Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

Abstract

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

G-quadruplex based biosensor: A potential tool for SARS-CoV-2 detection

“…Targeting G-quadruplex structures appears to be a promising alternative method for SARS-CoV-2 detection ( Zhang et al, 2020 ). Credle et al (2020) showed that based on aptamer probe ligation test, SARS-CoV-2 can be detected and its genotype can be effectively determined. This method is simple and does not require the nucleic acid extraction step.…”

ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection

“…We believe that our approach, and future improvements and modifications thereof, might form the basis for such an improvement of several orders of magnitude relative to current testing strategies. Specifically, the ability to pool hundreds or thousands of samples as early as possible, without losing the sample-specific information, is a crucial improvement on all currently published NGS-based tests 6,23,24,25,26,27 . The early pooling approach proposed here, which might be applicable to other protocols, elicits proportional reductions in costs and labor that directly translate to higher throughput in testing.…”

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations

“…A review by Esbin et al describes alternative COVID-19 diagnostic protocols involving lysis reagents and direct addition of samples (extraction-free protocols) into the amplification reaction mix [22]. There have been recent advances in extraction-free protocols for COVID-19 amplification [154][155][156][157][158][159] and sequencing-based tests [160]. However, such protocols involve trade-offs between lower sensitivity and simplicity [22,161,162].…”

“…Hi-throughput pooled tests [23][24][25][26][27][28] Pooled sequencing using barcodes [93] Commercial platforms-Hi-throughput RT-PCR [220] Alternative reagents and protocols. for diagnostic testing to augment shortages and supply chain constraints during pandemic peak [22] Extraction-free protocols to augment shortage of RNA extraction kits for amplification-based tests [154][155][156][157][158][159] for sequencing-based tests [160] Drive-through collection of respiratory samples [142] At-home self-collection of clinical samples to augment conventional sample collection formats [147,149] Antibody tests to measure seroprevalence of SARS-CoV-2 antibodies and understand the extent of community transmission [133,134] Large scale population-wide screening for identification of asymptomatic cases in low-prevalence regions [23,25,26] Screening for infections at clinics/doctor's offices, airports, borders, etc.…”