Search results for "primers"
showing 10 items of 332 documents
Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology
2020
In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview r…
Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants.
2015
In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firm…
Exploring species-level taxonomy in the Cryptocephalus flavipes species complex (Coleoptera: Chrysomelidae)
2016
In insects, morphological species identification is often challenging. The discrimination of closely related species may be hampered when only subtle differences in phenotypic characters or a continuum in their variability are present. This is exemplified in the Cryptocephalus flavipes species complex (Coleoptera; Chrysomelidae) where, until now, the species have been discriminated only by the yellow pattern on frons, pronotum, and epipleurae. In the present study, the phylogeny of the C. flavipes species complex was resolved through a multi-locus sequence approach, and the inclusion in the group of the phenotypically similar Cryptocephalus quadripustulatus Gyllenhal, 1813 was evaluated. Su…
Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in si…
2008
International audience; Gene expression profiling based on tissue extracts gives only limited information about genes associated with complex developmental processes such as those implicated in fungal interactions with plant roots during arbuscular mycorrhiza development and function. To overcome this drawback, a direct fluorescent in situ RT-PCR methodology was developed for spatial mapping of gene expression in different presymbiotic and symbiotic structures of an arbuscular mycorrhizal fungus. Transcript detection was optimized by targeting the LSU rRNA gene of Glomus intraradices and monitoring expression of a stearoyl-CoA-desaturase gene that is consistently expressed at high levels in…
Multilocus sequence typing confirms the close genetic inter-relatedness between three distinct flavescence doree phytoplasma strain clusters and grou…
2007
ABSTRACT Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus . To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB - degV , along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the…
Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment
2004
Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for t…
Identification of Candida auris and related species by multiplex PCR based on unique GPI protein‐encoding genes
2020
Background The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, however, the fungus has often been misidentified by commercial systems. Objectives To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol-modified protein-encoding genes. Methods Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, …
Directional high-throughput sequencing of RNAs without gene-specific primers.
2018
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5′-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding. The sequencing results of a phytoplankton mock community showed a highly similar profile to the biomass indicators. This method…
Generation of a novel next-generation sequencing-based method for the isolation of new human papillomavirus types
2018
Abstract With the advent of new molecular tools, the discovery of new papillomaviruses (PVs) has accelerated during the past decade, enabling the expansion of knowledge about the viral populations that inhabit the human body. Human PVs (HPVs) are etiologically linked to benign or malignant lesions of the skin and mucosa. The detection of HPV types can vary widely, depending mainly on the methodology and the quality of the biological sample. Next-generation sequencing is one of the most powerful tools, enabling the discovery of novel viruses in a wide range of biological material. Here, we report a novel protocol for the detection of known and unknown HPV types in human skin and oral gargle …
Fast detection of Southern tomato virus by one-step transcription loop-mediated isothermal amplification (RT-LAMP)
2017
Southern tomato virus (STV) is a double stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae) which has been detected in tomato plants showing stunting, fruit discoloration and size reduction. A one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of STV in total RNA or sap extracts (obtained just by grinding in buffer) from STV-infected tomato plants by using a set of three primers pairs which were designed to the sequence of the STV putative coat protein. Amplification products were visualized by gel electrophoresis or direct staining of DNA. The sensitivity of RT-LAMP was identical to that of th…