scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Effect of gamma irradiation on viability and DNA of Staphylococcus epidermidis and Escherichia coli

2006 ◽  
Vol 55 (9) ◽  
pp. 1271-1275 ◽  
Author(s):  
Andrej Trampuz ◽  
Kerryl E. Piper ◽  
James M. Steckelberg ◽  
Robin Patel
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Gamma Irradiation ◽  
Staphylococcus Epidermidis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Bacterial Cells ◽  
E Coli

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D 10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D 10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D 10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of DNA in viable bacterial cells has little effect on amplifiable DNA, was not able to eliminate amplifiable 16S rRNA genes at a dose of up to 12 kGy and cannot therefore be used for elimination of DNA contamination of PCR reaction components or laboratory equipment when this DNA is present in microbial cells. This finding has practical implications for those using molecular diagnostic techniques in microbiology.

scholarly journals Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

1998 ◽  
Vol 64 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Julian R. Marchesi ◽  
Takuichi Sato ◽  
Andrew J. Weightman ◽  
Tracey A. Martin ◽  
John C. Fry ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Amplification ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr

ABSTRACT We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

scholarly journals Target enrichment from a DNA mixture by oligoribonucleotide interference-PCR (ORNi-PCR)

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Daisuke Motooka ◽  
Hodaka Fujii
Keyword(s):  
16S Rrna ◽  
Genome Editing ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Detailed Assessment ◽  
Dna Mixture

Abstract Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method that suppresses PCR amplification of target DNA in an ORN-specific manner. In this study, we examined whether ORNi-PCR can be used to enrich desirable DNA sequences from a DNA mixture by suppressing undesirable DNA amplification. ORNi-PCR enriched edited DNA sequences from a mixture of genomic DNA subjected to genome editing. ORNi-PCR enabled more efficient analysis of the types of insertion/deletion mutations introduced by genome editing. In addition, ORNi-PCR reduced the detection of 16S ribosomal RNA (16S rRNA) genes in 16S rRNA gene-based microbiome profiling, which might permit a more detailed assessment of populations of other 16S rRNA genes. Enrichment of desirable DNA sequences by ORNi-PCR may be useful in molecular biology, medical diagnosis, and other fields.

scholarly journals Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

2005 ◽  
Vol 71 (3) ◽  
pp. 1405-1416 ◽  
Author(s):  
Xiaozhen Mou ◽  
Mary Ann Moran ◽  
Ramunas Stepanauskas ◽  
José M. González ◽  
Robert E. Hodson
Keyword(s):  
16S Rrna ◽  
Cell Sorting ◽  
Marine Ecosystem ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Coastal Marine ◽  
Four Seasons ◽  
Culture Independent

ABSTRACT Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

scholarly journals Identification and comparison of three carp fishes based on mitochondrial 16S rRNA gene

2017 ◽  
Vol 26 (2) ◽  
pp. 167-174
Author(s):  
Hawa Jahan ◽  
Maria Akter ◽  
Rowshan Ara Begum ◽  
Reza Md Shahjahan
Keyword(s):  
16S Rrna ◽  
Developmental Stages ◽  
Pcr Amplification ◽  
Morphological Features ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Rrna Gene ◽  
Multiple Sequence ◽  
Alternative Means

Identification of Labeo rohita, L. bata and L. gonius is sometimes problematic when usual morphological features are lost and it is difficult to differentiate them with traditional morphological features at their diverse developmental stages. PCR-sequencing provides an authentic alternative means of identification of individuals at species level. Three local carp fishes were collected and 16S rRNA genes were sequenced by sanger sequencing method after PCR amplification using universal primers. Obtained sequences were found accurate with blast search result which showed maximum range of similarity with the existing respective gene fragments present in GenBank database. Sequences were compared and multiple sequence alignment has revealed some polymorphic sites which can be used to differentiate these three species from one another. This study may provide valuable understanding to study their population in future. Dhaka Univ. J. Biol. Sci. 26(2): 167-174, 2017 (July)

scholarly journals Widespread Occurrence of a Novel Division of Bacteria Identified by 16S rRNA Gene Sequences Originally Found in Deep Marine Sediments

2004 ◽  
Vol 70 (9) ◽  
pp. 5708-5713 ◽  
Author(s):  
Gordon Webster ◽  
R. John Parkes ◽  
John C. Fry ◽  
Andrew J. Weightman
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Marine Sediments ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Sedimentary Environments

ABSTRACT Phylogenetic analysis of 16S rRNA gene sequences from deep marine sediments identified a deeply branching clade, designated candidate division JS1. Primers for PCR amplification of partial 16S rRNA genes that target the JS1 division were developed and used to detect JS1 sequences in DNA extracted from various sedimentary environments, including, for the first time, coastal marine and brackish sediments.

scholarly journals An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments

2006 ◽  
Vol 72 (11) ◽  
pp. 7218-7230 ◽  
Author(s):  
Karen G. Lloyd ◽  
Laura Lapham ◽  
Andreas Teske
Keyword(s):  
16S Rrna ◽  
Gulf Of Mexico ◽  
Microbial Community Composition ◽  
Archaeal Community ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Anaerobic Oxidation Of Methane ◽  
Clone Libraries

ABSTRACT Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 μM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.

scholarly journals Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Mohammad Mahmoodi ◽  
Kian Pahlevan Afshari ◽  
Hamid Reza Seyedabadi ◽  
Mehran Aboozari
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Conservation Strategies ◽  
12S Rrna ◽  
Rrna Gene ◽  
Salting Out ◽  
The Rich

Phylogenetic relationships and genetic variation in Iranian Afshari sheep breed were analyzed using 12S rRNA and 16S rRNA gene sequences. The genomic DNA was isolated by salting out method and amplified 12S rRNA and 16S rRNA genes using PCR method. PCR amplification of 12S and 16S rRNA generated PCR amplicons at 859 and 1053 bp lengths, respectively. Sequence analysis was performed using BioEdit software. Phylogenetic tree was constructed using MEGA software. Phylogenetic analysis of haplotype in the combination with the sheep from GenBank showed that Iranian Afshari sheep made a close to the Australian sheep cluster. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

Combinatorial Genetic Technology for the Development of New Anti-infectives

2004 ◽  
Vol 128 (12) ◽  
pp. 1351-1359
Author(s):  
Eleftheria Laios ◽  
Marny Waddington ◽  
Ashesh A. Saraiya ◽  
Kris Ann Baker ◽  
Elizabeth O'Connor ◽  
...  
Keyword(s):  
Drug Resistance ◽  
16S Rrna ◽  
High Throughput ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Evolution System ◽  
Genetic Technology ◽  
E Coli

Abstract Context.—We previously developed a novel technology known as instant evolution for high-throughput analysis of mutations in Escherichia coli ribosomal RNA. Objective.—To develop a genetic platform for the isolation of new classes of antiinfectives that are not susceptible to drug resistance based on the instant evolution system. Design.—Mutation libraries were constructed in the 16S rRNA gene of E coli and analyzed. In addition, the rRNA genes from a number of pathogenic bacteria were cloned and expressed in E coli. The 16S rRNA genes were incorporated into the instant-evolution system in E coli. Setting.—The Department of Biological Sciences, Wayne State University, Detroit, Mich. Main Outcome Measures.—Ribosome function was assayed by measuring the amount of green fluorescent protein produced by ribosomes containing mutant or foreign RNA in vivo. Results.—We have developed a new combinatorial genetic technology (CGT) platform that allows high-throughput in vivo isolation and analysis of rRNA mutations that might lead to drug resistance. This information is being used to develop anti-infectives that recognize the wild type and all viable mutants of the drug target. CGT also provides a novel mechanism for identifying new drug targets. Conclusions.—Antimicrobials produced using CGT will provide new therapies for the treatment of infections caused by human pathogens that are resistant to current antibiotics. The new therapeutics will be less susceptible to de novo resistance because CGT identifies all mutations of the target that might lead to resistance during the earliest stages of the drug discovery process.

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