Rapid and High-Throughput Reverse Transcriptase Quantitative PCR (RT-qPCR) Assay for Identification and Differentiation between SARS-CoV-2 Variants B.1.1.7 and B.1.351.
Oran Erster, Ella Mendelson, Virginia Levy, Areej Kabat, Batya Mannasse, Hadar Asraf, Roberto Azar, Yaniv Ali, Rachel Shirazi, Efrat Bucris, Dana Bar-Ilan, Orna Mor, Michal Mandelboim, Danit Sofer, Shai Fleishon, Neta S Zuckerman
Author Information
Oran Erster: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel. ORCID
Ella Mendelson: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Virginia Levy: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Areej Kabat: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Batya Mannasse: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Hadar Asraf: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Roberto Azar: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Yaniv Ali: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Rachel Shirazi: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Efrat Bucris: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Dana Bar-Ilan: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Orna Mor: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Michal Mandelboim: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Danit Sofer: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Shai Fleishon: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Neta S Zuckerman: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, Israel.
Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens ( = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.
