scholarly journals Variant Prediction by Analyzing RdRp/S Gene Double or Low Amplification Pattern in Allplex SARS-CoV-2 Assay

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1854
Author(s):  
Min-Kyung So ◽  
Sholhui Park ◽  
Kyunghoon Lee ◽  
Soo-Kyung Kim ◽  
Hae-Sun Chung ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Diagnostic Testing ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Rdrp Gene ◽  
Genomic Sequencing ◽  
Amplification Curve ◽  
S Gene ◽  
Cost Constraints ◽  
Global Threat

The spread of delta variants (B.1.671.2) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a severe global threat. Multiplex real-time PCR is a common method for confirming SARS-CoV-2 infection, however, additional tests, such as whole genomic sequencing, are required to reveal the presence or type of viral mutation. Moreover, applying whole genomic sequencing to all SARS-CoV-2 positive samples is challenging due to time and cost constraints. Here, we report that the double or low amplification curve observed during RNA-dependent RNA polymerase (RdRp) gene/S gene amplification in the Allplex SARS-CoV-2 Assay is related to delta/alpha variants. We analyzed the RdRp/S gene amplification curve using 94 samples confirmed as SARS-CoV-2 infection by the Allplex SARS-CoV-2 Assay from January to August, 2021. These positive samples identified variant types using the Novaplex SARS-CoV-2 Variants I and IV Assays. Overall, 17 samples showing a double curve and 11 samples showing a low amplification pattern were associated with alpha-/delta-type strains with variants in the P681 region. The double or low curve shown in the RdRp gene amplification curve had 100% sensitivity and 100% specificity for diagnosing delta/alpha variants. During the SARS-CoV-2 virus diagnostic RT-PCR test using the Allplex SARS-CoV-2 Assay, we could consider the presence of delta/alpha variants in the samples with double or low amplification curve of the RdRp/S gene channel. This PCR amplification curve abnormality enables rapid and cost-effective variant type prediction during SARS-CoV-2 diagnostic testing in clinical laboratories.

scholarly journals A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant

2021 ◽  
Vol 15 (02) ◽  
pp. 242-246
Author(s):  
Gabriele Ibba ◽  
Rosangela Sau ◽  
Flavia Angioj ◽  
Marcello Abbondio ◽  
Salvatore Rubino ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Cost Effective ◽  
Rt Pcr ◽  
Curve Pattern ◽  
Amplification Curve ◽  
Viral Genomes ◽  
S Gene ◽  
Thermo Fisher Scientific ◽  
S Genes ◽  
Fisher Scientific

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.

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

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.

scholarly journals GenomeDiver: A platform for phenotype-guided medical genomic diagnosis

2020 ◽  
Author(s):  
Nathaniel Pearson ◽  
Christian Stolte ◽  
Kevin Shi ◽  
Faygel Beren ◽  
Noura S. Abul-Husn ◽  
...  
Keyword(s):  
Diagnostic Testing ◽  
Diagnostic Process ◽  
Genomic Sequencing ◽  
Sequencing Data ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Phenotypic Data ◽  
Human Phenotype ◽  
Phenotype Data

ABSTRACTPurposeMaking a diagnosis from clinical genomic sequencing requires well-structured phenotypic data to guide genotype interpretation. A patient’s phenotypic features can be documented using the Human Phenotype Ontology (HPO), generating terms used to prioritize genes potentially causing the patient’s disease. We have developed GenomeDiver to provide a user interface for clinicians that allows more effective collaboration with the clinical diagnostic laboratory, with the goal of improving the success of the diagnostic process.MethodsGenomeDiver is designed to prompt reverse phenotyping of patients undergoing genetic testing, enriching the amount and quality of structured phenotype data for the diagnostic laboratory, and helping clinicians to explore and flag diseases potentially causing their patient’s presentation.ResultsWe show how GenomeDiver communicates the clinician’s informed insights to the diagnostic lab in the form of HPO terms for interpretation of genomic sequencing data. We describe our user-driven design process, the engineering of the software for efficiency, security and portability, and an example of the performance of GenomeDiver using simulated genomic testing data.ConclusionsGenomeDiver is a first step in a new approach to genomic diagnostics that enhances laboratory-clinician interactions, with the goal of directly engaging clinicians to improve the outcome of genomic diagnostic testing.

scholarly journals NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2577
Author(s):  
Imogen A. Wright ◽  
Kayla E. Delaney ◽  
Mary Grace K. Katusiime ◽  
Johannes C. Botha ◽  
Susan Engelbrecht ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Cost Effective ◽  
Full Length ◽  
Sequencing Error ◽  
Consensus Building ◽  
Genome Sequences ◽  
Bioinformatics Pipeline ◽  
Oxford Nanopore ◽  
Single Genome ◽  
Hiv 1

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

scholarly journals First Report of Cymbidium mosaic virus and Odontoglossum ringspot virus in Orchids in Mexico

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 464-464
Author(s):  
A. G. Soto-Valladares ◽  
R. De La Torre-Almaraz ◽  
B. Xoconostle-Cazares ◽  
R. Ruíz-Medrano
Keyword(s):  
Mosaic Virus ◽  
Pcr Amplification ◽  
Positive Sample ◽  
Ringspot Virus ◽  
Rdrp Gene ◽  
Mixed Infections ◽  
Cymbidium Mosaic Virus ◽  
First Report ◽  
Odontoglossum Ringspot Virus ◽  
Cp Gene

In 2010, a survey for viral diseases in commercial, orchid-producing greenhouses was carried out in Morelos, Mexico. Many symptomatic plants were observed. The most common leaf symptoms were yellow mottle, yellow streaks, and chlorotic and necrotic ringspots. Leaf samples were collected from eight symptomatic plants from the following genera: Encyclia, Oncidium, Shomburghia, Brassia, Guarianthe, Cattleya, Epidendrum, Vanilla, Xilobium, Laelia, and Brassocattleya. Samples were tested using double-antibody sandwich (DAS)-ELISA (Agdia, Elkhart, IN) with antiserum for Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cymbidium ringspot mosaic virus, and Tobacco mosaic virus (TMV) and a general antiserum for potyviruses. At least one plant from each genus was positive to CymMV and ORSV as individual or mixed infections. Encyclia and Laelia plants were the most frequently found with mixed infections by both viruses. All genera were negative for TMV and potyviruses. Total RNA extracts were obtained from all ELISA-positive samples by a modified silica capture protocol (2). Reverse transcription (RT)-PCR was carried out with general polymerase (RdRp) gene primers corresponding to the Potexvirus group (3) and specific primers for the coat protein gene (CP) of CymMV and ORSV (1). The PCR amplification from a positive sample of each genus was resolved in agarose gels. Amplification products of the expected size were obtained for CymMV and ORSV. Five CymMV RdRp gene clones from five different plants of Laelia (GenBank Accession Nos. HQ393958, HQ393959, HQ393960, HQ393961, and HQ393962), two CP gene clones of CP gene of CymMV from two different plants of Oncidium (GenBank Accession Nos. HQ393956 and HQ393957), and three CP clones of CP of ORSV from three different plants of Encyclia (GenBank Accession Nos. HQ393953, HQ393954, and HQ393955) were sequenced. The nucleotide sequences of the Mexican orchid CymMV isolates were 96 to 97% identical to CymMV sequences in the GenBank, while those of ORSV were 99 to 100% identical to deposited ORSV sequences. To our knowledge, this is the first report of CymMV and ORSV in orchids in Mexico, which are two of the most important quarantine virus in orchids in Mexico. References: (1) P. Ajjikuttira et al. J. Gen. Virol. 86:1543, 2005. (2) J. R. Thompson et al. J. Virol. Methods 111:85, 2003. (3) R. A. A. van der Vlugt and M. Berendsen. Eur. J. Plant Pathol. 108:367, 2002.

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

Abstract W P183: Testing the Utility of an Emergency Room-Based Stroke Evaluation Protocol

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Gyeong-Moon Kim ◽  
Johanna Helenius ◽  
E Murat Arsava ◽  
Hakan Ay
Keyword(s):  
Emergency Department ◽  
Clinical Evaluation ◽  
Diagnostic Testing ◽  
Cost Effective ◽  
Vascular Imaging ◽  
Cardiac Monitoring ◽  
Evaluation Protocol ◽  
Evaluation Strategies ◽  
Fundamental Goal ◽  
Major Stroke

Background and purpose: A fundamental goal in diagnostic stroke evaluation is to identify the underlying etiology. We sought to determine the yield of an emergency department-based diagnostic evaluation protocol for identifying the etiology of stroke. Methods: We determined etiologic stroke subtypes using the automated Causative Classification System (CCS, available at https://ccs.mgh.harvard.edu) in 2422 consecutive patients with ischemic stroke at admission and discharge. Admission assessment was based on information from clinical evaluation, ECG, brain imaging (CT or MRI), and vascular imaging (CTA/MRA). Discharge CCS was performed blinded to the admission CCS subtype using information from additional tests such as echocardiography, cardiac monitoring, and special blood and CSF tests. Results: Table 1 shows the distribution of CCS subtypes. Overall, admission and discharge CCS subtypes were different in 29% of the patients. The size of “undetermined” category decreased from 37% at admission to 12% at discharge. The shift from “undetermined” to a known etiology was primarily due to detection of cardiac sources with low or uncertain risk of stroke (94%). The yield of investigations performed after admission in identifying a major known subtype was only 4.1% (p=0.008). Conclusions: A careful clinical evaluation and first-line diagnostic testing including brain and vascular imaging in the emergency department identify > 90% of those with a major stroke etiology. The low yield of additional testing suggests a need for developing cost-effective evaluation strategies in suspected patients.

scholarly journals Assessing National Antimicrobial Resistance Campaigns Using a Health Equity Assessment Tool (HEAT)

Antibiotics ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 121 ◽  
Author(s):  
Hood ◽  
Toleikyte ◽  
Ashiru-Oredope
Keyword(s):  
Public Health ◽  
Antimicrobial Resistance ◽  
Health Equity ◽  
Assessment Tool ◽  
Significant Proportion ◽  
Cost Effective ◽  
Health Campaigns ◽  
Quality Healthcare ◽  
Public Health Campaigns ◽  
Global Threat

It has been widely recognised that a significant proportion of the world’s population suffer inequalities in accessing high quality healthcare and wider services. Within healthcare, antimicrobial resistance (AMR) is a global threat to public health affecting all healthcare systems and growing at an alarming pace. To ensure that national AMR campaigns developed by Public Health England are inclusive of all populations within the target audience a health equity assessment tool (HEAT) was used. The project leads for each campaign completed the HEAT independently with a follow up meeting with the study team to discuss and clarify the responses. A trend analysis was carried out with common themes being used to provide recommendations. The campaigns have demonstrated equality and diversity based on the requirements of the Equality Act 2010, particularly age, sex, and race protected characteristics. Some notable results include the translation of website materials in over 30 languages and reaching individuals in 122 countries. It was however noted that several of the protected characteristics were not applicable. The continuous development of resources with collaboration from a variety of diverse user groups would be advantageous towards aiding future campaign reach. The use of the HEAT has demonstrated the ease and cost-effective way to assess any health inequalities and would be a useful addition to antimicrobial stewardship and public health campaigns.

scholarly journals Pooled Testing for Expanding COVID-19 Mass Surveillance

2020 ◽  
Vol 14 (3) ◽  
pp. e42-e43 ◽  
Author(s):  
Angela Felicia Sunjaya ◽  
Anthony Paulo Sunjaya
Keyword(s):  
Diagnostic Testing ◽  
Pcr Amplification ◽  
The United States ◽  
Positive Sample ◽  
Pooled Testing ◽  
Blood Banks ◽  
Massive Scale ◽  
Polymerase Chain ◽  
Pool Test ◽  
Mass Surveillance

ABSTRACTDiagnostic testing to identify patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role to control the coronavirus disease (COVID-19) pandemic. While several countries have implemented the use of diagnostic testing in a massive scale as a cornerstone for infection control and surveillance, other countries affected by the pandemic are hampered by its limited testing capacity. Pooled testing was first introduced in the 1940s and is now used for screening in blood banks. Testing is done by pooling multiple individual samples together. Only in the case of a positive pool test would individual samples of the pool be tested, thus substantially reducing the number of tests needed. Several studies regarding their use for SARS CoV-2 have been done in the United States, Israel, and Germany. Studies have shown that an individual positive sample can still be detected in pools of up to 32 samples, and possibly even 64 samples, provided that additional polymerase chain reaction (PCR) amplification cycles are conducted with a sensitivity of 96%. Simulation studies to determine optimal pool size and pooling techniques have also been conducted. Based on these studies, pooled testing is shown to be able to detect positive samples with sufficient accuracy and can easily be used with existing equipment and personnel for population-wide screening.

Cost Analysis in the Diagnosis of Chronic Rhinosinusitis

2003 ◽  
Vol 17 (3) ◽  
pp. 139-142 ◽  
Author(s):  
James A. Stankiewicz ◽  
James M. Chow
Keyword(s):  
Antibiotic Therapy ◽  
Chronic Rhinosinusitis ◽  
Medical Therapy ◽  
Ct Scanning ◽  
Cost Effective ◽  
Subjective Symptoms ◽  
Cost Constraints ◽  
Health Care Expense ◽  
Charge Analysis ◽  
A Charge

Background The present treatment regimen for a diagnosis of chronic rhinosinusitis involves a prolonged course of antibiotic therapy along with other adjunctive therapy. The decision to start treatment is made after diagnosis of chronic rhinosinusitis, which is based on subjective symptoms. The working hypothesis of this study is that the diagnosis based on subjective symptoms is inaccurate, leading to inappropriate antibiotic therapy and unnecessary health care expense. Methods One hundred patients were evaluated prospectively to determine which patients qualified for this study. Seventy-eight patients satisfied current criteria for a diagnosis of rhinosinusitis. Results Fifty-three percent (41 patients) of the 78 patients did not have a diagnosis of chronic sinusitis based on same-day computed tomography (CT) scanning. A charge analysis comparing treatment after diagnosis with medical therapy alone and CT scan for failures versus CT scanning with medical treatment for positive scans was performed. Although the most economical method of treatment was initiating medical therapy, it was also the least sensitive and specific in that 52% of patients didn't require the treatment. Endoscopy and/or CT screening with medical therapy were much better at appropriate diagnosis and targeted therapy but charge analysis indicated a much higher cost. Conclusion Presently, the current subjective diagnostic paradigm for chronic rhinosinusitis is most cost-effective but least accurate. Objective evaluations (endoscopy and CT scanning) to aid in diagnosis are more accurate but more costly. Where cost constraints are important, careful considerations of alternatives are important.

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