scholarly journals Procalcitonin Detection in Veterinary Species: Investigation of Commercial ELISA Kits

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1511
Author(s):  
Federica Battaglia ◽  
Valentina Meucci ◽  
Rosalba Tognetti ◽  
Francesca Bonelli ◽  
Micaela Sgorbini ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Recombinant Proteins ◽  
Limit Of Detection ◽  
Rapid Identification ◽  
Human Medicine ◽  
Clinical Samples ◽  
Reference Ranges ◽  
Plasma Samples ◽  
Elisa Kit ◽  
Species Specific

In human medicine, procalcitonin (PCT), the precursor of calcitonin, is used for the rapid identification of the origin and severity of sepsis. In veterinary medicine, PCT has been studied in horses, cattle, and dogs, but the use of PCT in diagnostic and/or prognostic settings is not possible because of the lack of validated assays to obtain reference ranges. The aim of the present study was the investigation of commercially available ELISA kits for the detection of canine and equine PCT in plasma samples. Validation of the ELISA kits was performed by using species-specific recombinant proteins spiked both in plasma and buffer samples; linearity, limit of detection (LOD), recovery, and intra-assay and inter-assay variability were calculated. Moreover, clinical samples obtained from sick and healthy animals were also analyzed with the tested kits. Canine PCT was measured with a recombinant canine and a canine PCT ELISA kit. Equine PCT was measured with an equine and a human ELISA PCT kit. Our data demonstrate that the canine recombinant PCT ELISA kit can be used to measure canine PCT in plasma samples, showing an intra-assay and inter-assay coefficient of variation less than 20% and a LOD of 11 pg/mL, whereas the present results do not support the use of the canine PCT ELISA kit. The human PCT ELISA kit is suitable to detect equine PCT with a LOD of 56 ng/mL, whereas the equine PCT ELISA kit did not detect recombinant equine PCT.

scholarly journals Detection of Candida albicans DNA from blood samples using a novel electrochemical assay

2011 ◽  
Vol 60 (4) ◽  
pp. 467-471 ◽  
Author(s):  
Alastair Muir ◽  
Gordon Forrest ◽  
John Clarkson ◽  
Alan Wheals
Keyword(s):  
Genomic Dna ◽  
Limit Of Detection ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Electrochemical Assay ◽  
Blood Samples ◽  
Resistant Species ◽  
Life Threatening ◽  
Appropriate Therapy ◽  
Species Specific

The genus Candida contains a number of yeast species which are opportunistic pathogens and are associated with life-threatening infections in immunocompromised individuals. Provision of appropriate therapy relies on the rapid identification of the infecting species, and existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. We have previously developed a system for the rapid detection of yeast pathogens in clinical samples using PCR followed by hybridization with a suite of five species-specific, electrochemically labelled DNA probes. The limit of detection of the assay was shown to be 37 fg (∼1 genome) per reaction using extracted genomic DNA. We carried out a study to test the limit of detection of one of the probes, CA PR3, using blood samples from a healthy donor that were spiked with genomic DNA or with C. albicans cells. Our results demonstrated a limit of detection of 37 fg (ml blood)−1 (∼1 genome ml−1) using extracted DNA or 10 c.f.u. (ml blood)−1 using C. albicans cells, indicating that the assay is capable of detecting C. albicans nucleic acid at levels that are encountered in clinical samples.

scholarly journals SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades

2021 ◽  
Vol 7 (6) ◽  
pp. 433
Author(s):  
Ahmad Ibrahim ◽  
Lucie Peyclit ◽  
Rim Abdallah ◽  
Saber Khelaifia ◽  
Amanda Chamieh ◽  
...  
Keyword(s):  
Culture Medium ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Candida Auris ◽  
Phenotypic Profile ◽  
Stool Samples ◽  
Selective Culture Medium

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

scholarly journals In Situ Amplified Chemiluminescent Detection of DNA and Immunoassay of IgG Using Special-Shaped Gold Nanoparticles as Label

2006 ◽  
Vol 52 (10) ◽  
pp. 1958-1961 ◽  
Author(s):  
Zhouping Wang ◽  
Jianqiang Hu ◽  
Yan Jin ◽  
Xin Yao ◽  
Jinghong Li
Keyword(s):  
Gold Nanoparticles ◽  
Human Plasma ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Dna Detection ◽  
Clinical Samples ◽  
Plasma Samples ◽  
Au Nps ◽  
Human Plasma Samples

Abstract Background: Au(III) catalyzed luminol chemiluminescence (CL) is classic in luminescence analysis. Recently, spherical gold nanoparticles (Au-NPs) were found displaying far stronger catalytic activity on luminol CL than that of Au(III). Some methods based on Au-NPs probes have been developed for DNA detection or immunoassay. However, more complicated labeling or stripping procedures are often inescapable in these protocols. Methods: We synthesized specially shaped, irregular gold nanoparticles (IGNPs) and found their catalytic efficiency on luminol CL to be 100-fold greater than that of spherical Au-NPs. Using the IGNPs-functionalized DNA oligomers and the IGNPs-modified anti-IgG as in situ chemiluminescent probes, we established sandwich-type analytic methods for rapid, simple, selective, and sensitive sequence-specific DNA detection and for human plasma IgG immunoassay, respectively. We used 12 clinical human plasma samples to examine the precision and accuracy of the proposed method for IgG content determination. Results: Calibration curves for the oligonucleotide [ΔI = 15.73 + 27.55 (DNA) × 1010 (mol/L); R2 = 0.9936] and IgG [ΔI = 48.84 + 30.23 (IgG) × 1010 (mol/L); R2 = 0.9964] show good correlation, demonstrating the linear response over the concentrations tested (0.04–10 nmol/L for DNA, 0.05–10 nmol/L for IgG). The limit of detection, calculated based on 50 μL of a solution of calibrators, was 13 pmol/L for DNA and 17 pmol/L for IgG, with a signal-to-noise ratio of 3. We obtained good intra-and interassay reproducibility. The IgG contents in 12 human plasma samples obtained by the proposed method are identical with the data of clinical laboratory. Conclusions: We developed a simple and sensitive method for in situ amplified chemiluminescence detection of sequence-specific DNA and immunoassay of IgG by use of highly active, specially shaped, irregular gold nanoparticles (IGNPs) as label and confirmed by clinical samples test. This method has many desirable features including rapid detection, selectivity, and little required instrumentation. This new protocol may be quite promising, with potentially broad applications for clinical immunoassays and DNA hybridization analysis.

scholarly journals SCA Medium: A New Culture Medium for the Isolation of all Candida auris Clade

2021 ◽  
Author(s):  
Ahmad Ibrahim ◽  
Lucie Peyclit ◽  
Rim Abdallah ◽  
Saber Khelaifia ◽  
Amanda Chamieh ◽  
...  
Keyword(s):  
Culture Medium ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Candida Auris ◽  
Phenotypic Profile ◽  
Stool Samples ◽  
Selective Culture Medium

Candida auris is an emerging multidrug resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation is necessary for its diagnosis and containing spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection of 102 CFU/ml. The 100% specificity of SCA (Specific C. auris) medium is confirmed on a set of 134 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, and allows studying its phenotypic profile.

scholarly journals Diagnostic Performance and Clinical Feasibility of a Novel One-Step RT-qPCR for Detecting Multiple Severe Acute Respiratory Syndrome Coronavirus

2021 ◽  
Author(s):  
Tran Bac Le ◽  
Hye Kwon Kim ◽  
Min-Ju Ahn ◽  
Mark Zanin ◽  
Van Thi Lo ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Limit Of Detection ◽  
Laboratory Diagnosis ◽  
Rapid Identification ◽  
Qpcr Assay ◽  
Clinical Samples ◽  
Reverse Transcription Pcr ◽  
One Step ◽  
Human Coronaviruses ◽  
Culture Supernatants

Abstract The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 100 TCID50/ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

Inactivation of Factor XIa in Vivo: Studies in Chimpanzees and in Humans

1996 ◽  
Vol 76 (04) ◽  
pp. 549-555 ◽  
Author(s):  
Walter A Wuillemin ◽  
C Erik Hack ◽  
Wim K Bleeker ◽  
Bart J Biemond ◽  
Marcel Levi ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Life Time ◽  
In Vivo Studies ◽  
Clinical Samples ◽  
Plasma Samples ◽  
C1 Inhibitor ◽  
Elimination Kinetics ◽  
Factor Xia ◽  
Best Parameter

SummaryC1-inhibitor (C1Inh), antithrombin III (ATIII), α1-antitrypsin (a1AT), and α2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation.Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1 AT complexes due to their relatively long t1/2 may be the best parameter to assess FXI activation in clinical samples.

scholarly journals Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexia Bordigoni ◽  
Anne Motte ◽  
Hervé Tissot-Dupont ◽  
Philippe Colson ◽  
Christelle Desnues
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Papillomaviruses ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Gene Encoding ◽  
Hpv Dna ◽  
E6 And E7 ◽  
High Risk Hpv ◽  
Development And Validation

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

scholarly journals 423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S279
Author(s):  
Dorottya Nagy-Szakal ◽  
Mara Couto-Rodriguez ◽  
Joseph Barrows ◽  
Heather L Wells ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Viral Genome ◽  
Board Member ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analysis Tool ◽  
Viral Titer ◽  
Target Enrichment ◽  
Library Preparation ◽  
Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

scholarly journals Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anita G. Amin ◽  
Prithwiraj De ◽  
Barbara Graham ◽  
Roger I. Calderon ◽  
Molly F. Franke ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sample Pretreatment ◽  
Positive Association ◽  
Proteinase K ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Smear Negative ◽  
Tuberculostearic Acid ◽  
Negative Sputum ◽  
Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

scholarly journals Multispecies Identification of Oilseed- and Meat-Specific Proteins and Heat-Stable Peptide Markers in Food Products

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1577
Author(s):  
Klaudia Kotecka-Majchrzak ◽  
Natalia Kasałka-Czarna ◽  
Agata Sumara ◽  
Emilia Fornal ◽  
Magdalena Montowska
Keyword(s):  
Limit Of Detection ◽  
Raw Materials ◽  
Meat Products ◽  
Food Products ◽  
Plant Raw Materials ◽  
Heat Stable ◽  
2S Albumins ◽  
Specific Proteins ◽  
Meat Species ◽  
Species Specific

Consumer demand for both plant products and meat products enriched with plant raw materials is constantly increasing. Therefore, new versatile and reliable methods are needed to find and combat fraudulent practices in processed foods. The objective of this study was to identify oilseed species-specific peptide markers and meat-specific markers that were resistant to processing, for multispecies authentication of different meat and vegan food products using the proteomic LC-MS/MS method. To assess the limit of detection (LOD) for hemp proteins, cooked meatballs consisting of three meat species and hemp cake at a final concentration of up to 7.4% were examined. Hemp addition at a low concentration of below 1% was detected. The LOD for edestin subunits and albumin was 0.9% (w/w), whereas for 7S vicilin-like protein it was 4.2% (w/w). Specific heat-stable peptides unique to hemp seeds, flaxseed, nigella, pumpkin, sesame, and sunflower seeds, as well as guinea fowl, rabbit, pork, and chicken meat, were detected in different meat and vegan foods. Most of the oilseed-specific peptides were identified as processing-resistant markers belonging to 11S globulin subunits, namely conlinin, edestin, helianthinin, pumpkin vicilin-like or late embryogenesis proteins, and sesame legumin-like as well as 2S albumins and oleosin isoforms or selected enzymic proteins.

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