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Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities.

ABSTRACT: The world is currently facing a novel viral pandemic (SARS-CoV-2), and large-scale testing is central to decision-making for the design of effective policies and control strategies to minimize its impact on the global population. However, testing for the presence of the virus is a major bottleneck in tracking the spreading of the disease. Given its adaptability regarding the nucleotide sequence of target regions, RT-qPCR is a strong ally to reveal the rapid geographical spreading of novel viruses. We assessed PCR variations in the SARS-CoV-2 diagnosis taking into account public genome sequences and diagnosis kits used by different countries. We analyzed 226 SARS-CoV-2 genome sequences from samples collected by March 22, 2020. Our work utilizes a phylogenetic approach that reveals the early evolution of the virus sequence as it spreads around the globe and informs the design of RT-qPCR primers and probes. The quick expansion of testing capabilities of a country during a pandemic is largely impaired by the availability of adequately trained personnel on RNA isolation and PCR analysis, as well as the availability of hardware (thermocyclers). We propose that rapid capacity development can circumvent these bottlenecks by training medical and non-medical personnel with some laboratory experience, such as biology-related graduate students. Furthermore, the use of thermocyclers available in academic and commercial labs can be promptly calibrated and certified to properly conduct testing during a pandemic. A decentralized, fast-acting training and testing certification pipeline will better prepare us to manage future pandemics.

SUBMITTER: Barreto HG

PROVIDER: S-EPMC7567654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

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Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities.

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