Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR

Abstract

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

Positive impact of oral hydroxychloroquine and povidone-iodine throat spray for COVID-19 prophylaxis: An open-label randomized trial

“…Fifth, for resource, microbiological and logistical reasons, we did not perform nasopharyngeal swabs for the entire cohort and therefore could not exclude the possibility that some participants could harbor asymptomatic infection on recruitment. However, diagnosing asymptomatic infection can be challenging as the sensitivity of quantitative PCR could be as low as 30% in asymptomatic individuals depending on whether the test is performed during the incubation, presymptomatic, or recovery phases of the disease (Dong et al, 2021;Ternovoi et al, 2020). To further improve its accuracy, repeated PCR tests may be required.…”

Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies

“…ddPCR outperforms other molecular approaches in terms of repeatability, stability, sensitivity, and specificity [ 49 ]. It enables physicians to diagnose COVID-19 infection in individuals who are asymptomatic or paucisymptomatic by accurately detecting extremely low viral loads [ 50 ]. According to a recent study, 63 samples that had previously tested negative for SARS-CoV-2 by RT-qPCR were positive for the virus using ddPCR, and 55% of those samples displayed COVID-19 symptoms [ 51 ].…”

Point-of-Care PCR Assays for COVID-19 Detection

“…However, most of the reported ddPCR procedures included an RNA extraction and purification step, which can lead to potential amplification errors [ 38 ]. Moreover, direct quantification by ddPCR targeting the envelope (E) gene [ 39 ], ORF1ab gene [ 40 ], and nucleocapsid (N) [ 41 ] region have also been reported. The viral load can be quantified in throat swabs, sputum, nasal swabs, blood, and urine [ 37 ].…”