LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons

Abstract

SARS-CoV-2 has caused a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and Loop Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses alternative and often cheaper reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however a common artifact is nonspecific DNA amplification, complicating detection. Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions.

Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

“…Using simple isothermal amplification of unpurified saliva and endpoint fluorescence, the genotype of a viral sample can be determined visually without expensive equipment. With locally-purified enzymes 12 , the reagents costs can be reduced to pennies per test. Combined with the easily parallelizable nature of isothermal incubation, scaling is effectively limited only by sample collection and processing.…”

“…We then tested the S6970 primers and beacons on synthetic RNA from wild-type (Twist Australia/VIC01/2020) and B.1.1.7 (Twist England/205041766/2020) SARS-CoV-2 variants. For comparison, we also multiplexed S6970 with the N2 primer set 11 and molecular beacon 12 . For these experiments, S6970 B117 MB was synthesized with a fluorescein (Fam) label, S6970 WT MB with a cyanine 3 (Cy3) label and N2 MB with a cyanine (Cy5) label.…”

“…Reactions were performed using locally produced enzyme mix equivalent to commercial RT-LAMP mix 12 . Reactions consisted of 10 µL of 2x RT-LAMP enzyme mix, 2 µL of 10x primer-beacon mix and 8 µL of water doped with varying amounts of synthetic RNA.…”

“…IBFQ indicates Iowa Black FQ and IBRQ indicates Iowa Black RQ dark quenchers. ‡ N2 LB 2 and N2 MB are from Sherrill-Mix et al12 Other N2 primers are from Zhang et al11…”

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

“…The most common targets chosen as endogenous controls for RT-PCR and RT-LAMP reactions are housekeeping genes such as RNAse P [22] and GAPDH [23] which do not have tissue-specific expression patterns. Recently, an RT-LAMP primer set has been designed to detect the saliva specific expression of STATH [24] to work in conjunction with saliva-based detection of SARS-CoV-2. We have added to an increasing number of tissue-specific mRNA RT-LAMP primer sets by designing one specific to nasopharynx mRNA.…”