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 nonmedical 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.

Assessment of 12 qualitative RT‐PCR commercial kits for the detection of SARS‐CoV‐2

“…Moreover, the nucleotide mutation rate reported for SARS‐CoV‐2 was 8E‐04 substitution per site per year 13,14 . Many studies have already shown the high evolution rate of SAR‐CoV‐2 in many cases and linked its evolution with its origin 5 . Detection methods using RT‐PCR are based on a fixed target, however, as the pandemic proceeds the virus is changing and many cases are missed.…”

“…These measurements rely on an accurate diagnosis of individuals infected with the virus by using real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) 4 . The most common targets for detecting SARS‐CoV‐2 by (RT‐PCR) assays in diagnostic laboratories are the ORF1ab gene, the RdRP gene, the E gene, the N gene, and the S gene 5 …”

Pneumothorax in a patient with pneumonia caused by SARS-CoV-2: A case report

“…In very severe cases, the disease can progress rapidly and become complicated by acute respiratory distress syndrome (ARDS) and coagulopathies 11 . To date, it is recommended that the definitive diagnosis of SARS-CoV-2 infection be confirmed by a positive RT-PCR test or genetic sequencing 12 .…”

Development of RT-PCR Based Diagnosis of SARS-CoV-2

“…This RT-qPCR is currently a reliable test commonly used for the diagnosis of SARS-CoV-2 infected symptomatic and asymptomatic patients [56]. Several scientific and clinical institutions around the world have produced molecular assays to diagnose SARS-CoV-2 and have made RT-qPCR primers and sample sets available to the public [57].…”

“…As the genetic code of coronaviruses consists of RNA, its purification of the test samples is a crucial step in RT-qPCR protocols. Officially, institutions in some countries have suggested unique RNA isolation kits for SARSCoV-2 detection [57]. Various virus extraction and detections kits are available in market as shown in Table 3.…”