Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection

AbstractObjectiveThe ongoing COVID-19 pandemic continues imposing a demand for diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, we assessed the sensitivity of a number of one-step retrotranscription and quantitative PCR (RT-qPCR) solutions to detect SARS-CoV-2.MethodsWe evaluated six different RT-qPCR alternatives for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. That of best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating, overcoming the RNA extraction step.ResultsWe found a wide variability in the sensitivity of RT-qPCR solutions that associated with a range of false negatives from as low as 2% (0.3-7.9%) to as much as 39.8% (30.2-50.2). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0), in the range of some of the solutions based on standard RNA extractions.ConclusionsWe evidenced sensitivity limitations of currently used RT-qPCR solutions. Our results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.

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Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

Viruses ◽

10.3390/v13091712 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1712

Author(s):

Theresa Mair ◽

Maja Ivankovic ◽

Christian Paar ◽

Helmut J. F. Salzer ◽

Angelika Heissl ◽

...

Keyword(s):

Internal Control ◽

Multiple Testing ◽

Rna Extraction ◽

P Gene ◽

Human Rnase ◽

Extraction Step ◽

One Step ◽

And Control ◽

Digestion Step ◽

Pcr Test

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

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Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

10.1101/2020.04.08.20058495 ◽

2020 ◽

Cited By ~ 4

Author(s):

Julia Alcoba-Florez ◽

Rafaela González-Montelongo ◽

Antonio Íñigo-Campos ◽

Diego García-Martínez de Artola ◽

Helena Gil-Campesino ◽

...

Keyword(s):

Supply Chain ◽

Quantitative Pcr ◽

High Throughput ◽

Diagnostic Tests ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Average Increase ◽

Extraction Step ◽

One Step ◽

Standard Tests

AbstractThe current reference for COVID-19 diagnosis is based on the detection of SARS-CoV-2 on RNA extracts using one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput COVID-19 screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, overcoming the long and tedious RNA extraction step. Although with an average increase of 6.1 (± 1.6) cycles compared to standard tests with RNA extracts, we show that RT-qPCR yielded consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. Our findings provide viable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests by using any qPCR device, thereby complementing standard COVID-19 testing.

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Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples

10.1101/2020.04.26.20081307 ◽

2020 ◽

Cited By ~ 2

Author(s):

◽

Xabier Guruceaga ◽

Amanda Sierra ◽

Daniel Marino ◽

Izortze Santin ◽

...

Keyword(s):

Rna Extraction ◽

Policy Decision ◽

Cost Effective ◽

Diagnostic Process ◽

Nasopharyngeal Swab ◽

Effective Manner ◽

Extraction Step ◽

Simple Protocol ◽

One Step ◽

Commercial Kits

ABSTRACTThe SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

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Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽

10.3390/molecules26216617 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6617

Author(s):

Eva Rajh ◽

Tina Šket ◽

Arne Praznik ◽

Petra Sušjan ◽

Alenka Šmid ◽

...

Keyword(s):

Oral Cavity ◽

Reverse Transcription ◽

Screening Program ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Diagnostic Sensitivity ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Spread ◽

One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction

10.1101/2020.03.28.013508 ◽

2020 ◽

Cited By ~ 14

Author(s):

Carolina Beltrán-Pavez ◽

Chantal L. Márquez ◽

Gabriela Muñoz ◽

Fernando Valiente-Echeverría ◽

Aldo Gaggero ◽

...

Keyword(s):

Rna Extraction ◽

Nasopharyngeal Swab ◽

Short Supply ◽

Extraction Step

AbstractThe ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of an RNA extraction step should be considered in SARS-CoV-2 diagnosis.

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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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Development and Validation of Two In-house, Low-Cost SARS-CoV-2 Detection Assays

10.1101/2020.05.18.20105510 ◽

2020 ◽

Author(s):

Fatimah Alhamlan ◽

Ahmed Alqahtani ◽

Dana Bakheet ◽

Marie Bohol ◽

Sahar Althawadi ◽

...

Keyword(s):

Low Cost ◽

False Negative ◽

Rna Extraction ◽

Melting Curve ◽

Sybr Green ◽

Heat Processing ◽

Nasopharyngeal Swab ◽

Level 2 ◽

Biosafety Level ◽

Biosafety Level 2

Background One major challenge for detecting the virus that causes COVID19 is commercial SARSCoV2 testing kit or reagent availability. To allow every laboratory or hospital access to an inhouse assay, we developed two low cost SARSCoV2 detection assay protocols using inhouse primers and reagents equipment on hand in most biology or diagnostic laboratories a SYBR Green based RTPCR and PCR assays. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated alternative RNA extraction protocols. Methods SARSCoV2 genome sequences deposited into the GISAID database were retrieved to design and synthesize inhouse primers. Forty patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocols. Both assays used TRIzol and heat-processing techniques to extract RNA from patient samples and to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using samples previously confirmed positive for SARSCoV2. The positive amplicons were sequenced to confirm the results. The assay protocols were developed, and the specificity of each PCR product was confirmed using melting curve analyses. The most accurate heat processing technique for primers with short amplicon lengths was 95C for 15 mins. Of 40 samples, both the SYBR Green based quantitative RTPCR assay and the PCR assay detected SARSCoV2 target genes in 28 samples, with no false positive or false-negative results. These findings were concordant with those of the diagnostic laboratory that tested the same samples using a Rotor Gene PCR cycler with an Altona Diagnostics SARSCoV2 kit (R2=0.889). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low cost tools for the detection of SARSCoV2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or cost ineffective.

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

10.1101/2020.05.28.20115220 ◽

2020 ◽

Cited By ~ 3

Author(s):

Shan Wei ◽

Esther Kohl ◽

Alexandre Djandji ◽

Stephanie Morgan ◽

Susan Whittier ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Diagnostic Testing ◽

Rna Extraction ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Transport ◽

Extraction Step ◽

Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

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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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In silico evaluation of the impact of the Omicron variant on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing

10.21203/rs.3.rs-1220446/v1 ◽

2022 ◽

Author(s):

Divya Sharma ◽

Chengjin Ye ◽

Giusppe Lippi ◽

Jordi B. Torrelles ◽

Luis Martinez-Sobrido ◽

...

Keyword(s):

In Silico ◽

False Negative ◽

Whole Genome ◽

False Negatives ◽

Negative Results ◽

False Negative Results ◽

The World ◽

Reverse Primer ◽

Primer Annealing ◽

The Impact

Abstract Background The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world presenting a new threat to global public human health. Due to the large number of mutations possessed by Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for SARS-CoV-2. Here, we aimed to assess the impact of Omicron on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease-2019 (COVID-19) diagnosis via in silico analysis employing whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and viral genome. Methods In silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 reported by the World Health Organization (WHO) or available in the literature, was assessed for use in detecting SARS-CoV-2 Omicron BA.1 and BA.2 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. The mismatches between the amplicon regions of the SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated, and the clustering analysis of the corresponding amplicon sequences was carried out. Results From the 232 representative SARS-CoV-2 BA.1 Omicron sublineage genomes analyzed, 229 showed substitutions at the forward primer annealing site for assay China-CDC N, 226 showed mismatches in the reverse primer annealing site for assay Thai N, and all 232 had substitution at the 3’ end of the reverse primer annealing site for assay HKUniv RdRp/Hel. Therefore, the lowest sensitivity was observed for assay ChinaCDC N, Thai N and HKUniv RdRp/Hel for SARS-CoV-2 BA.1 sublineage genomes. For 5 SARS-CoV-2 BA.2 Omicron sublineage genomes, false negative results were observed for assays ChinaCDC N, Thai N, HKUniv RdRp/Hel, SigmAldr S5, SigmAldr S6 and HKUniv S. Conclusion In this study, we observed three (25%) assays (ChinaCDC N, Thai N, and HKUniv RdRp/Hel) demonstrated potential for false negatives for the SARS-CoV-2 Omicron BA.1 sublineage, while four (33.3%) assays (ChinaCDC N, Thai N, HKUniv RdRp/Hel, HKUniv S, SigmAldr S5 and SigmAldr S6) demonstrated potential false negative results for the for SARS-CoV-2 Omicron BA.2 sublineage, which also has the potential for Spike (S) gene dropout despite lacking 69-70 deletion in the S gene. Further, amplicon clustering and additional substitutions analysis along with the sensitivity analysis could be used for modification and development of RT-qPCR assays for detection of SARS-CoV-2 Omicron VoC lineages.

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