scholarly journals Extraction-free rapid cycle RT-qPCR and extreme RT-PCR for SARS-CoV-2 virus detection

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S139-S139
Author(s):  
J C Lownik ◽  
J S Farrar ◽  
G Way ◽  
R K Martin
Keyword(s):  
Supply Chain ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Virus Detection ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Sample Collection ◽  
Rt Pcr ◽  
Hydrolysis Probe ◽  
Time Requirements

Abstract Introduction/Objective Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. In this study, we explored the kinetic limitations of extraction-free rapid cycle RT-qPCR for SARS-CoV-2 virus detection using the commercially available capillary based LightCycler. Methods/Case Report We optimized reverse transcription and PCR under extraction-free and rapid thermocycling conditions utilizing hydrolysis probe-based detection methods using a Roche LightCycler. Results (if a Case Study enter NA) This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n= 40/41) and negative patient samples was 100% (40/40). We further demonstrate that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR and product verification by melting can be completed in less than 3 minutes. Conclusion We developed a protocol for sensitive and specific RT-qPCR of SARS-CoV-2 RNA from nasopharyngeal swabs in less than 20 minutes, with minimal hands-on time requirements. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.

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

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.

scholarly journals Defining the analytical and clinical sensitivity of the ARTIC method for the detection of SARS-CoV-2

2021 ◽  
Author(s):  
Nabil-Fareed Alikhan ◽  
Joshua Quick ◽  
Alexander J. Trotter ◽  
Samuel C. Robson ◽  
Matthew Bashton ◽  
...  
Keyword(s):  
Performance Metrics ◽  
Limit Of Detection ◽  
Diagnostic Testing ◽  
Virus Detection ◽  
Detection Methods ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Clinical Sensitivity ◽  
Diagnostic Assays ◽  
Sequencing Method

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly-available genome sequences. The protocol utilises 98 primers to amplify ~400bp fragments of the SARS-CoV-2 genome covering all 30,000 bases. Understanding the analytical performance metrics of this protocol will improve how the data is used and interpreted. Different concentrations of SARS-CoV-2 control material were used to establish the limit of detection (LoD) of the ARTIC protocol. Results demonstrated the LoD was a minimum of 25-50 virus particles per mL. The sensitivity of ARTIC was comparable to the published sensitivities of commercial diagnostics assays and could therefore be used to confirm diagnostic testing results. A set of over 3,600 clinical samples from three UK regions were then evaluated to compare the protocols performance to clinical diagnostic assays (Roche Lightcycler 480 II, AusDiagnostics, Roche Cobas, Hologic Panther, Corman RdRp, Roche Flow, ABI QuantStudio 5, Seegene Nimbus, Qiagen Rotorgene, Abbott M2000, Thermo TaqPath, Xpert). We developed a Python tool, RonaLDO, to perform this validation (available under the GNU GPL3 open-source licence from https://github.com/quadram-institute-bioscience/ronaldo). Positives detected by diagnostic platforms were generally supported by sequencing data; platforms that used RT-qPCR were the best predictors of whether the sample would subsequently sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples with Ct <31 should be chosen. For diagnostic tests that do not provide a quantifiable Ct value, adding a quantification step is recommended. The ARTIC SARS-CoV-2 sequencing protocol is highly sensitive, capable of detecting SARS-CoV-2 in samples with Cts in the high 30s. However, to routinely obtain whole genome coverage, samples with Ct <31 are recommended. Comparing different virus detection methods close to their LoD was challenging and significant discordance was observed.

scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

scholarly journals An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

2022 ◽  
Author(s):  
Bongkot Ngamsom ◽  
Alexander Iles ◽  
Moses Kamita ◽  
Racheal Kimani ◽  
Pablo Rodriguez-Mateos ◽  
...  
Keyword(s):  
Saliva Sample ◽  
Rna Extraction ◽  
Lab On A Chip ◽  
Cross Reactivity ◽  
Contact Tracing ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Sample Collection ◽  
Middle Income ◽  
Specificity And Sensitivity

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

scholarly journals Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

2020 ◽  
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
J. Berger ◽  
M. Aydin ◽  
F. Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Rna Extraction ◽  
Clinical Diagnostics ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Rt Pcr ◽  
Point Of Use ◽  
Viral Transport Medium

AbstractThe COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

scholarly journals SalivaSTAT: Direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance

2020 ◽  
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwali ◽  
...  
Keyword(s):  
Clinical Sample ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Community Setting ◽  
Absolute Quantification ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Sample Pooling ◽  
Saliva Collection

BackgroundThe limitations of widespread current COVID-19 diagnostic testing lie at both pre-analytical and analytical stages. Collection of nasopharyngeal swabs is invasive and is associated with exposure risk, high cost, and supply-chain constraints. Additionally, the RNA extraction in the analytical stage is the most significant rate-limiting step in the entire testing process. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction free RT-PCR test using any of the commercially available RT-PCR kits.MethodsWe optimized saliva collection devices, heat-shock treatment and homogenization. The effect of homogenization on saliva samples for extraction-free RT-PCR assay was determined by evaluating samples with and without homogenization and preforming viscosity measurements. Saliva samples (872) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. Further, a five-sample pooling strategy was evaluated as per FDA guidelines using the SalivaSTAT protocol.ResultsThe saliva collection (done without any media) performed comparable to the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95°C for 30-minutes and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreement (NPA) of 95.8% and 100%, respectively. The LoD was established as ∼20-60 copies/ml by absolute quantification. Further, a five-sample pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively.ConclusionWe have optimized an extraction-free direct RT-PCR assay for saliva samples that demonstrated comparable performance to FDA-EUA assay (Extraction and RT-PCR). The SalivaSTAT protocol is a rapid, sensitive, and cost-effective method that can be adopted globally, and has the potential to meet testing needs and may play a significant role in management of the current pandemic.

scholarly journals Nucleic Acid Testing of SARS-CoV-2

2021 ◽  
Vol 22 (11) ◽  
pp. 6150
Author(s):  
Hee-Min Yoo ◽  
Il-Hwan Kim ◽  
Seil Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Testing ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

scholarly journals Comparative analysis of the diagnostic performance of five commercial COVID-19 qRT PCR kits used in India

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. Singh ◽  
A. K. Yadav ◽  
A. Pakhare ◽  
P. Kulkarni ◽  
L. Lokhande ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Diagnostic Performance ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Control Group ◽  
Rt Pcr ◽  
Short Period ◽  
Commercial Kits

AbstractTo meet the unprecedented requirement of diagnostic testing for SARS-CoV-2, a large number of diagnostic kits were authorized by concerned authorities for diagnostic use within a short period of time during the initial phases of the ongoing pandemic. We undertook this study to evaluate the inter-test agreement and other key operational features of 5 such commercial kits that have been extensively used in India for routine diagnostic testing for COVID-19. The five commercial kits were evaluated, using a panel of positive and negative respiratory samples, considering the kit provided by National Institute of Virology, Indian Council of Medical Research (2019-nCoV Kit) as the reference. The positive panel comprised of individuals who fulfilled the 3 criteria of being clinically symptomatic, having history of contact with diagnosed cases and testing positive in the reference kit. The negative panel included both healthy and disease controls, the latter being drawn from individuals diagnosed with other respiratory viral infections. The same protocol of sample collection, same RNA extraction kit and same RT-PCR instrument were used for all the kits. Clinical samples were collected from a panel of 92 cases and 60 control patients, who fulfilled our inclusion criteria. The control group included equal number of healthy individuals and patients infected with other respiratory viruses (n = 30, in each group). We observed varying sensitivity and specificity among the evaluated kits, with LabGun COVID-19 RT-PCR kit showing the highest sensitivity and specificity (94% and 100% respectively), followed by TaqPath COVID-19 Combo and Allplex 2019-nCoV assays. The extent of inter-test agreement was not associated with viral loads of the samples. Poor correlation was observed between Ct values of the same genes amplified using different kits. Our findings reveal the presence of wide heterogeneity and sub-optimal inter-test agreement in the diagnostic performance of the evaluated kits and hint at the need of adopting stringent standards for fulfilling the quality assurance requirements of the COVID-19 diagnostic process.

scholarly journals Asymptomatic screening of SARS-CoV-2 (COVID-19) virus RNA using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)

2021 ◽  
Author(s):  
Rebecca Allsopp ◽  
Caroline Cowley ◽  
Ruth Barber ◽  
Carolyn Jones ◽  
Christopher Holmes ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
Screening Program ◽  
False Negative ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Clinical Patient ◽  
The University

Abstract This study demonstrates the diagnostic performance of SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. An RT-LAMP workflow was developed using synthetic positive control RNA and the diagnostic sensitivity and specificity was then determined using clinical patient samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. 92 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples along with 78 clinically positive and 63 clinically negative saliva samples were equally detected at 100% DSe and 100% DSp for all samples reporting a Ct < 20. DSe for all samples reporting a Ct < 30 reduced slightly to around 95% (100% DSp) for both the single genomic (large open reading frame; orf1a) and dual sub-genomic (nucleocapsid plus envelope) targeting RT-LAMP assays. Lastly, the diagnostic performance of a saliva direct workflow was only about 50% that of the saliva RNA extraction workflow. Subsequently, a swab based RNA -RT-LAMP assay was implemented to ISO 15189:2012 standards supporting an advisory COVID-19 screening program for staff and students at the University of Leicester between October and December 2020. Within a 24-hour period, total nucleic acid extraction was followed by genomic target RT-LAMP plus an internal total RNA control to mitigate the possibility of false negative reporting. SARS-CoV-2 RT-LAMP positive samples were confirmed by an RT-PCR test in an NHS diagnostic laboratory and results were included within national statistics. Nine confirmed positive samples were detected in 1680 symptom free individuals (equivalent to 540 cases per 100,000) thus demonstrating the utility of RT-LAMP molecular diagnostic tool for the detection of SARS-CoV-2 in an asymptomatic population.

scholarly journals The Use of Saliva as a Diagnostic Specimen for SARS CoV-2 Molecular Diagnostic Testing for Pediatric Patients

2020 ◽  
Author(s):  
Meghan Delaney ◽  
Joelle Simpson ◽  
Bobbe Thomas ◽  
Christal Ralph ◽  
Michael Evangalista ◽  
...  
Keyword(s):  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Test Results ◽  
Sample Collection ◽  
Specimen Collection ◽  
Collection Device ◽  
Viral Transport Medium ◽  
Study Population ◽  
Saliva Collection ◽  
Pcr Assays

ABSTRACTBackgroundChildren are an important population to test for COVID-19 infection, particularly because they may shed the virus without displaying symptoms. Testing children for COVID-19 via sensitive molecular methods is important, although collecting nasopharyngeal (NP) specimens can be challenging. A less invasive mode of specimen collection that yields test results comparable to those from NP specimens would be beneficial to simplify sample collection.MethodsTo demonstrate that saliva is a suitable specimen for collection from children, the clinical usability/acceptability and the analytic performance of saliva were compared to NP specimens suspended in viral transport medium. Four different FDA EUA-approved real-time RT-PCR assays and one EUA approved saliva collection device were investigated.ResultsThe study population included 526 patients between the ages of 3 and 61 years, 461 (88%) were <18 years, 425 were asymptomatic (81.1%), 92 were symptomatic (17.6%). Saliva mixed with saliva stabilizing buffer was found to yield comparable sensitivity to NP specimens when tested on the AllPlex SARS-CoV-2 molecular test (Seegene Inc). The analytic sensitivity of the AllPlex assay during testing of spiked saliva mixed with SpectrumDNA saliva stabilizer was found to be 250 genomic copies/mL.ConclusionsOf the four FDA EUA-approved SARS-CoV-2 PCR assays studied, we found the AllPlex assay to be best suited for testing saliva specimens collected from children 5 years of age or older. The sensitivity of viral detection was equivalent to NP specimens when saliva specimens were mixed with the saliva stabilizer.

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