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

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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Improved Sensitivity, Safety, and Rapidity of COVID-19 Tests by Replacing Viral Storage Solution with Lysis Buffer

10.1101/2020.09.25.20201921 ◽

2020 ◽

Author(s):

Oran Erster ◽

Omer Shkedi ◽

Gil Benedek ◽

Eyal Zilber ◽

Itay Varkovitzky ◽

...

Keyword(s):

Rna Extraction ◽

Detection Threshold ◽

Test Tube ◽

Clinical Samples ◽

Sample Collection ◽

Standard Guideline ◽

Viral Transport Medium ◽

Two Samples ◽

Lysis Buffer ◽

And Control

AbstractConducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic.Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling.We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB).We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

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Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-24463-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Duane S. Juang ◽

Terry D. Juang ◽

Dawn M. Dudley ◽

Christina M. Newman ◽

Molly A. Accola ◽

...

Keyword(s):

Infectious Disease ◽

Large Scale ◽

Rna Extraction ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Total Analysis System ◽

Total Analysis ◽

Analysis System ◽

Disease Testing

AbstractThe COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

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Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer

PLoS ONE ◽

10.1371/journal.pone.0249149 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0249149

Author(s):

Oran Erster ◽

Omer Shkedi ◽

Gil Benedek ◽

Eyal Zilber ◽

Itay Varkovitzky ◽

...

Keyword(s):

Rna Extraction ◽

Detection Threshold ◽

Test Tube ◽

Clinical Samples ◽

Sample Collection ◽

Standard Guideline ◽

Viral Transport Medium ◽

Two Samples ◽

Lysis Buffer ◽

And Control

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

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

10.1101/2020.11.09.20228437 ◽

2020 ◽

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 ◽

Rna Purification ◽

Large Populations ◽

Reaction Volumes

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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Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

Future Microbiology ◽

10.2217/fmb-2020-0094 ◽

2020 ◽

Vol 15 (15) ◽

pp. 1483-1487

Author(s):

Nikhil S Sahajpal ◽

Ashis K Mondal ◽

Allan Njau ◽

Sudha Ananth ◽

Kimya Jones ◽

...

Keyword(s):

Supply Chain ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Sample Collection ◽

Rt Pcr ◽

Pcr Assay ◽

Short Supply ◽

Viral Transport ◽

3D Printed ◽

Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

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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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Diagnosis of SARS-Cov-2 Infection by RT-PCR Using Specimens Other Than Naso- and Oropharyngeal Swabs: A Systematic Review and Meta-Analysis

Diagnostics ◽

10.3390/diagnostics11020363 ◽

2021 ◽

Vol 11 (2) ◽

pp. 363

Author(s):

Vânia M. Moreira ◽

Paulo Mascarenhas ◽

Vanessa Machado ◽

João Botelho ◽

José João Mendes ◽

...

Keyword(s):

Systematic Review ◽

Clinical Performance ◽

Point Of Care ◽

Meta Analysis ◽

High Sensitivity ◽

Nasopharyngeal Swab ◽

Sample Collection ◽

Rt Pcr ◽

Oropharyngeal Swab ◽

Nucleic Acid Assays

The rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0–98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4–88.8) and specificity of 96.4% (95% CI: 89.5–98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3–95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3–96.9) and specificity of 63.1% (95% CI: 36.8–89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes).

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