A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

Abstract

The COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with a low limit of detection (5–10 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here, we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS calibration standard. The approach was validated on clinical saliva samples, where it showed excellent agreement with RT-qPCR. COV-ID can also be performed directly on saliva absorbed on filter paper, simplifying collection logistics and sample handling.

Fatores associados à percepção do risco biológico e aos acidentes com material biológico em Agentes Comunitários de Saúde

“…A saliva foi o fluido biológico mais envolvido nos acidentes relatados pelos ACS e, apesar do seu baixo potencial de veicular microrganismos de importância infecciosa em comparação ao sangue, apresenta risco potencial de transmissão de vírus, tais como herpes vírus, vírus da hepatite A e E (19) , vírus influenza A subtipo H1N1 (20) e o SARS-CoV-2 (21) . Além dos vírus, as partículas de saliva aerossolizadas têm potencial para carrear o Micobacterium tuberculosis, agente infeccioso causador da tuberculose, doença com alta prevalência entre os ACS (22) .…”

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

“…Several studies have developed methods to distinguish among different strains of SARS-CoV-2. Isothermal amplification techniques such as Recombinase Polymerase Amplification (RPA), 15,16 Recombinase-Aided Amplification (RAA), 17 and Loop-mediated Isothermal Amplification (LAMP) 18 are rapid and simple methods that can be used for POC detection. On the contrary, there are some limitations of isothermal amplification methods, such as the secondary structure of primers (longer than 30 bp for RPA and RAA), complicated primer design (at least three primer pairs for LAMP), optimized primer concentration, suitable incubation temperature, and downstream detection method.…”

A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

“…The barcoding step can be achieved either using commercial kits, like the kits from Illumina and Oxford Nanopore Technologies, or a customized design ( Table 1 ). In the latter case, a sequence of a barcode is often embedded in primers as an overhang at the step of reverse transcription of viral RNA or PCR amplification of the RT product ( Figure 1B ) ( Bhoyar et al, 2021 ; Bloom et al, 2021 ; Duan et al, 2021 ; Gauthier et al, 2021 ; Ludwig et al, 2021 ; Stüder et al, 2021 ; Wu et al, 2021 ; Cohen-Aharonov et al, 2022 ; Credle et al, 2022 ; Gallego-García et al, 2022 ; Palmieri et al, 2022 ; Warneford-Thomson et al, 2022 ; Yermanos et al, 2022 ). Barcoded primers used in SwabSeq ( Bloom et al, 2021 ) and by Cohen-Aharonoc et al (2022) ( Cohen-Aharonov et al, 2022 ) are compatible with one-step RT-PCR.…”

“…For example, amplicons from SwabSeq ( Bloom et al, 2021 ) are subjected to barcodes (i5 and i7) used to maximize the specificity and avoid false-positive results. Amplicons from LAMP-Seq ( Ludwig et al, 2021 ) and COV-ID ( Warneford-Thomson et al, 2022 ) contain one LAMP barcode (10 bp used in LAMP-Seq and 5 bp used in COV-ID) and two standard PCR barcodes (Illumina i5 and i7) to scale up the deep sequencing capacity. Gauthier et al (2021) ( Gauthier et al, 2021 ) employed SISPA barcoded primers ( Reyes and Kim, 1991 ) to detect and assemble genomes of SARS-CoV-2 and Oxford Nanopore barcodes to multiplex samples.…”