Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens

Abstract

The pandemic novel coronavirus infection, Coronavirus Disease 2019 , has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID 50 /mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed Int.

Six decades of lateral flow immunoassay: from determining metabolic markers to diagnosing COVID-19

“…It is known that COVID-19 belongs to the Coronaviridae family of RNA-containing coronaviruses that cause acute respiratory infections in humans of varying severity (from asymptomatic or mild to severe pneumonia) [84] , [86] , [87] . In this case, the main pathogenetic targets are the respiratory system, the gastrointestinal tract, the liver and the central nervous system, the defeat of which has become a characteristic feature and others coronavirus infections (Middle East Respiratory Syndrome, MERS, and Severe Acute Respiratory Syndrome, SARS) [85] , [88] , [89] .…”

“…Before the emergence in the XXI century of epidemic outbreaks of infections (SARS in 2002–2004 and MERS in 2012 caused by β-coronaviruses MERS-CoV [80] , [81] , [84] and SARS-CoV [86] , [87] , these pathogens were not considered highly pathogenic to humans. They circulated in the human population, causing only sporadic cases of diseases that occur, as a rule, in a mild form [85] , [88] .…”

“…Like other β-coronaviruses, the SARS-CoV-2 gene contains specific RNA sequences encoding 27 proteins. Among them, 15 non-structural proteins that provide virus replication [87] , [92] – [94] and 12 structural proteins are distinguished. For SARS-CoV-2, antibodies have been detected that recognize three of the four SARS-CoV-2 proteins exposed on the surface of the viral capsid: the nucleocapsid (N), envelope (E), and spike (S) proteins [95] .…”

“…One of the most important directions in the strategy of combating a new infection has become the need for mass laboratory screening of populations at high risk of infection. The need for timely and high-quality laboratory diagnosis of patients infected with SARS-CoV-2 has become the main priority in eliminating the pandemic and introducing quarantine measures [87] , [90] , [96] , [97] .…”

“…However, both in cases of elimination of SARS and MERS infections, and in the diagnosis of COVID-19, public health faces the same task. It is associated with determining the role and place of various diagnostic platforms for screening, diagnosis and monitoring of new coronavirus infections: RT-PCR, RT-LAMP, ELISA and LFIA, taking into account the advantages [87] , [89] , [91] , [98] .…”