scholarly journals Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

Plant Disease ◽  
2020 ◽  
Vol 104 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Joseph D. Carrillo ◽  
Joey S. Mayorquin ◽  
Jason E. Stajich ◽  
Akif Eskalen
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Absolute Quantification ◽  
Coefficient Of Determination ◽  
Fungal Isolation ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Symbiotic Fungi ◽  
Fusarium Dieback ◽  
Platanus Racemosa

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
Author(s):  
Hanah Kim ◽  
Mina Hur ◽  
Eunsin Bae ◽  
Kyung-A Lee ◽  
Woo-In Lee
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Nucleic Acid Amplification ◽  
Coefficient Of Determination ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Cobas Taqman ◽  
Limit Of Quantitation ◽  
Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

scholarly journals Detection of Differential Host Susceptibility to the Marine Oomycete Pathogen Eurychasma dicksonii by Real-Time PCR: Not All Algae Are Equal

2008 ◽  
Vol 75 (2) ◽  
pp. 322-328 ◽  
Author(s):  
Claire M. M. Gachon ◽  
Martina Strittmatter ◽  
Dieter G. Müller ◽  
Julia Kleinteich ◽  
Frithjof C. Küpper
Keyword(s):  
Real Time ◽  
Brown Algae ◽  
Large Scale ◽  
Natural Populations ◽  
Host Susceptibility ◽  
Ecosystem Structure ◽  
Ectocarpus Siliculosus ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Oomycete Pathogen

ABSTRACT In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen Eurychasma dicksonii in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against Eurychasma, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of Eurychasma in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed Ectocarpus siliculosus to improve our understanding of brown algal diseases.

scholarly journals Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies

2010 ◽  
Vol 77 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Ana Palacio-Bielsa ◽  
Jaime Cubero ◽  
Miguel A. Cambra ◽  
Raquel Collados ◽  
Isabel M. Berruete ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Plant Material ◽  
Pathogen Detection ◽  
Plant Protection ◽  
The European Union ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Mediterranean Plant ◽  
Different Origins

ABSTRACTXanthomonas arboricolapv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as beingX. arboricolapv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system inX. arboricolapv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed forX. arboricolapv. pruni strains from different origins as well as for closely relatedXanthomonasspecies, non-Xanthomonasspecies, saprophytic bacteria, and healthyPrunussamples. The efficiency of the developed protocol was evaluated with field samples of 14Prunusspecies and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, andX. arboricolapv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test forX. arboricolapv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

scholarly journals Comparison of RNA-Based Versus DNA-Based Quantitative KIT D816V Mutation Analysis Reveals Significant Disparity in Patients with Advanced Systemic Mastocytosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2955-2955
Author(s):  
Mohamad Jawhar ◽  
Nicole Naumann ◽  
Johannes Lübke ◽  
Juliana Schwaab ◽  
Oliver Hofmann ◽  
...  
Keyword(s):  
Real Time ◽  
Research Funding ◽  
Limit Of Detection ◽  
Systemic Mastocytosis ◽  
Digital Pcr ◽  
Coefficient Of Determination ◽  
Lower Percentage ◽  
Molecular Profile ◽  
Number Of Patients ◽  
Kit D816v

Systemic mastocytosis (SM) is characterized by activating mutations in KIT, usually KIT D816V in >90% of patients. According to the WHO classification, detection of KIT D816V is a minor diagnostic criterion. Moreover, a reduction of the RNA-based KIT D816V expressed allele burden (EAB) of ≥25% at month 6 is a favorable marker for improved survival in midostaurin-treated advanced SM (AdvSM) patients. However, only limited data exist upon the comparison between RNA-based and DNA-based quantitative KIT D816V mutation analyses. We therefore collected peripheral blood samples from 161 SM patients (indolent SM [ISM], n=40; AdvSM, n=121) at time of referral. We applied a real time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for assessment of the KIT D816V EAB and a chip-based digital PCR (dPCR) for the quantification of the DNA-based KIT D816V variant allele frequency (VAF, QuantStudio 3D dPCR System, ThermoFisher Scientific, Massachusetts, USA). The limit of detection (LOD) was assessed through serial dilution experiments with DNA from healthy individuals and DNA from a SM patient with a KIT D816V VAF of approximately 50%. All samples were analyzed in two independent dPCR runs. In an inital round-robin test for an inter-laboratory (n=4) comparison on 30 DNA samples (ISM, n=7; AdvSM, n=19), we found a very good correlation between 3 different chip-based digital PCR systems (dPCR; droplet-based dPCR, ddPCR, BioRad Laboratories, California, USA; quantitative real-time PCR, qPCR, California, USA; dPCR vs. ddPCR r=0.99, R2=0.99; dPCR vs. qPCR r=0.99, R2=0.98). In ISM patients, the comparison between EAB and VAF revealed a strong linear relationship with a correlation (r) of 0.91 and a coefficient of determination (R2) of 0.84, which was significantly inferior (r=0.71; R2=0.5) in AdvSM patients. In 45/121 (37%) and 76/121 (63%) of AdvSM patients, the EAB/VAF ratio was ≤2 (cohort A) or >2 (cohort B). To confirm the significant disparity between EAB and VAF in individual patients, serial analyses of at least 3 samples in 12 patients revealed a stable EAB/VAF ratio during follow-up. The comparison between cohort A and cohort B revealed significant differences in terms of a higher median hemoglobin level (p=0.006), a lower percentage of patients with hemoglobin <10g/dL (p=0.02), a lower median monocyte level (p=0.01), a lower median alkaline phosphatase level (p=0.03), and a lower number of patients with a high risk molecular profile (at least one gene mutation in SRSF2, ASXL1, and/or RUNX1, S/A/R, p=0.02) in cohort A. Moreover, patients of cohort A had a significantly better overall survival (OS) (median OS 12.9 versus 3.3 years; hazard ratio 2.1; 95% confidence interval 1.2-3.5; p=0.005; Figure). We conclude that a) KIT D816V EAB and VAF are significantly different in AdvSM patients but not in ISM and b) AdvSM patients with an EAB/VAF ratio >2 present with an aggressive phenotype and adverse prognosis as compared to patients with EAB/VAF ratio ≤2. We therefore recommend to routinely determine KIT D816V EAB and VAF in AdvSM patients. Disclosures Fabarius: Novartis: Research Funding. Reiter:Blueprint: Consultancy, Honoraria, Other: Travel reimbursement; Deciphera: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement, Research Funding.

scholarly journals Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens

2020 ◽  
Author(s):  
Estefania Nunez-Bajo ◽  
Michael Kasimatis ◽  
Yasin Cotur ◽  
Tarek Asfour ◽  
Alex Collins ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Low Cost ◽  
High Specificity ◽  
Negative Temperature ◽  
Sample Solution ◽  
Rapid Screening ◽  
Battery Capacity ◽  
Field Samples ◽  
Silicon Based

AbstractRapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention e.g., drug therapy, quarantine, no action etc. when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2 (the pathogen causing COVID-19), and shows no or similar symptoms to other common infections. We report a silicon-based integrated Point-of-Need (PoN) transducer (TriSilix) that can chemically-amplify and detect pathogen-specific sequences of nucleic acids (NA) quantitatively in real-time. Unlike other silicon-based technologies, TriSilix can be produced at wafer-scale in a standard laboratory; we have developed a series of methodologies based on metal-assisted chemical (wet) etching, electroplating, thermal bonding and laser-cutting to enable a cleanroom-free low-cost fabrication that does not require processing in an advanced semiconductor foundry. TriSilix is, therefore, resilient to disruptions in the global supply chain as the devices can be produced anywhere in the world. To create an ultra-low-cost device, the architecture proposed exploits the intrinsic properties of silicon and integrates three modes of operation in a single chip: i) electrical (Joule) heater, ii) temperature sensor (i.e. thermistor) with a negative temperature coefficient that can provide the precise temperature of the sample solution during reaction and iii) electrochemical sensor for detecting target NA. Using TriSilix, the sample solution can be maintained at a single, specific temperature (needed for isothermal amplification of NA such as Recombinase Polymerase Amplification (RPA) or cycled between different temperatures (with a precision of ±1.3°C) for Polymerase Chain Reaction (PCR) while the exact concentration of amplicons is measured quantitatively and in real-time electrochemically. A single 4-inch Si wafer yields 37 TriSilix chips of 10×10×0.65 mm in size and can be produced in 7 hours, costing ~US $0.35 per device. The system is operated digitally, portable and low power – capable of running up to 35 tests with a 4000 mAh battery (a typical battery capacity of a modern smartphone). We were able to quantitatively detect a 563-bp fragment (Insertion Sequence IS900) of the genomic DNA of M. avium subsp. paratuberculosis (extracted from cultured field samples) through PCR in real-time with a Limit-of-Detection of 20 fg, equivalent to a single bacterium, at the 30th cycle. Using TriSilix, we also detected the cDNA from SARS-CoV-2 (1 pg), through PCR, with high specificity against SARS-CoV (2003).

scholarly journals Identification of Selected Tuna Species in Commercial Products

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1137
Author(s):  
Eliska Servusova ◽  
Zora Piskata
Keyword(s):  
Real Time ◽  
Muscle Tissue ◽  
Internal Control ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Fish Muscle ◽  
Coefficient Of Determination ◽  
12S Rrna ◽  
Rrna Gene ◽  
Additional Species

This study was conducted to develop systems for the identification of four tuna species (skipjack tuna Katsuwonus pelamis, yellowfin tuna Thunnus albacares, bullet tuna Auxis sp. and Atlantic bonito Sarda sp). At first, raw samples of these species and a mix intended as internal control were prepared for the authentication of fish muscle tissue of the genus Thunnus sp., Auxis sp. and Sarda sp. DNA from raw muscle tissue, the mix and samples was extracted with the DNeasy mericon Food Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of DNA in raw samples were evaluated using a spectrophotometer. Primers and probe sequences were specifically designed to identify the selected species. In addition, primers and a probe for the endogenous 12S rRNA gene were designed to determine the presence of amplifiable fish (especially tuna) DNA in samples. Furthermore, the species specificity of the designed primers and probes was verified in DNA samples of various tuna and bonito species. Limit of detection for the selected species was calculated as well as the coefficient of determination R2 and efficiency of real-time PCR testing was determined. To evaluate the developed real-time PCR methods, 70 commercial tuna products were analysed. The results show that mislabelling of fish products can still be encountered and, moreover, the presence of an additional species can be identified.

scholarly journals Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus

2021 ◽  
Vol 8 ◽  
Author(s):  
Yi-Lun Xie ◽  
Dian-Hong Lv ◽  
Xiao-Hui Wen ◽  
Qi Zhai ◽  
Man-Lin Luo ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Foot And Mouth Disease ◽  
Coefficient Of Determination ◽  
Potential Contribution ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Standard Curve ◽  
Mouth Disease Virus ◽  
B Virus

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/μl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

Sign in / Sign up

Export Citation Format

Share Document