Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR tests

Abstract

The rapid onset of the global COVID-19 pandemic has led to multiple challenges for accurately diagnosing the infection. One of the main bottlenecks for COVID-19 detection is reagent and material shortages for sample collection, preservation, and purification prior to testing. Currently, most authorized diagnostic tests require RNA extraction from patient samples and detection by reverse transcription polymerase chain reaction (RT-PCR). However, RNA purification is expensive, time consuming, and requires technical expertise to perform. Additionally, there have been reported shortages of the RNA purification kits needed for most tests. With these challenges in mind, we report on extraction-free amplification of SARS-CoV-2 RNA directly from patient samples. In addition, we have developed a multiplex RT-PCR using the CDC singleplex targets. This multiplex has a limit of detection of 2 copies/μL. We have demonstrated these improvements to the current diagnostic workflow, which reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation

“…As a prelude to understanding sample preparation and controls for assay development, we similarly assessed the sensitivity of an extraction-free RT-qPCR assay for naked genomic SARS-CoV-2 RNA (ATCC). Naked RNA was serially diluted in saline and directly subjected to RT-qPCR without RNA extraction, and could be detected at a concentration of 50 copies per 20 microliter qPCR reaction (approximately 2.5 copies per microliter) with the N1 primer set, which amplifies the nucleocapsid (N) gene of SARS-CoV-2 (Figure 1), confirming extraction-free RT-qPCR as a plausible diagnostic tool [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] .…”

“…In particular, while RT-qPCR remains the gold standard for analysis, the requirement of RNA purification prior to enzymatic amplification slows the overall throughput of the assay and limits the number of samples that can be tested. In consequence, many groups have reported on "direct RT-PCR" assays for CoV-2 RNA that bypass the RNA purification [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . As a prelude to understanding sample preparation and controls for assay development, we similarly assessed the sensitivity of an extraction-free RT-qPCR assay for naked genomic SARS-CoV-2 RNA (ATCC).…”

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.…”

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

“…Indeed, while several groups have demonstrated RNA amplification by direct addition of swab samples in the widely used viral transport medium (VTM), inhibition of RT-PCR by VTM typically leads to a significant delay in amplification [10][11][12][13][14][15]. A comparison of commercial master mixes found that the commonly used TaqPath master mix is particularly susceptible to inhibition by VTM [16].…”