Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2.
Anal Chem
; 92(13): 9399-9404, 2020 07 07.
Article
in English
| MEDLINE | ID: covidwho-342738
ABSTRACT
Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Pneumonia, Viral
/
Coronavirus Infections
/
Real-Time Polymerase Chain Reaction
Type of study:
Diagnostic study
/
Etiology study
/
Experimental Studies
/
Prognostic study
Limits:
Adult
/
Aged
/
Female
/
Humans
/
Male
/
Middle aged
Language:
English
Journal:
Anal Chem
Year:
2020
Document Type:
Article
Affiliation country:
Acs.analchem.0c01884
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