scholarly journals COMPARISON EFFICACY OF ITS AND 18S rDNA PRIMERS FOR DETECTION OF FUNGAL DIVERSITY IN COMPOST MATERIAL BY PCR-DGGE TECHNIQUE

2018 ◽  
Vol 15 (4) ◽  
pp. 729-735
Author(s):  
Pham Ngoc Tu Anh ◽  
Pham Thi Thu Hang ◽  
Le Thi Quynh Tram ◽  
Nguyen Thanh Minh ◽  
Dinh Hoang Dang Khoa
Keyword(s):  
Fungal Diversity ◽  
Pcr Amplification ◽  
Fungal Communities ◽  
Variable Regions ◽  
Pcr Inhibitors ◽  
Composting Process ◽  
Pcr Products ◽  
Pure Cultures ◽  
Primer Sets ◽  
Aspergillus Sp

Through composting process, biosolid wastes are gradually transformed into compost material which can be used as soil fertilizer. Among microorganisms involved in composting process, fungi play important roles because they break down complex substrates, such as ligno-cellulose. Recently, PCR-DGGE technique has been considered as a useful tool for analysis of fungal diversity in environmental samples. Among other factors, primer set selection is necessary for successful of the PCR-DGGE analysis. There are several PCR primer sets targeting fungal variable regions of 18S ribosomal DNA (rDNA) and internal transcribed spacer (ITS) for the use in community analyses, however there exist just few reports on efficacy of these primers in studying fungal communities in compost materials. In this study, four different primer sets were tested, including EF4/Fung5 (followed by EF4/NS2-GC), EF4/ITS4 (followed by ITS1F-GC/ITS2), NS1/GC-Fung, and FF390/FR1-GC. Extracted DNA from compost materials often contains co-extracted humic substances and other PCR inhibitors. Therefore, the primers were tested for (i) tolerance to the PCR inhibitors presenting in the DNA extracted from compost materials, and (ii) efficacy and specificity of the PCR. The results showed that of the four primer sets, only FF390/FR1-GC achieved both criteria tested whereas the other three did not, i.e. primer EF4/ITS4 had low tolerance to PCR inhibitors, primers EF4/Fung5 was low in PCR amplification efficacy, whereas primers EF4/ITS4 created unspecific products. DGGE analyses of PCR products amplified with the primer set FF390/FR1-GC showed single bands for reference pure cultures Penicillium sp., Aspergillus sp., and Trichoderma sp., as well as distinctly separated bands for the fungal communities of three different composting materials. Thus, the primer set FF390/FR1-GC could be suitable for studying structure and dynamic of fungal communities in compost materials.

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

1999 ◽  
Vol 65 (6) ◽  
pp. 2307-2311 ◽  
Author(s):  
R. R. de Moraes ◽  
J. E. Maruniak ◽  
J. E. Funderburk
Keyword(s):  
Laboratory Experiments ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Soil Samples ◽  
Anticarsia Gemmatalis ◽  
Isolation Method ◽  
Viral Dna ◽  
Pcr Inhibitors ◽  
Magnetic Capture ◽  
Pcr Products

ABSTRACT Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.

scholarly journals A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato

Plant Disease ◽  
2016 ◽  
Vol 100 (8) ◽  
pp. 1660-1668 ◽  
Author(s):  
A. L. Strayer ◽  
A. Jeyaprakash ◽  
G. V. Minsavage ◽  
S. Timilsina ◽  
G. E. Vallad ◽  
...  
Keyword(s):  
Real Time ◽  
Infected Plant ◽  
Multiplex Assay ◽  
Bacterial Spot ◽  
Pcr Assay ◽  
Tomato Production ◽  
Xanthomonas Species ◽  
Pcr Products ◽  
Pure Cultures ◽  
Primer Sets

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

scholarly journals Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

2005 ◽  
Vol 71 (9) ◽  
pp. 5494-5500 ◽  
Author(s):  
Susan M. Barns ◽  
Christy C. Grow ◽  
Richard T. Okinaka ◽  
Paul Keim ◽  
Cheryl R. Kuske
Keyword(s):  
Related Species ◽  
Environmental Samples ◽  
Pcr Amplification ◽  
Soil Samples ◽  
Rrna Gene ◽  
New Subspecies ◽  
Pcr Products ◽  
Primer Sets ◽  
Initial Survey ◽  
Close Relatives

ABSTRACT Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

scholarly journals Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

1997 ◽  
Vol 87 (12) ◽  
pp. 1192-1196 ◽  
Author(s):  
M. Sato ◽  
K. Watanabe ◽  
M. Yazawa ◽  
Y. Takikawa ◽  
K. Nishiyama
Keyword(s):  
Pseudomonas Syringae ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enzyme Gene ◽  
Indigenous Plasmid ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Primer Sets

Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the “kudzu strain”) and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

scholarly journals Genome size and microsatellites: the effect of nuclear size on amplification potential

Genome ◽  
2002 ◽  
Vol 45 (1) ◽  
pp. 212-215 ◽  
Author(s):  
Trenton WJ Garner
Keyword(s):  
Genome Size ◽  
Microsatellite Dna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Amplification Success ◽  
Pcr Products ◽  
Negative Effect ◽  
C Value ◽  
Primer Sets ◽  
Using Data

Although the frequency of microsatellite DNA regions generally increases with increasing genome size, genome size has a negative effect on polymerase chain reaction (PCR) amplification. Thus, researchers developing sets of PCR primers, as is commonly done for microsatellite DNA regions, may encounter greater difficulty when working with species that have larger genomes. I investigated the effect of genome size on overall amplification success using data from nine different metazoan taxa. The proportion of primer sets that did not amplify PCR products was strongly and positively correlated with the haploid C value of the target species. Increasing genome size may affect amplification success negatively because of a decrease in target:nontarget DNA or by dilution of the available primer pool by nonspecific binding.Key words: microsatellites, genome size, amplification success.

scholarly journals Selection and Experimental Evaluation of Universal Primers to Study the Fungal Microbiome of Higher Plants

2018 ◽  
Vol 2 (4) ◽  
pp. 225-236 ◽  
Author(s):  
Silvia Scibetta ◽  
Leonardo Schena ◽  
Ahmed Abdelfattah ◽  
Sonia Pangallo ◽  
Santa O. Cacciola
Keyword(s):  
Fungal Diversity ◽  
Higher Plants ◽  
Fungal Communities ◽  
Universal Primers ◽  
Its2 Region ◽  
Its1 Region ◽  
Perfect Set ◽  
Fungal Microbiome ◽  
Primer Sets ◽  
The Impact

The impact of primer choice on results of metabarcoding studies was experimentally evaluated by analyzing fungal communities associated with leaves of four plant species. Significant differences in target specificity of primers were highlighted by a percentage of plant reads ranging from almost nothing to 30 to 35% of the total detected sequences. Overall, primer sets targeting the internal transcribed spacer 1 (ITS1) region proved to be more specific than those targeting the ITS2 region. A comparable taxa coverage was revealed for all investigated primer sets. However, each primer set detected only around 50% of the overall detected taxa highlighting that a consistent part of the actual fungal diversity remains undetected in studies conducted using a single couple of primers. The coverage was increased to 70 to 80% by combining results from two different primer sets. Some fungal taxa were preferentially or exclusively detected by certain primer sets and this association between primers and taxa was generally recurrent on several plant hosts. Data highlighted that a perfect set of primers to investigate the whole fungal diversity does not exist and that whatever the choice, only a fraction of the actual microbial diversity will be investigated. However, provided information may be valuable to select the best primers according to the objective of the analysis.

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

scholarly journals Are Wildfires a Threat to Fungi in European Pinus Forests? A Case Study of Boreal and Mediterranean Forests

Forests ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 309 ◽  
Author(s):  
Iván Franco-Manchón ◽  
Kauko Salo ◽  
Juan Oria-de-Rueda ◽  
José Bonet ◽  
Pablo Martín-Pinto
Keyword(s):  
Boreal Forests ◽  
Fungal Diversity ◽  
Community Dynamics ◽  
Forest Products ◽  
Climatic Conditions ◽  
Fungal Communities ◽  
Mediterranean Forests ◽  
Natural Forests ◽  
Edible Fungi ◽  
Pinus Species

Natural forests and plantations of Pinus are ecologically and economically important worldwide, producing an array of goods and services, including the provision of non-wood forest products. Pinus species play an important role in Mediterranean and boreal forests. Although Pinus species seem to show an ecological adaptation to recurrent wildfires, a new era of mega fires is predicted, owing to climate changes associated with global warming. As a consequence, fungal communities, which are key players in forest ecosystems, could be strongly affected by these wildfires. The aim of this study was to observe the fungal community dynamics, and particularly the edible fungi, in maritime (Pinus pinaster Ait.), austrian pine (Pinus nigra J.F. Arnold), and scots pine (Pinus sylvestris L.) forests growing under wet Mediterranean, dry Mediterranean, and boreal climatic conditions, respectively, by comparing the mushrooms produced in severely burned Pinus forests in each area. Sporocarps were collected during the main sampling campaigns in non-burned plots, and in burned plots one year and five years after fire. A total of 182 taxa, belonging to 81 genera, were collected from the sampled plots, indicating a high level of fungal diversity in these pine forests, independent of the climatic conditions. The composition of the fungal communities was strongly affected by wildfire. Mycorrhizal taxa were impacted more severely by wildfire than the saprotrophic taxa, particularly in boreal forests—no mycorrhizal taxa were observed in the year following fire in boreal forests. Based on our observations, it seems that fungal communities of boreal P. sylvestris forests are not as adapted to high-intensity fires as the Mediterranean fungal communities of P. nigra and P. pinaster forests. This will have an impact on reducing fungal diversity and potential incomes in rural economically depressed areas that depend on income from foraged edible fungi, one of the most important non-wood forest products.

scholarly journals Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

2006 ◽  
Vol 72 (7) ◽  
pp. 4995-5001 ◽  
Author(s):  
Feng Chen ◽  
Kui Wang ◽  
Jeneen Stewart ◽  
Robert Belas
Keyword(s):  
Mitomycin C ◽  
Marine Bacterium ◽  
Pcr Amplification ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Genomes ◽  
Dominant Type ◽  
Transmission Electron ◽  
Viral Particles ◽  
Genomic Approach ◽  
Primer Sets

ABSTRACT Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2630-2640 ◽  
Author(s):  
J. T. Tambong ◽  
R. Xu ◽  
E. S. P. Bromfield
Keyword(s):  
Root Zone ◽  
Phylogenetic Analyses ◽  
Melting Curve ◽  
Pcr Amplification ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Its1 Region ◽  
A Genome ◽  
Pure Cultures ◽  
Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

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