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.

Development of Ancient DNA Isolation Method for Improved PCR Amplification

2007 ◽  
Vol 20 (4) ◽  
pp. 267
Author(s):  
Ki Jeong Kim ◽  
Ariunaa Togloom ◽  
Eun Hee Jeon ◽  
Min Soo Lee ◽  
Youn Ock Cho ◽  
...  
Keyword(s):  
Ancient Dna ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Isolation Method

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.

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.

Detection ofEchinococcus multilocularisDNA in fox faeces using DNA amplification

Parasitology ◽  
1993 ◽  
Vol 106 (2) ◽  
pp. 193-199 ◽  
Author(s):  
S. Bretagne ◽  
J. P. Guillou ◽  
M. Morand ◽  
R. Houin
Keyword(s):  
Dna Isolation ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Target Sequence ◽  
Microscopical Examination ◽  
Dna Variation ◽  
Faecal Samples ◽  
U1 Snrna ◽  
Ethidium Bromide Staining ◽  
Pcr Products

SUMMARYIn order to identifyEchinococcus multilocularisDNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol–chloroform extraction and a purification step on a matrix (Prep-A-Gene®). The target sequence for amplification was theE. multilocularisU1 snRNA gene. PCR products were indistinguishable for 32 differentE. multilocularisisolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, includingE. granulosus.The sensitivity of amplification was monitored by the addition ofE. multilocularisDNA or eggs to faeces free ofE. multilocularisand was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which noE. multilocularisadult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouringE. multilocularisadult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosusandE. multilocularis), or to DNA variation between different isolates ofE. multilocularis. Further work in the field should be initiated in order to confirm these results.

scholarly journals A Begomovirus Isolated from Chlorotic and Stunted Soybean Plants in Mexico is a New Strain of Rhynchosia golden mosaic virus

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 972-972 ◽  
Author(s):  
J. Méndez-Lozano ◽  
L. L. Perea-Araujo ◽  
R. D. Ruelas-Ayala ◽  
N. E. Leyva-López ◽  
J. A. Mauricio-Castillo ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Dna Sequences ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Nucleotide Identity ◽  
Degenerate Primers ◽  
Viral Dna ◽  
Field Samples ◽  
Pcr Products ◽  
Soybean Plants

Soybean (Glycine max Merr.) is an alternative crop during the summer in Sinaloa, a northern state of Mexico. During the last 4 years, symptoms of yellowing, curled leaves, and stunting have been observed on soybean plantings, and a scrutiny of field samples collected in 2003 identified a begomovirus related to Pepper golden mosaic virus in symptomatic plants (4). A new survey was conducted during the summer of 2004 when the soybean disease was prevalent in the region. Affected plants appeared as patches displaying symptoms ranging from mild to severe yellow mosaic with leaf deformation and stunted growth in several parcels of commercial fields of northern Sinaloa. More than 100 samples from symptomatic soybean plants and weeds growing within the same fields were collected and analyzed for the presence of begomoviruses using DNA hybridization with the coat protein gene of Pepper huasteco yellow vein virus as a probe. Thirty-eight soybean, 12 Rhynchosia sp., and 14 sunflower hybridization-positive samples were subsequently used for polymerase chain reaction (PCR) amplification with the degenerate primers pRep-DGR and pCP70-Mot (1). PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced. The amplified viral DNA (915 nt) from two soybean plants, Sb1 and Sb2 (GenBank Accession Nos. AY955101 and AY957561, respectively), one isolate from Rhynchosia minima (GenBank Accession No. AY955102), and one from Heliantus annum (GenBank Accession No. AY957560) were sequenced and compared with DNA sequences available at NCBI database using BLAST. The highest sequence similarity was obtained with the two known isolates of Rhynchosia golden mosaic virus, RhGMV [Honduras] (GenBank Accession No. AF239671), and RhGMV [Chiapas] (GenBank Accession No. AF408199), displaying a nucleotide identity of approximately 89% with the Sinaloa isolates. Sequence comparisons of the latter isolates showed that viruses in the weeds were 97% identical to one of the soybean isolates, RhGMV-Sb1, but differed significantly (88% of nucleotide identity) from the second soybean isolate, RhGMV-Sb2. The complete genome A sequence of RhGMV-Sb1 was determined using PCR amplification of viral DNA with four degenerate primers recently described (2), cloning of overlapping PCR products into pGEM-T Easy vector (Promega) and sequencing. The 2,604-bp DNA-A of RhGMV-Sb1 (GenBank Accession No. DQ347950) was compared with the homologous genome of RhGMV [Chiapas] and RhGMV [Honduras] using the CLUSTAL alignment method (MegAlign, DNASTAR software, London) and an overall nucleotide identity of 89.2 and 88.6%, respectively, was determined. Current taxonomic criteria for begomoviruses establish that a DNA-A sequence identity lower than 93% with other isolates of a virus is indicative of a separate strain (3). Therefore, the virus identified in this study is a new strain of RhGMV that is provisionally named Rhynchosia golden mosaic virus-Soybean [Mexico:Sinaloa:2004]. This is the first soybean-infecting begomovirus from the American continent whose genome A has been completely characterized as of today. References: (1) J. T. Ascencio-Ibañez et al. Plant Dis. 86:692, 2002. (2) R. De La Torre-Almaraz et al. Plant Dis. 90:378, 2006. (3) C. Fauquet et al. Arch. Virol. 150:2151, 2005. (4) J. Mendez-Lozano et al. Plant Dis. 90:109, 2006.

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 Sorption and Desorption of Vanadate, Arsenate and Chromate by Two Volcanic Soils of Equatorial Africa

Soil Systems ◽  
2021 ◽  
Vol 5 (2) ◽  
pp. 22
Author(s):  
Sara Gonzalez-Rodriguez ◽  
Maria Luisa Fernandez-Marcos
Keyword(s):  
Laboratory Experiments ◽  
Sorption Isotherms ◽  
Soil Samples ◽  
Mineral Surfaces ◽  
Volcanic Soils ◽  
Iron Species ◽  
Freundlich Model ◽  
Equatorial Africa ◽  
Relevant Role ◽  
Icp Mass Spectrometry

Sorption of oxyanions by soils and mineral surfaces is of interest due to their role as nutrients or pollutants. Volcanic soils are variable charge soils, rich in active forms of aluminum and iron, and capable of sorbing anions. Sorption and desorption of vanadate, arsenate, and chromate by two African andosols was studied in laboratory experiments. Sorption isotherms were determined by equilibrating at 293 K soil samples with oxyanion solutions of concentrations between 0 and 100 mg L−1 V, As, or Cr, equivalent to 0−2.0 mmol V L−1, 0−1.3 mmol As L−1, and 0−1.9 mmol Cr L−1, in NaNO3; V, As, or Cr were determined by ICP-mass spectrometry in the equilibrium solution. After sorption, the soil samples were equilibrated with 0.02 M NaNO3 to study desorption. The isotherms were adjusted to mathematical models. After desorption with NaNO3, desorption experiments were carried out with a 1 mM phosphate. The sorption of vanadate and arsenate was greater than 90% of the amount added, while the chromate sorption was much lower (19–97%). The sorption by the Silandic Andosol is attributed to non-crystalline Fe and Al, while in the Vitric Andosol, crystalline iron species play a relevant role. The V and Cr sorption isotherms fitted to the Freundlich model, while the As sorption isotherms conformed to the Temkin model. For the highest concentrations of oxyanions in the equilibrating solution, the sorbed concentrations were 37–38 mmol V kg−1, 25 mmol As kg−1, and 7.2–8.8 mmol Cr kg−1. The desorption was low for V and As and high for Cr. The comparison of the sorption and desorption isotherms reveals a pronounced hysteresis for V in both andosols and for Cr in the Silandic Andosol. Phosphate induced almost no V desorption, moderate As desorption, and considerable Cr desorption.

Sign in / Sign up

Export Citation Format

Share Document