Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection

Abstract

Objectives: The ongoing COVID-19 pandemic continues imposing a demand for diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, we assessed the sensitivity of a number of one-step retrotranscription and quantitative PCR (RT-qPCR) solutions to detect SARS-CoV-2. Methods: We evaluated six different RT-qPCR alternatives for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. That of best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating, overcoming the RNA extraction step. Results: We found a wide variability in the sensitivity of RT-qPCR solutions that associated with a range of false negatives from as low as 2% (0.3-7.9%) to as much as 39.8% (30.2-50.2). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0), in the range of some of the solutions based on standard RNA extractions. Conclusions: We evidenced sensitivity limitations of currently used RT-qPCR solutions. Our results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.

Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis

“…The global spread of a new type of coronavirus, SARS-CoV-2, causing the respiratory disease COVID-19 [ 1 , 2 , 3 , 4 ] mobilized both the public and private sector and resulted in a rapid development of solutions focused on SARS-CoV-2 detection and analysis. Next to a number of solutions utilizing the advantages of RT-qPCR techniques for SARS-CoV-2 detection [ 5 , 6 ], the next-generation sequencing (NGS)-based protocols allows the analysis of SARS-CoV-2 genome evolution and variability and the monitoring of its spread within the global population (Nextstrain; ) [ 7 , 8 ]. These knowledges address the need to elucidate its genomic characteristics (GISAID; ) in order to ensure the efficiency of RT-qPCR testing, assess its transmission through clonal events, and develop a reliable vaccination protocols future therapies, especially considering the fact that RNA viruses are prone to accumulate variants in its genome in a relatively short timeline, which in the case of SARS-CoV-2 is also related to its capacity to proofread and remove mismatched nucleotides during genome replication and transcription [ 9 , 10 , 11 , 12 ].…”

In-House Development, Standardization, and Validation of a RT-qPCR Assay for the Detection of SARS-CoV-2 Virus in Ecuador.

“…The duplex set-up reduced reagents usage as well as processing time, while maintaining a high detection rate, modest analytical sensitivity and a much lower cost. Even though the test still has room for improvement, it showed a high sensitivity which is well within the parameters for diagnostics and comparable to other commercial kits [16][17]. Many in-house tests can be found in literature, however, to our knowledge their viability has not been explored in Ecuador.…”

“…3). An excellent starting point would be an exploratory study on selection of RT-qPCR mixes which has been shown to signi cantly alter the sensitivity of a COVID diagnostic [17].…”

“…We present an in-house protocol which is primarily based on the envelope protein gene E for COVID-19 detection. This gene target is highly recommended by the WHO and the Pan American Health Organization (PAHO) as a diagnostic target and it is considered a highly sensitive probe [11,[15][16][17]. The in-house protocol also includes an internal control for quality of genetic material, the human ribonuclease P (RP).…”

Asymptomatic COVID-19 in hospital visitors: The underestimated potential of viral shedding

“…Samples’ RNA were purified with Quick-RNA Viral Kit (Zymo Research, Irvine, CA, USA), and viral detection was performed with AgPath-ID One-Step RT-PCR reagents (ThermoFisher Scientific, Austin, TX, USA), according to manufacturers’ instructions. A 20 µL total reaction volume containing 5.0 µL purified RNA, 400 nM primers, and 200 nM probes following the CDC USA protocol targeting the N1 and N2 sequences of the SARS-CoV-2 nucleoprotein gene [8] , with a sensitivity of 75-80% [9] . Cycle thresholds < 40 were considered positive.…”