Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

AbstractThe emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

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Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

PLoS ONE ◽

10.1371/journal.pone.0243333 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0243333

Author(s):

Julien Fassy ◽

Caroline Lacoux ◽

Sylvie Leroy ◽

Latifa Noussair ◽

Sylvain Hubac ◽

...

Keyword(s):

High Throughput ◽

Cellular Response ◽

Rna Extraction ◽

Reference Method ◽

Proteinase K ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Extraction Step ◽

Large Populations ◽

Reaction Volumes

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

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Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.638902 ◽

2021 ◽

Vol 9 ◽

Author(s):

Sofía N. Rodríguez Flores ◽

Luis Mario Rodríguez-Martínez ◽

Bernardita L. Reyes-Berrones ◽

Nadia A. Fernández-Santos ◽

Elthon J. Sierra-Moncada ◽

...

Keyword(s):

Rna Extraction ◽

Proteinase K ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Sample Collection ◽

Extraction Protocol ◽

Assay Performance ◽

Two Samples ◽

Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P > 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P < 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

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Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS ONE ◽

10.1371/journal.pone.0247792 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247792

Author(s):

Valeria Genoud ◽

Martin Stortz ◽

Ariel Waisman ◽

Bruno G. Berardino ◽

Paula Verneri ◽

...

Keyword(s):

Rna Extraction ◽

Standard Technique ◽

Proteinase K ◽

Clinical Samples ◽

Heat Inactivation ◽

Nasopharyngeal Swab ◽

Reverse Transcription Pcr ◽

Extraction Step ◽

Commercial Kits ◽

Inexpensive Procedure

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

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

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

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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Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

Scientific Reports ◽

10.1038/s41598-021-81487-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shan Wei ◽

Esther Kohl ◽

Alexandre Djandji ◽

Stephanie Morgan ◽

Susan Whittier ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Diagnostic Testing ◽

Rna Extraction ◽

Cost Effective ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Percentage Agreement ◽

Testing Method ◽

Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

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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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Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

Pathogens and Disease ◽

10.1093/femspd/ftaa061 ◽

2020 ◽

Vol 78 (8) ◽

Cited By ~ 1

Author(s):

Onya Opota ◽

René Brouillet ◽

Gilbert Greub ◽

Katia Jaton

Keyword(s):

High Throughput ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Test Results ◽

Rt Pcr ◽

Clinical Specimens ◽

Rapid Spread ◽

Bronchial Aspirate ◽

Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 > 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

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Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: comparison with automated systems

10.1101/2020.05.26.20099440 ◽

2020 ◽

Author(s):

Henrik Dimke ◽

Sanne L Larsen ◽

Marianne N Skov ◽

Hanne Larsen ◽

Gitte N Hartmeyer ◽

...

Keyword(s):

Low Cost ◽

Rna Extraction ◽

Diagnostic System ◽

Nasopharyngeal Swab ◽

Analytical Sensitivity ◽

Automated Systems ◽

Suitable Alternative ◽

Fully Integrated ◽

Rna Purification ◽

Sensitivity Specificity

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 remains essential for tracking and containing the rapid spread of the virus. However, due to increased global demand, kits and proprietary reagents for RNA extraction are limited, which markedly reduce SARS-CoV-2 testing capabilities in many countries. Here, we explore the use of conventional acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA purification as an alternative to commercial automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical oropharyngeal or nasopharyngeal swab specimens were extracted by AGPC and compared to the commercial platforms, the Maxwell® RSC 48 instrument for automated RNA extraction and the fully integrated diagnostic system, the Cobas®6800 apparatus. Our results show that RNA extracted using the AGPC method is fully comparable to modern automated systems regarding analytical sensitivity, specificity and accuracy with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, we find that the AGPC method is easily scalable and implemented in conventional laboratories. Taken together, these data identify conventional AGPC-based RNA extraction as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2, when automated systems, kits and reagents are not readily available.

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Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.716436 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ruth E. Thom ◽

Lin S. Eastaugh ◽

Lyn M. O’Brien ◽

David O. Ulaeto ◽

James S. Findlay ◽

...

Keyword(s):

Heat Treatment ◽

High Throughput ◽

Rna Extraction ◽

Clinical Samples ◽

Sample Type ◽

Diagnostic Laboratory ◽

Viral Pathogen ◽

Infectious Dose ◽

Viral Transport Medium ◽

Lysis Buffer

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

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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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