Search results for " PCR"
showing 10 items of 153 documents
A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis.
2012
The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogen-infected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a h…
Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction.
2006
Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, wh…
Phenotypic and genotypic evaluation of slime production by conventional and molecular microbiological techniques.
2009
Twenty-nine staphylococcal isolates from different clinical samples were tested for slime production: phenotypic characterization was carried out using Christensen test (tube test) and Congo red agar plate test (CRA plate test), while the presence and expression of icaA and icaD genes were evaluated by real-time PCR. In 79.3% of studied strains there was a concordance between slime production and presence of icaA and icaD genes, and between lack of slime production and absence of both or only one of the tested genes. In four of five strains where positive phenotype was not associated with the presence of ica genes, gene co-expression (evaluated by mRNA determination) was lacking, while in o…
Comparative evaluation of molecular methods for the quantitative measure of torquetenovirus (TTV) viremia, the new surrogate marker of immune compete…
2019
Background Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune-related complications, and eventually to customize immunosuppression. Methods In this study, 327 blood samples were tested using two real-time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in-house rtPCR developed by our group, the second one was the recently marketed TTV R-GENE assay. Results In the validation study, the TTV R-GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in-house rtPCR. The Bl…
The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics
2014
The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. …
Genetic potential, diversity and activity of an atrazine-degrading community enriched from a herbicide factory effluent
2008
Aims: To characterize an atrazine-degrading bacterial community enriched from the wastewater of a herbicide factory. Methods and Results: The community mineralized 81·4 ± 1·9% of [14C-ring]atrazine and 31·0 ± 1·8% of [14C-ethyl]atrazine within 6 days of batch cultivation in mineral salts medium containing atrazine as the sole nitrogen source. Degradation activity of the community towards different chloro- and methylthio-substituted s-triazine compounds was also demonstrated. Restriction analysis of amplified 16S rDNA revealed high diversity of bacterial populations forming the community, with Pseudomonas species dominating in the clone library. Atrazine-degrading genetic potential of the …
PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.
2004
D . E R C O L I N I , G . M A U R I E L L O , G . B L A I O T T A , G . M O S C H E T T I A N D S . C O P P O L A . 2003. Aims: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. Methods and Results: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates…
Fast and low-cost decentralized surveillance of transmission of tuberculosis based on strain-specific PCRs tailored from whole genome sequencing data…
2015
Molecular epidemiology has transformed our knowledge of how tuberculosis (TB) is transmitted. Whole genome sequencing (WGS) has reached unprecedented levels of accuracy. However, it has increased technical requirements and costs, and analysis of data delays results. Our objective was to find a way to reconcile speed and ease of implementation with the high resolution of WGS. The targeted regional allele-specific oligonucleotide PCR (TRAP) assay presented here is based on allele-specific PCR targeting strain-specific single nucleotide polymorphisms, identified from WGS, and makes it possible to track actively transmitted Mycobacterium tuberculosis strains. A TRAP assay was optimized to track…
Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR.
2005
ABSTRACT A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum , Lactobacillus…
Impact of maize mucilage on atrazine mineralization andatzC abundance
2005
Soil was amended with maize mucilage, a major rhizodeposit, to study its role on the number of culturable soil micro-organisms, the structure of the bacterial community, atrazine mineralization and atzC abundance. The maximal percentage of atrazine mineralization was lower for mucilage-amended than for water-amended soil. Total culturable soil bacteria and 16S rDNA copy number, measured by RT-PCR, presented similar values and were not significantly (P < 0.05) different among treatments. Mucilage applied at a rate of 70 mu g C g(-1) dry soil day(-1) over two weeks did not modify the abundance of the total soil microflora. Global structure of soil bacterial communities revealed by RISA analys…