2 and compared it to the performance of RT-qPCR.METHODS:
The analytical performances, including limit of blank and limit of detection, were established using positive and negative SARS-CoV-2 reference materials. A total of 366 RNA extracts (173 positive and 193 negative by RT-qPCR) were collected from four institutions and tested with a Bio-Rad SARS-CoV-2 ddPCR kit that detects the SARS-CoV-2 genome using primers for N1 and N2.RESULTS:
Limit of blank was set at 0, and the limits of detection of N1 and N2 were 1.99 copies/µL and 5.18 copies/µL, respectively. Linearity was evaluated using serial dilution samples, which demonstrated good results (R2 0.999, linear range 5.88-6825.25 copies/µL for N1 and R2 0.999, 5.53-5855.47 copies/µL for N2). The results of ddPCR and RT-qPCR revealed substantial agreement (Cohen's kappa 0.639, p < 0.01). The 63 samples with positive ddPCR but negative RT-qPCR showed low copy numbers, and 55% of them had COVID-19-related symptoms.CONCLUSIONS:
Droplet digital polymerase chain reaction demonstrated excellent sensitivity for SARS-Cov-2 detection and consistently agreed with the results from conventional RT-qPCR. Furthermore, ddPCR provided quantitative data that can be used to monitor changes in the viral load of patients with COVID-19.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA, Viral
/
COVID-19 Nucleic Acid Testing
/
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
/
Experimental Studies
/
Prognostic study
Topics:
Long Covid
Limits:
Humans
Language:
English
Journal:
Mol Diagn Ther
Journal subject:
Molecular Biology
/
Pharmacology
/
Laboratory Techniques and procedures
Year:
2021
Document Type:
Article
Similar
MEDLINE
...
LILACS
LIS