scholarly journals LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258263
Author(s):  
Elizabeth C. Stahl ◽  
Allan R. Gopez ◽  
Connor A. Tsuchida ◽  
Vinson B. Fan ◽  
Erica A. Moehle ◽  
...  
Keyword(s):  
Large Scale ◽  
Local Community ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
Clinical Surveillance ◽  
Sample Pooling ◽  
Wastewater Samples

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

scholarly journals IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

2020 ◽  
Author(s):  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Jennifer R. Hamilton ◽  
Enrique Lin-Shiao ◽  
Shana L. McDevitt ◽  
...  
Keyword(s):  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
False Negative Results ◽  
Single Well ◽  
Sample Pooling ◽  
One Step ◽  
Viral Target

AbstractCommonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

scholarly journals Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 594 ◽  
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Sou Yamura ◽  
Rikiya Takeuchi ◽  
Noriko Aoki ◽  
...  
Keyword(s):  
De Novo ◽  
Limit Of Detection ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Virus Rna ◽  
Antigen Tests ◽  
Spike Proteins

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

scholarly journals SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 863 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Müller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
N Gene ◽  
Extraction Protocol ◽  
Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

scholarly journals Combined Newborn Screening for Succinylacetone, Amino Acids, and Acylcarnitines in Dried Blood Spots

2008 ◽  
Vol 54 (4) ◽  
pp. 657-664 ◽  
Author(s):  
Coleman Turgeon ◽  
Mark J Magera ◽  
Pierre Allard ◽  
Silvia Tortorelli ◽  
Dimitar Gavrilov ◽  
...  
Keyword(s):  
Amino Acids ◽  
Newborn Screening ◽  
False Negative ◽  
Internal Standard ◽  
Early Death ◽  
Cost Effective ◽  
Dried Blood Spots ◽  
Type I ◽  
Blood Spots ◽  
Negative Results

Abstract Background: Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). Methods: We extracted 3/16-inch DBS punches with 300 μL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 μL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155–137) and 13C5-SUAC (m/z 160–142). Analysis time was 1.2 min. Results: SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13–81 μmol/L), with Tyr concentrations ranging from 65 to 293 μmol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 μmol/L. Conclusion: The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

scholarly journals Shine: To explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations.

2021 ◽  
Author(s):  
Cong Ji ◽  
Junbin Jack Shao
Keyword(s):  
False Negative ◽  
Genomic Data ◽  
Cost Effective ◽  
Pcr Primers ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Pathogenic Microorganisms ◽  
New Strategy ◽  
Microbial Genomic ◽  
Species Specific

To improve the quality of nucleic acid detection reagents, we provided a new strategy, Shine, to explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations in order to improve detection sensitivity and accuracy. It is obvious that the more comprehensive genomic data are, the more effective the detection biomarkers. Here, we demonstrated that our method could detect undiscovered multicopy conserved species-specific or even subspecies-specific target fragments, according to several clinical projects. In particular, this approach was effective for any pathogenic microorganism even in incompletely assembled motifs. Based on our strategy, the detection device designed with quantitative PCR primers and probes for systematic and automated detection of pathogenic microorganisms in biological samples may cover all pathogenic microorganisms without limits based on genome annotation. On the website https://bioinfo.liferiver.com.cn, users may select different configuration parameters depending on the purpose of the project to realize routine clinical detection practices. Therefore, it is recommended that our strategy is suitable to identify shared universal phylogenetic markers with few false positive or false negative errors and to automate the design of minimal primers and probes to detect pathogenic communities with cost-effective predictive power.

scholarly journals Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

2021 ◽  
Author(s):  
Enos C Kline ◽  
Nuttada Panpradist ◽  
Ian T Hull ◽  
Qin Wang ◽  
Amy K Oreskovic ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
Detection System ◽  
Point Of Care ◽  
False Negative ◽  
Cost Effective ◽  
Internal Amplification Control ◽  
Robust Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Target Redundancy

AbstractThe increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

scholarly journals Development and Multicentric Validation of a Lateral Flow Immunoassay for Rapid Detection of MCR-1-Producing Enterobacteriaceae

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Hervé Volland ◽  
Laurent Dortet ◽  
Sandrine Bernabeu ◽  
Hervé Boutal ◽  
Marisa Haenni ◽  
...  
Keyword(s):  
Rapid Detection ◽  
False Negative ◽  
Cost Effective ◽  
Resistance Mechanisms ◽  
Lateral Flow ◽  
Colistin Resistance ◽  
Multicentric Study ◽  
Bacterial Colony ◽  
Content Type ◽  
Negative Results

ABSTRACT Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene mcr-1 (mostly in Escherichia coli) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals.

scholarly journals Extracting Coastal Raft Aquaculture Data from Landsat 8 OLI Imagery

Sensors ◽  
2019 ◽  
Vol 19 (5) ◽  
pp. 1221 ◽  
Author(s):  
Jun Wang ◽  
Lichun Sui ◽  
Xiaomei Yang ◽  
Zhihua Wang ◽  
Yueming Liu ◽  
...  
Keyword(s):  
Satellite Imagery ◽  
Large Scale ◽  
Vegetation Index ◽  
Normalized Difference Vegetation Index ◽  
Eastern China ◽  
Chlorophyll Concentration ◽  
False Negative ◽  
Distribution Data ◽  
Landsat 8 ◽  
Negative Results

Information, especially spatial distribution data, related to coastal raft aquaculture is critical to the sustainable development of marine resources and environmental protection. Commercial high spatial resolution satellite imagery can accurately locate raft aquaculture. However, this type of analysis using this expensive imagery requires a large number of images. In contrast, medium resolution satellite imagery, such as Landsat 8 images, are available at no cost, cover large areas with less data volume, and provide acceptable results. Therefore, we used Landsat 8 images to extract the presence of coastal raft aquaculture. Because the high chlorophyll concentration of coastal raft aquaculture areas cause the Normalized Difference Vegetation Index (NDVI) and the edge features to be salient for the water background, we integrated these features into the proposed method. Three sites from north to south in Eastern China were used to validate the method and compare it with our former proposed method using only object-based visually salient NDVI (OBVS-NDVI) features. The new proposed method not only maintains the true positive results of OBVS-NDVI, but also eliminates most false negative results of OBVS-NDVI. Thus, the new proposed method has potential for use in rapid monitoring of coastal raft aquaculture on a large scale.

scholarly journals The Role of FNAC in the Diagnosis and Management of Warthin Tumour: Analysis of 74 Cases

2020 ◽  
Author(s):  
Mohamed Zahran ◽  
Sundus Alsedra ◽  
Daron Cope ◽  
Ahmed Youssef
Keyword(s):  
Parotid Gland ◽  
False Negative ◽  
Benign Neoplasm ◽  
Cost Effective ◽  
Needle Aspiration ◽  
Warthin Tumor ◽  
True Negative ◽  
Warthin Tumour ◽  
Negative Results ◽  
Histopathological Correlation

Abstract Introduction After pleomorphic adenoma, Warthin tumor gets its popularity as the second most common benign neoplasm of the parotid gland. Fine-needle aspiration cytology (FNAC) is the most cost-effective and minimally-invasive way to determine the histological character of a parotid gland tumor. Objective To determine the accuracy of FNAC in the diagnosis of Warthin Tumour. Methods A retrospective study conducted between 2014 and 2018. Out of 243 FNACs performed for parotid lesions, a histopathological correlation was established in 74 cases to reveal the accuracy of FNAC in the diagnosis of Warthin tumor. Results A total of 243 FNACs of parotid lesions were performed, and a histopathological correlation was established in 74 (30.4%) cases. Later on, we confirmed that 16 (21.6%) out of these 74 patients had cases of Warthin tumor. In total, 15 (20.3%) out of those 74 cases were confirmed as Warthin tumors on the initial cytology, which revealed a true positive concordance between the cytology and the final histological diagnosis; 55/74 (74%) were true negative results; on the other hand, 1/74 (1.4%) was a false negative, and 3/74 (4.1%) were false positive results. The sensitivity of the FNAC in the diagnosis of Warthin tumor was of 93%, while the specificity was of 94.8%, and the accuracy, of 94.6%. Conclusion In the present study, FNAC had a high diagnostic accuracy, reaching 94%.

scholarly journals Improved Real-Time PCR Diagnosis of Citrus Stubborn Disease by Targeting Prophage Genes of Spiroplasma citri

Plant Disease ◽  
2015 ◽  
Vol 99 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Xuefeng Wang ◽  
Harsha Doddapaneni ◽  
Jianchi Chen ◽  
Raymond K. Yokomi
Keyword(s):  
False Negative ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Detection Sensitivity ◽  
Spiroplasma Citri ◽  
Negative Results ◽  
Enhanced Sensitivity ◽  
Field Samples ◽  
Primer Sets ◽  
Citrus Stubborn Disease

Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is technically demanding and time consuming. S. citri is typically low in titer and unevenly distributed in citrus, making reliable detection challenging. The current preferred detection method is polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri housekeeping genes. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Therefore, targeting multicopy prophage genes was hypothesized to improve sensitivity of PCR detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. These primer sets were used to evaluate detection sensitivity in SYBR Green-based quantitative PCR (qPCR) assays with 18 S. citri in cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by 4.91 and 3.65 cycle threshold (Cq) units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Detection was slightly less sensitive for the Php-orf3 primer set at 3.02 and 1.76 Cq units, respectively, over the same housekeeping gene primers. The prophage primer sets were validated for qPCR detection with field samples from three citrus orchards in California's San Joaquin Valley collected from 2007 to 2013. The data showed that S. citri prophage sequences improved sensitivity for qPCR detection of S. citri-infected trees at least 10-fold and reduced the number of false-negative results. No false-positive samples were detected with any of the primer sets. The enhanced sensitivity resulted from the higher copy number of prophage genes in the S. citri genome and, thus, improved CSD diagnosis from field samples.

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