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.

Keywords: COVID-19; Coronavirus; Infrastructure; Polymerase chain reaction; Testing.

Fig. 1

Timeline of complete SARS-CoV-2 genome sequences deposited to GenBank, according to their collection dates. The viral structure image was designed using resources at Freepik.com

Fig. 2

Phylogenetic analysis of 14 complete SARS-CoV-2 genome sequences published by several countries. The molecular phylogenetic analysis was conducted in MEGA7 and inferred by the maximum likelihood method on the Hasegawa-Kishino-Yano model. For more complete studies on the evolution of SARS-CoV-2 during the pandemic and follow-up discussions on proper methodologies of phylogenetic analyses, please refer to [–21]

Fig. 3

a Representation of the SARS-CoV-2 genome sequence depicting annotated genes. This scheme uses the nucleotide coordinates of a virus sequenced in China (NC_045512). b Positions of the primer/probe targets used for detection by institutions in six countries. Colors of primers and probes correspond to countries: China, CDC (red); USA, CDC (blue); Germany (yellow); China/Hong Kong (gray); France (pink); Japan (black); and Thailand (orange). The numbers above each primer and probe indicate nucleotide position (coordinate) in the genome. Important details: France developed two assays for the RdRP gene (identified in the figure as ORF1a and ORF1b, according to the reference sequence [NC_045512] used). The assay developed by Germany for gene E was also tested in France. Germany developed two probes for the same RdRP gene region indicated in the image, one being specific for SARS-CoV-2 and another common for SARS-CoV-2, SARS-CoV, and bat SARS-related coronavirus, but only one assay is indicated in the image. Japan also indicated assays for nested RT-PCR, but the image above only represents RT-qPCR assays. An important aspect of RT-qPCR assay development for viral detection is confirming that primers and probes are specific to the virus of interest and do not detect viruses from the same family. This is particularly important for coronaviruses, since members of this group already circulate in the human population

Fig. 4

Variations of the polymerase chain reaction (PCR) used for virus diagnoses. In conventional PCR after reverse transcription (RT-PCR), the target DNA is detected at the end of PCR amplification, requiring a post-PCR process for visualization (e.g., DNA electrophoresis). The nested RT-PCR significantly enhances the sensitivity of conventional PCR, but it nearly doubles the amplification time. All qPCR protocols detect and measure target DNA after each polymerization (extension) cycle during the exponential amplification through fluorescence and do not require post-PCR processing. While two-step RT-qPCR involves two distinct stages of reverse transcription followed by qPCR, one-step RT-involves a single reaction in which cDNA synthesis and amplification occur successively