Assessment of the Diagnostic Ability of Four Detection Methods Using Three Sample Types of COVID-19 Patients

Abstract

BackgroundViral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. However, unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic.MethodsWe compared four nucleic acid detection methods [two kinds of reverse transcription polymerase chain reactions (RT-PCR A: ORF1ab and N testing; RT-PCRB: only ORF1ab testing), reverse transcription recombinase aided amplification (RT-RAA) and droplet digital RT-PCR (dd-RT-PCR)] using 404 samples of 72 hospitalized COVID-19 patients, including oropharyngeal swab (OPS), nasopharyngeal swabs (NPS) and saliva after deep cough, to evaluate the best sample type and method for SARS-CoV-2 detection.ResultsAmong the four methods, dd-RT-PCR exhibited the highest positivity rate (93.0%), followed by RT-PCR B (91.2%) and RT-RAA (91.2%), while the positivity rate of RT-PCR A was only 71.9%. The viral load in OPS [24.90 copies/test (IQR 15.58-129.85)] was significantly lower than that in saliva [292.30 copies/test (IQR 20.20-8628.55)] and NPS [274.40 copies/test (IQR 33.10-2836.45)]. In addition, if OPS samples were tested alone by RT-PCR A, only 21.4% of the COVID-19 patients would be considered positive. The accuracy of all methods reached nearly 100% when saliva and NPS samples from the same patient were tested simultaneously.ConclusionsSARS-CoV-2 nucleic acid detection methods should be fully evaluated before use. High-positivity rate methods such as RT-RAA and dd-RT-PCR should be considered when possible. Furthermore, saliva after deep cough and NPS can greatly improve the accuracy of the diagnosis, and testing OPS alone is not recommended.

Secondary Infection Surveillance with Metagenomic Next-Generation Sequencing in COVID-19 Patients: A Cross-Sectional Study

“…Samples with Ct values ≤38.0 for both ORFab1 and N genes were considered positive, specimens with Ct values >38.0 were repeated, specimens with repeated results of Ct values >38 and specimens with undetectable Ct values were considered negative. 19 …”

Understanding viruses and viral infections by biophotonic methods

“…PCR-based methods are considered to be the current gold standard for virus detection [37,38]. Due to the amplification of specific nucleic acid sequences, viruses can be identified in a sensitive and highly specific manner [38,39]. By employing certain fluorescent dyes (or pairing them with quenchers) even quantification is possible [40].…”

“…Therefore, it is difficult to isolate them from the sample fluid and as described above they are often bound to nanoparticles or antibodies for isolation and quantification. In clinical surroundings, standardized tests based on PCR methods are used for viral load determination [37,39]. Since a higher viral load correlates with the severity of the infection, many different optical approaches-like fluorescence microscopy, vibrational spectroscopy-are being explored to detect viral particles [83].…”

Advances in droplet digital polymerase chain reaction on microfluidic chips

“…Polymerase chain reaction (PCR) has been crucial in these situations owing to the advantages of high detection sensitivity and capacity to amplify signals from samples with low amounts of genetic material via repeated cycles of denaturation, annealing, and extension. 1–5…”