A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

ABSTRACTEffective interventions are mandatory to control the transmission and spread of SARS-CoV-2, a highly contagious virus causing devastating effects worldwide. Cost-effective approaches are pivotal tools required to increase the detection rates and escalate further in massive surveillance programs, especially in countries with limited resources that most of the efforts have focused on symptomatic cases only. Here, we compared the performance of the RT-qPCR using an intercalating dye with the probe-based assay. Then, we tested and compared these two RT-qPCR chemistries in different pooling systems: after RNA extraction (post-RNA extraction) and before RNA extraction (pre-RNA extraction) optimizing by pool size and template volume. We evaluated these approaches in 610 clinical samples. Our results show that the dye-based technique has a high analytical sensitivity similar to the probe-based detection assay used worldwide. Further, this assay may also be applicable in testing by pool systems post-RNA extraction up to 20 samples. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 µL of RNA template. The low cost and the potential use in pre-RNA extraction pool systems, place of this assays as a valuable resource for scalable sampling to larger populations. Implementing a pool system for population sampling results in an important savings of laboratory resources and time, which are two key factors during an epidemic outbreak. Using the pooling approaches evaluated here, we are confident that it can be used as a valid alternative assay for the detection of SARS-CoV-2 in human samples.

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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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Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

10.1101/2020.08.01.20166538 ◽

2020 ◽

Author(s):

Pierre Garneret ◽

Etienne Coz ◽

Elian Martin ◽

Jean-Claude Manuguerra ◽

Elodie Brient-Litzler ◽

...

Keyword(s):

Point Of Care ◽

Low Cost ◽

Rna Extraction ◽

Dna Amplification ◽

Developed Countries ◽

Temperature Cycling ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Molecular Testing

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

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A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.632646 ◽

2021 ◽

Vol 11 ◽

Author(s):

Iqbal Azmi ◽

Md Imam Faizan ◽

Rohit Kumar ◽

Siddharth Raj Yadav ◽

Nisha Chaudhary ◽

...

Keyword(s):

Data Storage ◽

Limit Of Detection ◽

Rna Extraction ◽

Smartphone Application ◽

Lateral Flow ◽

Clinical Samples ◽

Heat Inactivation ◽

Analytical Sensitivity ◽

Test Results ◽

Extraction Step

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13AssistedSaliva-based &SmartphoneIntegratedTesting), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

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Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses

Animals ◽

10.3390/ani11102976 ◽

2021 ◽

Vol 11 (10) ◽

pp. 2976

Author(s):

Edson Kinimi ◽

Mana Mahapatra ◽

Tebogo Kgotlele ◽

Mariam R. Makange ◽

Chandana Tennakoon ◽

...

Keyword(s):

Low Cost ◽

Viral Evolution ◽

Cost Effective ◽

Nucleotide Identity ◽

Clinical Samples ◽

Depth Information ◽

Peste Des Petits Ruminants ◽

Sequencing Technology ◽

Oxford Nanopore ◽

Complete Genomes

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

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Border security and surveillance System using IoT

International Journal of Information Retrieval Research ◽

10.4018/ijirr.289953 ◽

2022 ◽

Vol 12 (1) ◽

pp. 0-0

Keyword(s):

Low Cost ◽

Cost Effective ◽

Surveillance Systems ◽

Security Assessment ◽

Efficient System ◽

Human Gestures ◽

Set Up ◽

International Border ◽

Border Surveillance

Security along the international border is a critical process in security assessment; It must be exercised the 24x7. With the advancements in wireless IoT technology, it has become much easier to design, develop and deploy a cost-effective, automatic and efficient system for intrusion detection in the context of surveillance. This paper set up to set up the most efficient surveillance solution, we propose a Border Surveillance Systems and sensitive sites. this surveillance and security system is to detect and track intruders trespassing into the monitoring area along the border, it able which triggers off precocious alerts and valuation necessary for the catch of efficient measurements in case of a threat. Our system is based on the classification of the human gestures drawn from videos envoy by Drones equipped with cameras and sensors in real-time. All accomplished experimentation and acquired results showed the benefit diverted from the use of our system and therefore it enables our soldiers to watch the borders at each and every moment to effectively and at low cost.

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A simple and rapid method for isolating high-quality RNA from kenaf containing high polysaccharide and polyphenol contents

10.1101/2020.07.06.189506 ◽

2020 ◽

Author(s):

Xiaofang Liao ◽

Hongwei Li ◽

Aziz Khan ◽

Yanhong Zhao ◽

Wenhuan Hou ◽

...

Keyword(s):

Low Cost ◽

Rna Extraction ◽

Rna Isolation ◽

Cost Effective ◽

Spectrophotometric Analysis ◽

Rt Pcr ◽

High Quality ◽

Time Saving ◽

Ctab Method ◽

Commercial Kits

AbstractThe isolation of high-quality RNA from kenaf is crucial for genetic and molecular biology studies. However, high levels of polysaccharide and polyphenol compounds in kenaf tissues could irreversibly bind to and coprecipitate with RNA, which complicates RNA extraction. In the present study, we proposed a simplified, time-saving and low-cost extraction method for isolating high quantities of high-quality RNA from several different kenaf tissues. RNA quality was measured for yield and purity, and the proposed protocol yielded high quantities of RNA (10.1-12.9 μg/g·FW). Spectrophotometric analysis showed that A260/280 ratios of RNA samples were in the range of 2.11 to 2.13, and A260/230 ratios were in the range of 2.04-2.24, indicating that the RNA samples were free of polyphenols, polysaccharides, and protein contaminants after isolation. The method of RNA extraction presented here was superior to the conventional CTAB method in terms of RNA isolation efficiency and was more sample-adaptable and cost-effective than commercial kits. Furthermore, to confirm downstream amenability, the high-quality RNA obtained from this method was successfully used for RT-PCR, real-time RT-PCR and Northern blot analysis. We provide an efficient and low-cost method for extracting high quantities of high-quality RNA from plants that are rich in polyphenols and polysaccharides, and this method was also validated for the isolation of high-quality RNA from other plants.

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Detecting SARS-CoV-2 variants with SNP genotyping

10.1101/2020.11.18.388140 ◽

2020 ◽

Author(s):

Helen Harper ◽

Amanda J. Burridge ◽

Mark Winfield ◽

Adam Finn ◽

Andrew D. Davidson ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Snp Genotyping ◽

Clinical Samples ◽

Marker Panel ◽

Qrt Pcr ◽

Crucial Information ◽

The Uk ◽

Genotyping Panel

AbstractTracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

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Multiplex Solid-Phase RPA Coupled CRISPR-Based Visual Detection of SARS-CoV-2

10.1101/2021.11.02.21265831 ◽

2021 ◽

Author(s):

Xiaochen Qin ◽

Yuyuan Zhou ◽

Ratul Paul ◽

Yue Wu ◽

Yaling Liu

Keyword(s):

Solid Phase ◽

Dna Detection ◽

Point Of Care ◽

Low Cost ◽

Control Sample ◽

Virus Detection ◽

Cost Effective ◽

Analytical Sensitivity ◽

One Pot ◽

Hpv Dna

COVID-19 has challenged the world's public health and led to over 4.5 million deaths. A rapid, sensitive, and cost-effective point-of-care virus detection device is crucial to the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Here we demonstrate a solid phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive, and visual COVID-19 DNA detection. By targeting the SARS-CoV-2 structure protein encoded genomes, two specific genes were simultaneously detected with the control sample without cross-interaction with other sequences. The endpoint signal can be directly visualized for rapid detection of COVID-19. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter released fluorescent signal when specific genes were recognized. The system was tested with samples of a broad range of concentrations (20 to 2x105 copies) and showed analytical sensitivity down to 20 copies per reaction. Furthermore, a low-cost LED UV flashlight (~$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method with the capacity to specifically identify low concentration genes. Similar CRISPR biosensor chips can support a broad range of applications such as HPV DNA detection, influenza SARS-CoV-2 multiplex detection, and other infectious disease testing assays.

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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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Detecting SARS-CoV-2 variants with SNP genotyping

PLoS ONE ◽

10.1371/journal.pone.0243185 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0243185 ◽

Cited By ~ 2

Author(s):

Helen Harper ◽

Amanda Burridge ◽

Mark Winfield ◽

Adam Finn ◽

Andrew Davidson ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Snp Genotyping ◽

Clinical Samples ◽

Marker Panel ◽

Qrt Pcr ◽

Crucial Information ◽

The Uk ◽

Genotyping Panel

Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system, we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020 and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 61.9% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with a marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

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