scholarly journals Detection of Toxoplasmic Encephalitis in HIV Positive Patients in Urine with Hydrogel Nanoparticles.

2020 ◽  
Author(s):  
Hannah Steinberg ◽  
Natalie M Bowman ◽  
Andrea Diestra ◽  
Cusi Ferradas ◽  
Paul Russo ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Toxoplasmic Encephalitis ◽  
Proof Of Concept ◽  
Resource Limited ◽  
Parallel Reaction Monitoring ◽  
Promising Target ◽  
Urine Diagnostics ◽  
Blotting Technique ◽  
Polymerase Chain

Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-isoproplyacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Our results demonstrate nanoparticle technology potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

scholarly journals Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

2021 ◽  
Vol 15 (3) ◽  
pp. e0009199
Author(s):  
Hannah E. Steinberg ◽  
Natalie M. Bowman ◽  
Andrea Diestra ◽  
Cusi Ferradas ◽  
Paul Russo ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Toxoplasmic Encephalitis ◽  
Clinical Sensitivity ◽  
Resource Limited ◽  
Parallel Reaction Monitoring ◽  
Promising Target ◽  
Urine Diagnostics ◽  
Blotting Technique ◽  
Polymerase Chain

BackgroundDiagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) forToxoplasmain blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment.Methology/principle findingsHere we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml ofT.gondiiantigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1)T.gondiiserology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected,T.gondiiseropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence ofT.gondiiantigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic.Conclusion/significancesOur results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

scholarly journals Tracing Septic Pollution Sources Using Synthetic DNA Tracers: Proof of Concept

2019 ◽  
Vol 12 ◽  
pp. 117862211986379 ◽  
Author(s):  
Christine B Georgakakos ◽  
Paul L Richards ◽  
M Todd Walter
Keyword(s):  
Preferential Flow ◽  
Quantitative Polymerase Chain Reaction ◽  
Breakthrough Curves ◽  
Septic Systems ◽  
Proof Of Concept ◽  
Septic System ◽  
Flow Paths ◽  
Synthetic Dna ◽  
Preferential Flow Paths ◽  
Polymerase Chain

Contamination from septic systems is one of the most difficult sources of nonpoint source (NPS) pollution to quantify. Quantification is difficult in part because locating malfunctioning septic systems within a watershed is challenging. This study used synthetic-DNA-based tracers to track flows from 2 septic systems. Sample DNA was quantified using quantitative polymerase chain reaction (qPCR). This technology could be especially useful for simultaneously assessing multiple septic systems because there are essentially infinite unique combinations of DNA bases such that unique tracers could be engineered for each septic system. Two studies were conducted: the first, to determine whether the tracers move through septic systems (experiment 1), and the second, to determine whether the tracers were detectable at watershed scales (experiment 2). In both cases, clear, although complex, breakthrough curves were detected. Experiment 1 revealed possible preferential flow paths that might not have been otherwise obvious, indicative of short circuiting systems. This proof of concept suggests that these tracers could be applied to watersheds suspected of experiencing NPS septic system pollution.

scholarly journals EUAdb: a resource for COVID-19 test development

2020 ◽  
Author(s):  
Alyssa Woronik ◽  
Henry W. Shaffer ◽  
Karin Kiontke ◽  
Jon M. Laurent ◽  
Ronald Zambrano ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Test ◽  
Original Source ◽  
Data Architecture ◽  
Polymerase Chain ◽  
Chain Reaction Test ◽  
Modified Forms ◽  
Sheer Number

AbstractDue to the sheer number of COVID-19 (coronavirus disease 2019) cases, the prevalence of asymptomatic cases and the fact that undocumented cases appear to be significant for transmission of the causal virus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), there is an urgent need for increased SARS-CoV-2 testing capability that is both efficient and effective1. In response to the growing threat of the COVID-19 pandemic in February, 2020, the FDA (US Food and Drug Administration) began issuing Emergency Use Authorizations (EUAs) to laboratories and commercial manufacturers for the development and implementation of diagnostic tests1. So far, the gold standard assay for SARS-CoV-2 detection is the RT-qPCR (real-time quantitative polymerase chain reaction) test2. However, the authorized RT-qPCR test protocols vary widely, not only in the reagents, controls, and instruments they use, but also in the SARS-CoV-2 genes they target, what results constitute a positive SARS-CoV-2 infection, and their limit of detection (LoD). The FDA has provided a web site that lists most of the tests that have been issued EUAs, along with links to the authorization letters and summary documents describing these tests1. However, it is very challenging to use this site to compare or replicate these tests for a variety of reasons. First, at least 12 of 18 tests for EUA submissions made prior to March 31, 2020, are not listed there. To our knowledge, no EUAs have been issued for these applications. Second, the data are not standardized and are only provided as longhand prose in the summary documents. Third, some details (e.g. primer sequences) are absent from several of the test descriptions. Fourth, for tests provided by commercial manufacturers, summary documents are completely missing. To address at least the first three issues, we have developed a database, EUAdb (EUAdb.org), that holds standardized information about EUA-issued tests and is focused on RT-qPCR diagnostic tests, or “high complexity molecular-based laboratory developed tests”1. By providing a standardized ontology and curated data in a relational architecture, we seek to facilitate comparability and reproducibility, with the ultimate goal of consistent, universal and high-quality testing nationwide. Here, we document the basics of the EUAdb data architecture and simple data queries. The source files can be provided to anyone who wants to modify the database for his/her own research purposes. We ask that the original source of the files be made clear and that the database not be used in its original or modified forms for commercial purposes.

scholarly journals Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Unoh Ki ◽  
Takeru Suzuki ◽  
Satoshi Nakazawa ◽  
Yuuki Yonekawa ◽  
Kazuki Watanabe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Confidence Interval ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Low Copy Number ◽  
Polymerase Chain

AbstractRecently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

scholarly journals Homocysteine-Potentiated Keap1 Promotes Neuronal Senescence via Inhibiting Ubiquitination of β-Catenin

2022 ◽  
Author(s):  
Yao Zhang ◽  
Jiazhao Xie ◽  
Yan-li Jiang ◽  
Shao-juan Yang ◽  
Hui Wei ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Wilms Tumor ◽  
Quantitative Polymerase Chain Reaction ◽  
Elevated Serum ◽  
N2a Cells ◽  
Promising Target ◽  
Polymerase Chain ◽  
High Level ◽  
Related Proteins

Abstract Background Elevated serum homocysteine (Hcy) is an independent risk factor of Alzheimer’s disease (AD). It has been reported that Hcy dramatically accelerates the aging of endothelial progenitor cells or endothelial cells. However, whether and how Hcy produces neuronal senescence is largely unknown. Methods Mouse neuroblastma 2a (N2a) cells were treated with Hcy, and senescence-associated β-galactosidase (SA-β-gal) staining was applied to assay senescence. Senescent markers and related proteins were examined by western blot, quantitative Polymerase Chain Reaction (qPCR), immunofluorescence staining. Methylation of promoter was assay by bisulfite sequencing PCR (BSP). Immunoprecipitation (IP) was applied to examine association between proteins. Rats were injected with homocysteine and examined neuronal senescence. Results In this study, we observed that Hcy significantly promoted the senescence of N2a cells with elevated β-catenin and Kelch like ECH-associated protein 1 (Keap1). Intriguingly, Hcy increased the interaction between Keap1 and Wilms tumor gene on X chromosome (WTX), but decreased β-catenin-WTX interaction simultaneously. Mechanistically, Hcy attenuated the methylation level of Keap1 promoter’s CqG island and activated the transcription of Keap1. While, slow degradation rate rather than transcriptional activation contributed to the high level of β-catenin. Hcy-increased Keap1 competed with β-catenin to bind to WTX. Knockdown of β-catenin and Keap1 both attenuated Hcy-induced senescence of N2a cells. Hcy-induced rats model also showed neuronal senescence in cortex along with elevated senescent markers. Conclusions Our data highlight a crucial role of Keap1-β-catenin pathway in Hcy-induced neuronal-like senescence and provide a promising target for AD treatment.

scholarly journals Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

2021 ◽  
Author(s):  
Carlos dos Santos ◽  
Kézia de Oliveira ◽  
Geovana Mendes ◽  
Lívia Silva ◽  
Marcio de Souza Jr. ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Asymptomatic Carrier ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Diagnostic Methods ◽  
Public And Private ◽  
Polymerase Chain ◽  
Increasing Demand

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

scholarly journals Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

2020 ◽  
Author(s):  
Karel Bezstarosti ◽  
Mart M. Lamers ◽  
Jeroen J. A. van Kampen ◽  
Bart L. Haagmans ◽  
Jeroen A. A. Demmers
Keyword(s):  
Mass Spectrometry ◽  
Potential Role ◽  
Limit Of Detection ◽  
Viral Proteins ◽  
Clinical Samples ◽  
Proof Of Concept ◽  
Specific Detection ◽  
Clinical Specimens ◽  
Parallel Reaction Monitoring

ABSTRACTThe rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. Here, we explore the potential of targeted mass spectrometry based proteomics for the detection of SARS-CoV-2 proteins in both research and clinical samples. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) mass spectrometry. For Nucleocapsid the limit of detection was found to be in the mid-attomole range (0.9 x 10−12 g). Next, we apply this PRM assay to the detection of viral proteins in in vitro mucus substitutes, as well as in various clinical specimens such as nasopharyngeal swabs and sputum. In this proof-of-concept study SARS-CoV-2 proteins could unambiguously be detected in clinical samples, suggesting that the sensitivity of this technology may be sufficiently high to further explore its potential role in diagnostics.

scholarly journals Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259165
Author(s):  
Karel Bezstarosti ◽  
Mart M. Lamers ◽  
Wouter A. S. Doff ◽  
Peter C. Wever ◽  
Khoa T. D. Thai ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Viral Proteins ◽  
Reaction Monitoring ◽  
Proof Of Concept ◽  
Specific Detection ◽  
Clinical Specimens ◽  
Additional Tool ◽  
Parallel Reaction Monitoring

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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