Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

10.1101/2021.01.10.21249151 ◽

2021 ◽

Cited By ~ 1

Author(s):

Jennifer R. Hamilton ◽

Elizabeth C. Stahl ◽

Connor A. Tsuchida ◽

Enrique Lin-Shiao ◽

C. Kimberly Tsui ◽

...

Keyword(s):

Nucleic Acid ◽

Rna Extraction ◽

Detection Sensitivity ◽

Nasopharyngeal Swab ◽

Infection Prevalence ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Rapid Pcr ◽

Large Populations ◽

Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

PLoS ONE ◽

10.1371/journal.pone.0255690 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0255690

Author(s):

Jennifer R. Hamilton ◽

Elizabeth C. Stahl ◽

Connor A. Tsuchida ◽

Enrique Lin-Shiao ◽

C. Kimberly Tsui ◽

...

Keyword(s):

Nucleic Acid ◽

Rna Extraction ◽

Detection Sensitivity ◽

Nasopharyngeal Swab ◽

Infection Prevalence ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Rapid Pcr ◽

Large Populations ◽

Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48363.14842 ◽

2021 ◽

Author(s):

Santosh Karade ◽

Pratik Thosani ◽

Prashant Patil ◽

Kavita Bala Anand ◽

Sourav Sen ◽

...

Keyword(s):

Nucleic Acid ◽

Pilot Study ◽

Real Time ◽

Gold Standard ◽

Rna Extraction ◽

Molecular Diagnostic ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

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High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v2

10.17504/protocols.io.b2mjqc4n ◽

2021 ◽

Author(s):

Aaron Topol (Verily Life Sciences) ◽

marlene.wolfe not provided ◽

Brad White (Verily Life Sciences) ◽

Krista Wigginton ◽

Alexandria B Boehm

Keyword(s):

Quantitative Analysis ◽

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Digital Pcr ◽

N Gene ◽

Acid Extraction ◽

Rt Pcr ◽

Pcr Inhibitor ◽

Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

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Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.00077-11 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4336-4343 ◽

Cited By ~ 64

Author(s):

Akihiko Hata ◽

Hiroyuki Katayama ◽

Masaaki Kitajima ◽

Chettiyappan Visvanathan ◽

Chea Nol ◽

...

Keyword(s):

Nucleic Acid ◽

Water Samples ◽

Rna Extraction ◽

Environmental Water ◽

Environmental Water Samples ◽

Acid Extraction ◽

Murine Norovirus ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Viral Nucleic Acid

ABSTRACTInhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

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