SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Vogels, Chantal B.F.; Watkins, Anne E.; Harden, Christina A.; Brackney, Doug E.; Shafer, Jared; Wang, Jianhui; Caraballo, César; Kalinich, Chaney C.; Ott, Isabel M.; Fauver, Joseph R.; Kudo, Eriko; Lu, Peiwen; Venkataraman, Arvind; Tokuyama, Maria; Moore, Adam J.; Muenker, M. Catherine; Casanovas‐Massana, Arnau; Fournier, John; Bermejo, Santos; Campbell, Melissa; Datta, Rupak; Nelson, Allison; Cruz, Charles S. Dela; Ko, Albert Icksang; Iwasaki, Akiko; Krumholz, Harlan M.; Matheus, JD; Hui, Pei; Liu, Chen; Farhadian, Shelli; Sikka, Robby; Wyllie, Anne L.; Grubaugh, Nathan D.

doi:10.1101/2020.08.03.20167791

Cited by 105 publications

(140 citation statements)

References 30 publications

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“…The ability to reopen economies safely depends crucially on the testing capacity available. Efforts to increase testing capacity have included testing from saliva 4 , using nonstandard storage media or dry swabs 5 , and eliminating the normal RNA purification step from the standard RT-qPCR tests 6,7 . Strategies such as pooling samples followed by detection using traditional or high throughput sequencing approaches have also been proposed as a way to allow significantly more testing at a highly reduced cost 8,9 .…”

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confidence: 99%

An enhanced isothermal amplification assay for viral detection

et al. 2020

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Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

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confidence: 99%

An enhanced isothermal amplification assay for viral detection

et al. 2020

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“…The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.23.20236901 doi: medRxiv preprint sensitivity [23]. Hence, the goal of this study was to optimize both pre-analytical and analytical variables by developing a universal saliva processing protocol that would enable an extraction free RT-PCR test using any of the commercially available RT-PCR kits.…”

Section: Discussionmentioning

confidence: 99%

“…Several groups have demonstrated the feasibility of extraction free RT-PCR reaction maintaining the high sensitivity of the assay with NPS samples [19][20][21][22]. It is noteworthy that performing extraction free RT-PCR assay using saliva samples has been found to be a feasible method but only with the following caveats: a) effective for the asymptomatic population; b) requires early morning saliva (pure saliva); and, c) has a limit of detection (LoD) of 6000-12000 copies/ml [23]. Although the study is encouraging, the pre-requisite conditions render it unsuitable for mass population screening, especially because the precise sample collection requirement and the low test sensitivity would lead to a high percentage of false negative results.…”

Section: Introductionmentioning

confidence: 99%

SalivaSTAT: Direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance

Sahajpal

Mondal

Ananth

et al. 2020

Preprint

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BackgroundThe limitations of widespread current COVID-19 diagnostic testing lie at both pre-analytical and analytical stages. Collection of nasopharyngeal swabs is invasive and is associated with exposure risk, high cost, and supply-chain constraints. Additionally, the RNA extraction in the analytical stage is the most significant rate-limiting step in the entire testing process. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction free RT-PCR test using any of the commercially available RT-PCR kits.MethodsWe optimized saliva collection devices, heat-shock treatment and homogenization. The effect of homogenization on saliva samples for extraction-free RT-PCR assay was determined by evaluating samples with and without homogenization and preforming viscosity measurements. Saliva samples (872) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. Further, a five-sample pooling strategy was evaluated as per FDA guidelines using the SalivaSTAT protocol.ResultsThe saliva collection (done without any media) performed comparable to the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95°C for 30-minutes and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreement (NPA) of 95.8% and 100%, respectively. The LoD was established as ∼20-60 copies/ml by absolute quantification. Further, a five-sample pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively.ConclusionWe have optimized an extraction-free direct RT-PCR assay for saliva samples that demonstrated comparable performance to FDA-EUA assay (Extraction and RT-PCR). The SalivaSTAT protocol is a rapid, sensitive, and cost-effective method that can be adopted globally, and has the potential to meet testing needs and may play a significant role in management of the current pandemic.

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“…The use of saliva as sample has demonstrated not only to serve as an alternative upper respiratory tract specimen type for SARSCoV-2 detection [15][16][17] , but also saliva offers a number of advantages over nasopharyngeal (NP) and nasal swabs when considering the aforementioned criteria for mass testing efforts. The use of NP swabs can cause discomfort and irritation that could promote sneezing and coughing and increase the risk of exposure for the medical providers 18,19 . Collection of NP swabs has been associated with variable, inconsistent, and false negative test results 17 .…”

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confidence: 99%

RT-LAMP assay for ultra-sensitive detection of SARS-CoV-2 in saliva and VTM clinical samples

Ganguli

Mostafa

Berger

et al. 2020

Preprint

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The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostic platforms. Although several PCR-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid SARS-CoV-2 detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. There are limited reports of saliva as the sample source and some of these indicate inferior sensitivity when comparing RT-LAMP with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay to test saliva using a 2-step RT-LAMP assay, where a short 10-minute RT step is performed with only B3 and BIP primers before the final reaction. We show that while the 1-step RT-LAMP demonstrate satisfactory results, the optimized 2-step approach allows for single molecule sensitivity per reaction and performs significantly better than the 1-step RT-LAMP and conventional 2-step RT-LAMP approaches with all primers included in the RT Step. Importantly, we demonstrate RNA extraction-free RT-LAMP based assays for detection of SARS-CoV-2 from VTM and saliva clinical samples.

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SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Cited by 105 publications

References 30 publications

An enhanced isothermal amplification assay for viral detection

An enhanced isothermal amplification assay for viral detection

SalivaSTAT: Direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance

RT-LAMP assay for ultra-sensitive detection of SARS-CoV-2 in saliva and VTM clinical samples

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