Multiplex PCR Assays for Identifying All Major SARS-CoV-2 Variants

Abstract

Variants of Concern (VOC) of SARS-CoV-2, including Alpha, Beta, Gamma, and Delta, threaten to prolong the pandemic leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the development and evolution of the virus, but is costly, slow, and not easily accessible. A multiplex qRT-PCR assay for SARS-CoV-2 was developed, which identifies all VOC as well as other mutations of interest in the viral genome, eight mutations total, using single nucleotide discriminating molecular beacons in a two-tube assay. The sensitivity and specificity of the assay was tested using in vitro-transcribed targets. Twenty-six SARS-CoV-2 positive patient samples were blinded, then tested using this assay and compared with deep sequencing results. The presented variant molecular beacon assay showed high accuracy when testing in vitro-transcribed targets, down to a limit of detection of five copies of the viral RNA, with 100% specificity. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26/26). We have developed a qRT-PCR assay for the identification of currently circulating VOC of SARS-CoV-2 as well as other important mutations in its Spike protein coding sequence. This assay can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

OMICRON: Virology, immunopathogenesis, and laboratory diagnosis

“…Because variations propagate throughout populations over time, these techniques may allow for an improved search of variants, particularly in resource-limited contexts. 62,63 Genome sequencing is the gold standard for tracking the virus's genesis and evolution, but it is expensive, time-consuming and challenging to get.…”

“…Although PCR techniques target preselected mutations, they are less expensive and more convenient than sequencing for SARS‐CoV‐2 genotyping, producing more rapid results. Because variations propagate throughout populations over time, these techniques may allow for an improved search of variants, particularly in resource‐limited contexts 62,63 . Genome sequencing is the gold standard for tracking the virus’s genesis and evolution, but it is expensive, time‐consuming and challenging to get.…”

“…Because variations propagate throughout populations over time, these techniques may allow for an improved search of variants, particularly in resource‐limited contexts. 62 , 63 Genome sequencing is the gold standard for tracking the virus’s genesis and evolution, but it is expensive, time‐consuming and challenging to get. Using single‐nucleotide discriminating molecular beacons, multiplex quantitative RT‐PCR assays for SARS‐CoV‐2 have been created that identify all VOCs and additional variants of interest in the viral genome, for a total of nine mutations.…”

Sub‐lineages of the SARS‐CoV‐2 Omicron variants: Characteristics and prevention

“…56,57 For VOCs, including the Omicron variant, two-tube multiplex qRT-PCR can detect five copies of viral RNA. 58 Next-generation sequencing has become the gold standard for identifying Omicron. Scott et al 59 have shown that viral genome sequencing can be used for detecting apparent deletions of the S gene (S-).…”

“…In addition, RT‐PCR testing is necessary for potentially infected individuals with Omicron, such as a history of recent travel to the epidemic area and close contact with confirmed patients 56,57 . For VOCs, including the Omicron variant, two‐tube multiplex qRT‐PCR can detect five copies of viral RNA 58 . Next‐generation sequencing has become the gold standard for identifying Omicron.…”