Search results for " PCR"

Mixed infection of Pectobacterium carotovorum subsp. Carotovorum and P. carotovorum subsp. Brasiliensis in tomato stem rot in Italy

2016

Grafted tomato plants grown in a soilless culture system in heated greenhouses in Sicily were affected by stem rot disease. Symptoms consisted of dark brown longitudinal discoloration of the basal part of the stem and petioles. Longitudinal sections of the stem revealed brown watersoaked or soft-rotted pith tissue and internal vascular discoloration. Bacterial isolates with two different colony morphologies were obtained from symptomatic tissues. Isolates were identified as Pectobacterium spp. on the basis of biochemical and molecular analyses. The analysis of the 16S rRNA gene sequence and of the concatenated sequences of the housekeeping genes rpoS and pgi revealed that the isolates of th…

Seasonal Human Coronavirus Respiratory Tract Infection in Recipients of Allogeneic Hematopoietic Stem Cell Transplantation

2021

Abstract Background Little is known about characteristics of seasonal human coronaviruses (HCoVs) (NL63, 229E, OC43, and HKU1) after allogeneic stem cell transplantation (allo-HSCT). Methods This was a collaborative Spanish and European bone marrow transplantation retrospective multicenter study, which included allo-HSCT recipients (adults and children) with upper respiratory tract disease (URTD) and/or lower respiratory tract disease (LRTD) caused by seasonal HCoV diagnosed through multiplex polymerase chain reaction assays from January 2012 to January 2019. Results We included 402 allo-HSCT recipients who developed 449 HCoV URTD/LRTD episodes. Median age of recipients was 46 years (range,…

Environmental DNA analysis indicates that migration barriers are decreasing the occurrence of European eel (Anguilla anguilla) in distance from the s…

2020

Abstract The European eel (Anguilla L.) is considered critically endangered by the IUCN Red List, and recruitment remains low. One of the challenges for the species today is migration barriers that limit their habitat. Along the Norwegian coast, like in other countries, the abundance of eels appears to decrease with distance from the sea. This pattern may be a result of factors like water temperature, water quality, competition, and habitat suitability and availability. This study aims to use environmental DNA (eDNA) analysis to investigate the potential relationship between migration barriers and the decreasing occurrence of eels in distance from the sea by the coast of southern Norway. Si…

Estimating trematode prevalence in snail hosts using a single-step duplex PCR: how badly does cercarial shedding underestimate infection rates?

2014

Background: Trematode communities often consist of different species exploiting the same host population, with two or more trematodes sometimes co-occuring in the same host. A commonly used diagnostic method to detect larval trematode infections in snails has been based on cercarial shedding, though it is often criticized as inaccurate. In the present study we compare infection prevalences determined by cercarial emission with those determined, for the first time, by molecular methods, allowing us to quantify the underestimation of single and double infections based on cercarial emission. We thus developed a duplex PCR for two host-parasite systems, to specifically differentiate between sin…

Quantification of PCR products by phosphate measurement

2008

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as beta-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laborato…

Molecular diagnostic tools for the detection and characterization of Phoma tracheiphila

2011

In the recent years PCR-based techniques for the identification and detection of Phoma tracheiphila, the causal agent of citrus mal secco disease, have been evaluated aiming to provide tools for biological and epidemiological studies. A wide collection of P. tracheiphila strains was used to evaluate and validate diagnostic protocols and a fAFLP method for fungal characterization. Conventional and real-time PCR protocols were successfully tested for the specific identification of P. tracheiphila and its detection in planta. A further improvement of the real-time PCR protocol and the DNA extraction methods allowed the quantification of the fungus both from naturally infected and artificially …

Polymerase Chain Reaction (PCR)

2008

Quality controls for cell cultures: identification of interspecies cross-contamination by PCR-RFLP analysis of the cytochrome b gene

2012

Cross-contaminations of a cell line with cells of different species represent a potential risk in laboratories handling human and animal cells. Therefore, it is necessary to control such contaminations. Tests based on mitochondrial DNA (mtDNA) are used in forensic analysis, phylogenetic studies and in food authentication. However, the use of mtDNA in quality controls of cell cultures is recent. Mitochondrial sequence differences of closely related animal species are five- to tenfold higher than those of nuclear genes. On the contrary, intraspecies variation in mitochondrial sequences is low in most animal species. Moreover, each cell contains 100–10.000 mitochondrial genomes. The amount of …

Study of variability of an endophytic acremonium population in symptomless grapevine

2009

The endophytism of genus Acremonium in symptomless grapevines of several cultivars, grown both in pots or in the field, is reported. The no-tillage management of tested grapevines and the lack of chemical treatments facilitated the investigation of the ecological plant-fungus interaction. The isolates were differentiated at the strain level by vegetative compatibility assay and randomly amplified polymorphic DNA (RAPD) analyses, using a set of four oligo-nucleotide primers. The endophytic Acremonium population in the healthy grapevine object of the survey seemed to show a low variability; all the techniques agreed in grouping the Acremonium strains in three different clusters and no relatio…

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance

2022

Background & Aims Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen wi…