scholarly journals Rapid and Accurate Identification of SARS-CoV-2 Omicron Variants Using Droplet Digital PCR (RT-ddPCR)

2022 ◽  
Author(s):  
Margaret Mills ◽  
Pooneh Hajian ◽  
Shah Mohamed Bakhash ◽  
Hong Xie ◽  
Derrek Mantzke ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Washington State ◽  
Genome Sequences ◽  
Accurate Identification ◽  
Clinical Specimens ◽  
Public Health Professionals ◽  
Variant Detection ◽  
Ngs Data ◽  
Rna Concentration ◽  
Selection Of

Background Mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in the recently described Omicron variant. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals. Objective To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment. Study Design Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant. Results Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen. Conclusion We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.

scholarly journals Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens

2021 ◽  
Author(s):  
Kevin S Kuchinski ◽  
Jason Nguyen ◽  
Tracy D Lee ◽  
Rebecca Hickman ◽  
Agatha N Jassem ◽  
...  
Keyword(s):  
British Columbia ◽  
Laboratory Data ◽  
Genomic Sequencing ◽  
Genome Sequences ◽  
High Quality ◽  
Laboratory Methods ◽  
Clinical Specimens ◽  
Recent Outbreak ◽  
High Quality Genome

Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.

scholarly journals Draft Genome Sequences of 12 Clinical and Environmental Methicillin-Resistant Staphylococcus pseudintermedius Strains Isolated from a Veterinary Teaching Hospital in Washington State

2018 ◽  
Vol 6 (15) ◽  
pp. e00290-18
Author(s):  
Devendra H. Shah ◽  
Lisa P. Jones ◽  
Narayan Paul ◽  
Margaret A. Davis
Keyword(s):  
Teaching Hospital ◽  
Draft Genome ◽  
Multidrug Resistant ◽  
Washington State ◽  
Nosocomial Transmission ◽  
Whole Genome ◽  
Genome Sequences ◽  
Staphylococcus Pseudintermedius ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACT Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a globally emergent multidrug-resistant pathogen of dogs associated with nosocomial transmission in dogs and with potential zoonotic impacts. Here, we report the draft whole-genome sequences of 12 hospital-associated MRSP strains and their resistance genotypes and phenotypes.

scholarly journals Draft Genome Sequences of 64 Type Strains of 50 Species and 25 Subspecies of the Genus Staphylococcus Rosenbach 1884

2019 ◽  
Vol 8 (17) ◽  
Author(s):  
Kevin Cole ◽  
Dona Foster ◽  
Julie E. Russell ◽  
Tanya Golubchik ◽  
Martin Llewelyn ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Whole Genome ◽  
Genome Sequences ◽  
Accurate Identification ◽  
Access To Data ◽  
Type Strains

Members of the genus Staphylococcus have been isolated from humans, animals, and the environment. Accurate identification with whole-genome sequencing requires access to data derived from type strains.

scholarly journals Multimodal Approach to Assessment of Fecal Microbiota Donors based on Three Complementary Methods

2020 ◽  
Vol 9 (7) ◽  
pp. 2036
Author(s):  
Jaroslaw Bilinski ◽  
Mikolaj Dziurzynski ◽  
Pawel Grzesiowski ◽  
Edyta Podsiadly ◽  
Anna Stelmaszczyk-Emmel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fecal Microbiota ◽  
Dead Cell ◽  
Series Of Experiments ◽  
Multi Method Approach ◽  
The Stability ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing ◽  
Selection Of

Methods of stool assessment are mostly focused on next-generation sequencing (NGS) or classical culturing, but only rarely both. We conducted a series of experiments using a multi-method approach to trace the stability of gut microbiota in various donors over time, to find the best method for the proper selection of fecal donors and to find “super-donor” indicators. Ten consecutive stools donated by each of three donors were used for the experiments (30 stools in total). The experiments assessed bacterial viability measured by flow cytometry, stool culturing on different media and in various conditions, and NGS (90 samples in total). There were no statistically significant differences between live and dead cell numbers; however, we found a group of cells classified as not-dead-not-alive, which may be possibly important in selection of “good” donors. Donor C, being a regular stool donor, was characterized by the largest number of cultivable species (64). Cultivable core microbiota (shared by all donors) was composed of only 16 species. ANCOM analysis of NGS data highlighted particular genera to be more abundant in one donor vs. the others. There was a correlation between the not-dead-not-alive group found in flow cytometry and Anaeroplasma found by NGS, and we could distinguish a regular stool donor from the others. In this work, we showed that combining various methods of microbiota assessment gives more information than each method separately.

scholarly journals Draft Genome Sequences of Six Novel Picorna-Like Viruses from Washington State Spiders

2017 ◽  
Vol 5 (9) ◽  
Author(s):  
Ryan C. Shean ◽  
Negar Makhsous ◽  
Rodney L. Crawford ◽  
Keith R. Jerome ◽  
Alexander L. Greninger
Keyword(s):  
Amino Acid ◽  
Viral Genome ◽  
Draft Genome ◽  
Amino Acid Identity ◽  
Washington State ◽  
Genome Sequences ◽  
Acid Identity ◽  
Spider Species ◽  
Viral Sequences

ABSTRACT We report draft genome sequences of six novel Picornavirales members from six different spider species found in Washington state. These six viral sequences distinctly clustered together phylogenetically with less than 35% amino acid identity to the closest reference viral genome.

scholarly journals SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

2020 ◽  
Author(s):  
Renfei Lu ◽  
Jian Wang ◽  
Min Li ◽  
Yaqi Wang ◽  
Jia Dong ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pharyngeal Swab ◽  
Clinical Specimens ◽  
Positive Results ◽  
Negative Detection

SummaryBackgroundSARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results (for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis.MethodsDigital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes.ResultsThe lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment.ConclusionsDigital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

scholarly journals Aptamers selection for platelets using droplet digital PCR

2021 ◽  
pp. 100-103
Author(s):  
A.V. Blagodatova ◽  
◽  
K.V. Kochkina ◽  
M.A. Komarova ◽  
N.Y. Trofina ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Anticoagulant Activity ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Platelet Glycoprotein ◽  
Systematic Evolution ◽  
Selection For ◽  
Exponential Enrichment ◽  
Selection Of

The aim of the research. To obtain aptamers-inhibitors of platelet glycoprotein IIb / IIIa receptors, blocking platelet aggregation. Material and methods. Th e selection of aptamers for IIb / IIIa receptors of platelets was carried out according to the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment), modifi ed to select aptamers for a specifi c epitope. Th e method allows selection and in vitro evolution of aptamers with selectivity to a specifi c target from a large library of oligonucleotides. Th e affi nity of aptamers for platelet IIb / IIIa receptors was determined using fl ow cytometry. Results. Pools of aptamers of aptamers with high affi nity for IIb / IIIa platelet receptors were obtained. Th e study of the antiaggregation properties of the pools with the best binding showed that platelet aggregation was minimal when using the aptamers from the pool of the 5th round of selection. Th us, the aptamers of this pool have the greatest potential to be used as an analogue of a synthetic peptide that blocks thromboaggregation. Aptamers from this pool were taken for sequencing in order to obtain sequences of aptamers with the best antiaggregatory properties. Conclusion. Pools of aptamers with high affi nity for IIb / IIIa receptors of platelets and anticoagulant activity were obtained.

scholarly journals Virulence Factors and Antifungal Susceptibility Profile of C. tropicalis Isolated from Various Clinical Specimens in Alexandria, Egypt

2021 ◽  
Vol 7 (5) ◽  
pp. 351
Author(s):  
Mohammed A. El-Kholy ◽  
Ghada F. Helaly ◽  
Ebtisam F. El Ghazzawi ◽  
Gamal El-Sawaf ◽  
Sherine M. Shawky
Keyword(s):  
Virulence Factors ◽  
Biofilm Formation ◽  
Antifungal Susceptibility ◽  
Accurate Identification ◽  
Icu Patients ◽  
Clinical Specimens ◽  
High Incidence ◽  
Invasive Infections ◽  
Vitek 2 ◽  
Urinary Isolates

Background: The incidence of candidiasis caused by non-albicans Candida (NAC) species is increasing. Candida tropicalis has emerged as one of the most important NAC species. This study aims to examine the antifungal susceptibility profile and some virulence factors of C. tropicalis isolated from various clinical specimens. Methods: A total of 71 C. tropicalis isolates from various clinical specimens (69.01%, 18.31%, 9.86%, and 2.82% of isolates were collected from urine, respiratory samples, blood, and skin and soft tissue infections, respectively) from ICU patients in Alexandria, Egypt. The isolates were identified at species level by CHROMagar Candida and VITEK 2 compact system. Furthermore, the antifungal susceptibility was determined using the VITEK 2 system AST-YS07 card containing different antifungals. Hemolysin, phospholipase, and proteinase activity and biofilm formation were also tested as virulence factors. Results: Only 30 isolates (42.25%) were non-susceptible (MIC ≥ 4 µg/mL) to fluconazole, of which 28 isolates showed non-susceptibility (MIC ≥ 0.25 µg/mL) to voriconazole. All isolates showed both hemolysin and proteinase activities, while only 9 isolates (12.68%) showed phospholipase production and 70 isolates (98.59%) demonstrated biofilm formation. Strong biofilm production was observed among the blood culture isolates (85.71%), followed by the respiratory and urinary isolates (61.54% and 46.94%, respectively). Conclusions: This study sought to provide useful data on the antifungal susceptibility of C. tropicalis isolates from ICU patients suffering from invasive infections with an increased trend towards elevated MICs levels of both fluconazole and voriconazole. Due to the high incidence of systemic candidiasis and antifungal resistance, C. tropicalis is emerging as a serious root of infections. Therefore, early and accurate identification of Candida species along with susceptibility testing is of utmost importance.

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