scholarly journals Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohammad Arif ◽  
Grethel Y. Busot ◽  
Rachel Mann ◽  
Brendan Rodoni ◽  
James P. Stack
Keyword(s):  
Population Level ◽  
Illumina Miseq ◽  
Cross Reactivity ◽  
Internal Controls ◽  
Detection And Identification ◽  
Highly Sensitive ◽  
Whole Genomes ◽  
Genomic Regions ◽  
Taqman Qpcr ◽  
Support Research

AbstractRathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

scholarly journals Whole Genome Alignment Based Development of Molecular Marker for Detecting Leptosphaeria maculans and Leptosphaeria biglobosa, the Causal Agent of Blackleg Disease in Brassicas

2019 ◽  
Author(s):  
Mostari Jahan Ferdous ◽  
Mohammad Rashed Hossain ◽  
Denison Michael Immanuel Jesse ◽  
Jong-In Park ◽  
Hee-Jeong Jung ◽  
...  
Keyword(s):  
Leptosphaeria Maculans ◽  
Effector Proteins ◽  
Blackleg Disease ◽  
Target Species ◽  
Highly Sensitive ◽  
Whole Genomes ◽  
Primer Sets ◽  
Species Specific ◽  
Genomic Regions ◽  
Leptosphaeria Biglobosa

Background: Accurate diagnosis of the differentially aggressive fungus Leptosphaeria maculans and Leptosphaeria biglobosa causing Blackleg in crucifers is crucial. Available markers were designed decades ago which may become ineffective due to the ever evolving nature of the fungus, requiring the development of more precise molecular markers. Methods: The whole genomes of available isolates belonging to this two species were aligned using progressive MAUVE tool, species specific genomic regions were extracted and species specific primers were designed from the sequences that encode for effector proteins. Results: Three (Lm1, Lm2 and Lm5) and two (Lb3 and Lb3’) primer sets specifically detected the isolates of target species in PCR based assay, of which the primers Lm5 and Lb3’ were multiplexed for detection of Leptosphaeria maculans and Leptosphaeria biglobosa, generating PCR amplicons of 230 and 834 bp, respectively from a single PCR reaction. The markers were highly sensitive and were able to amplify target species from crude ‘pseudothecia and ascospores suspension’ without requiring DNA extraction. Conclusions: These markers, solitarily or in combination, designed from species specific genomic segments will serve as precise, sensitive and rapid detection of Leptosphaeria maculans and Leptosphaeria biglobosa species and will be helpful for surveillance, management and transboundary quarantine of the devastating disease.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

scholarly journals Genomic characterization of Vibrio parahaemolyticus from Pacific white shrimp and rearing water in Malaysia reveals novel sequence types and structural variation in genomic regions containing the Photorhabdus insect-related (Pir) toxin-like genes

2019 ◽  
Vol 366 (17) ◽  
Author(s):  
Chrystine Zou Yi Yan ◽  
Christopher M Austin ◽  
Qasim Ayub ◽  
Sadequr Rahman ◽  
Han Ming Gan
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Illumina Miseq ◽  
Pacific White Shrimp ◽  
White Shrimp ◽  
Phylogenomic Analysis ◽  
Genomic Characterization ◽  
Genomic Resource ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Genomic Regions

ABSTRACT The Malaysian and global shrimp aquaculture production has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND) typically caused by Vibrio parahaemolyticus harboring the pVA plasmid containing the pirAVp and pirBVp genes, which code for Photorhabdus insect-related (Pir) toxin. The limited genomic resource for V. parahaemolyticus strains from Malaysian aquaculture farms precludes an in-depth understanding of their diversity and evolutionary relationships. In this study, we isolated shrimp-associated and environmental (rearing water) V. parahaemolyticus from three aquaculture farms located in Northern and Central Malaysia followed by whole-genome sequencing of 40 randomly selected isolates on the Illumina MiSeq. Phylogenomic analysis and multilocus sequence typing (MLST) reveal distinct lineages of V. parahaemolyticus that harbor the pirABVp genes. The recovery of pVA plasmid backbone devoid of pirAVp or pirABVp in some V. parahaemolyticus isolates suggests that the toxin genes are prone to deletion. The new insight gained from phylogenomic analysis of Asian V. parahaemolyticus, in addition to the observed genomic instability of pVa plasmid, will have implications for improvements in aquaculture practices to diagnose, treat or limit the impacts of this disease.

scholarly journals Detection and identification of allergens from Canadian mustard varieties of Sinapis alba and Brassica juncea

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 489 ◽  
Author(s):  
L’Hocine ◽  
Pitre ◽  
Achouri
Keyword(s):  
Brassica Juncea ◽  
Protein Extraction ◽  
Immunoglobulin E ◽  
Sinapis Alba ◽  
Cross Reactivity ◽  
Protein Profiling ◽  
Ige Reactivity ◽  
Ige Binding ◽  
Reducing Conditions ◽  
Detection And Identification

Currently, information on the allergens profiles of different mustard varieties is rather scarce. Therefore, the objective of this study was to assess protein profiles and immunoglobulin E (IgE)-binding patterns of selected Canadian mustard varieties. Optimization of a non-denaturing protein extraction from the seeds of selected mustard varieties was first undertaken, and the various extracts were quantitatively and qualitatively analyzed by means of protein recovery determination and protein profiling. The IgE-binding patterns of selected mustard seeds extracts were assessed by immunoblotting using sera from mustard sensitized and allergic individuals. In addition to the known mustard allergens—Sin a 2 (11S globulins), Sin a 1, and Bra j 1 (2S albumins)—the presence of other new IgE-binding protein bands was revealed from both Sinapis alba and Brassica juncea varieties. Mass spectrometry (MS) analysis of the in-gel digested IgE-reactive bands identified the unknown ones as being oleosin, β-glucosidase, enolase, and glutathione-S transferase proteins. A bioinformatic comparison of the amino acid sequence of the new IgE-binding mustard proteins with those of know allergens revealed a number of strong homologies that are highly relevant for potential allergic cross-reactivity. Moreover, it was found that Sin a 1, Bra j 1, and cruciferin polypeptides exhibited a stronger IgE reactivity under non-reducing conditions in comparison to reducing conditions, demonstrating the recognition of conformational epitopes. These results further support the utilization of non-denaturing extraction and analysis conditions, as denaturing conditions may lead to failure in the detection of important immunoreactive epitopes.

scholarly journals Analytical and Clinical Evaluation of the Automated Elecsys Anti–SARS-CoV-2 Antibody Assay on the Roche cobas e602 Analyzer

2020 ◽  
Vol 154 (5) ◽  
pp. 620-626 ◽  
Author(s):  
Clarence W Chan ◽  
Kyle Parker ◽  
Vera Tesic ◽  
Angel Baldwin ◽  
Nga Yeung Tang ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Population Level ◽  
Cross Reactivity ◽  
Good Precision ◽  
Igg Antibody ◽  
Predictive Values ◽  
Serologic Testing ◽  
Automated Platform ◽  
Sensitivity Specificity ◽  
Roche Cobas

Abstract Objectives To evaluate the analytical and clinical performance of the automated Elecsys anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Elecsys Ab) assay on the Roche cobas e602 analyzer. With the ongoing global coronavirus disease 2019 (COVID-19) pandemic, widespread and routine serologic testing of SARS-CoV-2 remains a pressing need. To better understand its epidemiologic spread and to support policies aimed at curtailing further infections, reliable serologic testing is crucial for providing insight into the dynamics of the spread of COVID-19 on a population level. Methods The presence of anti–SARS-CoV-2 antibodies in polymerase chain reaction–positive, confirmed COVID-19 patient samples was determined using the Elecsys Ab assay on the Roche cobas e602 analyzer. The precision and cross-reactivity of the Elecsys Ab assay were characterized and its performance was compared against the EuroImmun IgA/IgG antibody (EuroImmun Ab) assay. Calculated sensitivity, specificity, and positive and negative predictive values were assessed. Results The Elecsys Ab assay demonstrated good precision, had no cross-reactivity with other viral samples, and showed 100% concordance with the EuroImmun Ab assay. Excellent clinical performance with respect to sensitivity, specificity, and positive and negative predictive values was observed. Conclusions The Elecsys Ab assay is a precise and highly reliable automated platform for clinical detection of seropositivity in SARS-CoV-2 infection.

Highly sensitive assays of autoantibodies to thyroglobulin and to thyroid peroxidase.

1989 ◽  
Vol 35 (9) ◽  
pp. 1949-1954 ◽  
Author(s):  
K Beever ◽  
J Bradbury ◽  
D Phillips ◽  
S M McLachlan ◽  
C Pegg ◽  
...  
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Cross Reactivity ◽  
Thyroid Peroxidase ◽  
Scatchard Analysis ◽  
Thyroid Autoantibodies ◽  
Serum Samples ◽  
Highly Sensitive ◽  
Phase Protein ◽  
High Concentrations

Abstract These highly sensitive assays are based on the interaction between thyroid autoantibodies and 125I-labeled autoantigens. Serum samples are incubated with labeled thyroid peroxidase (TPO) or thyroglobulin (Tg) to allow the formation of antibody-labeled antigen complexes. The complexes are then precipitated by addition of solid-phase Protein A. In the presence of high concentrations of TPO antibody or Tg antibody, more than 50% of the respective labeled antigen was precipitated, whereas only 1-2% was precipitated in the absence of autoantibody. Interassay CVs were 3.2% and 5.7%, respectively, for the anti-TPO and anti-Tg assays. There was no cross-reactivity between Tg antibody and TPO antibody. Results correlated highly significantly with results from other assay systems based on antigen-coated cells or plastic supports, but the assays described here were considerably more sensitive. Scatchard analysis of the assay data provided information on the affinity and serum concentration of TPO autoantibodies (ka approximately 10(9) L/mol and concentrations up to 1 g/L) and Tg autoantibodies (ka approximately 4 x 10(10) L/mol and concentrations up to 1 g/L). Overall, these assays provide a sensitive, precise, and convenient system for measuring and investigating the properties of thyroid autoantibodies.

Albatrosses bathe before departing on a foraging trip: implications for risk assessments and marine spatial planning

2017 ◽  
Vol 28 (2) ◽  
pp. 208-215 ◽  
Author(s):  
JOSÉ P. GRANADEIRO ◽  
LETIZIA CAMPIONI ◽  
PAULO CATRY
Keyword(s):  
Spatial Planning ◽  
Population Level ◽  
Sea Surface ◽  
Risk Assessments ◽  
Marine Spatial Planning ◽  
Large Numbers ◽  
Highly Sensitive ◽  
Order Of Magnitude ◽  
Thalassarche Melanophris ◽  
Marine Areas

SummaryTracking studies of seabirds have generally focused in identifying areas used for foraging, in the hope of highlighting regions of energy transfer which may be important for seabird and general ecosystem conservation and special management. However, some sea areas may serve functions other than providing nutritional resources, which may be equally relevant, particularly if used by large numbers of individuals. In this paper, based on a study of 4 breeding colonies in the Falkland Islands and on 314 individuals tracked, we show that virtually all (97.8%) black-browed albatrosses Thalassarche melanophris (BBA) bathe in the close vicinity of the colony, remaining in the area for nearly an hour, before departing on a foraging trip. This compares with only 20 to 40% of the individuals landing close to the colony at the end of a foraging trip. The observed utilization of marine areas by BBA in a radius of 1 to 5 km around the nesting colony is one order of magnitude higher than elsewhere, including foraging hotspots. Clearly, even long-range flying birds such as albatrosses can make an intensive use of the sea-surface in the immediate vicinity of the colonies, and therefore any threats to seabirds in these areas (disturbance, pollutants, collision with artificial structures and light attraction) can potentially have a major impact at the population level. As such, the close neighbourhood of seabird colonies are potentially highly sensitive areas, and this needs to be taken into account when carrying out risk assessments or during marine spatial planning exercises.

scholarly journals Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. eaam8419 ◽  
Author(s):  
Nathaniel D. Anderson ◽  
Richard de Borja ◽  
Matthew D. Young ◽  
Fabio Fuligni ◽  
Andrej Rosic ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Ewing Sarcoma ◽  
Rna Binding ◽  
Fusion Gene ◽  
Soft Tissue Tumors ◽  
Gene Fusions ◽  
Gene Encoding ◽  
Reciprocal Translocations ◽  
Whole Genomes ◽  
Genomic Regions

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between EWSR1, a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when EWSR1-ETS fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel.

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