Journal articles: 'T-RFLP'

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Nagashima, Koji, Takayoshi Hisada, Maremi Sato, and Jun Mochizuki. "Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces." Applied and Environmental Microbiology 69, no. 2 (February 2003): 1251–62. http://dx.doi.org/10.1128/aem.69.2.1251-1262.2003.

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ABSTRACT New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.

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Stępień-Słodkowska, Marta, Krzysztof Ficek, Mariusz Kaczmarczyk, Agnieszka Maciejewska-Karłowska, Marek Sawczuk, Agata Leońska-Duniec, Miłosz Stępiński, et al. "The Variants Within the COL5A1 Gene are Associated with Reduced Risk of Anterior Cruciate Ligament Injury in Skiers." Journal of Human Kinetics 45, no. 1 (March 1, 2015): 103–11. http://dx.doi.org/10.1515/hukin-2015-0011.

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Abstract The purpose of this study was to examine the association of the BstUI RFLP C/T (rs 12722) and DpnII RFLP C/T (rs 13946) COL5A1 polymorphisms, individually and as haplotypes, with anterior cruciate ligament ruptures in recreational skiers. Subjects were 138 male recreational skiers with surgically diagnosed primary anterior cruciate ligament ruptures. The control group consisted of 183 apparently healthy male recreational skiers, who were without any self-reported history of ligament or tendon injury. DNA was extracted from buccal cells donated by the subjects and genotyping was carried out using real-time PCR. The genotype distributions for both polymorphisms met Hardy- Weinberg expectations in both groups. There were no significant differences in genotype distribution of allele frequencies of COL5A1 BstUI RFLP C/T and COL5A1 DpnII RFLP C/T polymorphisms between the ACL rupture and control groups. The T-T (BstUI RFLP T, DpnII RFLP T) haplotype was the most common (55.6%). The haplotype T-C was not present in any of the subjects. There was an underrepresentation tendency of the C-T haplotype in the study group compared to controls under recessive mode of inheritance. Higher frequency of the COL5A1 BstUI RFLP C/T and COL5A1DpnII RFLP C/T polymorphisms haplotype is associated with reduced risk of anterior cruciate ligament injury in a group of apparently healthy male recreational skiers.

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ASHER, A. J., L. S. WALDRON, and M. L. POWER. "Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism." Parasitology 139, no. 8 (March 15, 2012): 1005–13. http://dx.doi.org/10.1017/s0031182012000388.

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SUMMARYHumans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.

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Råberg, Ulrika, Nils O. S. Högberg, and Carl Johan Land. "Detection and species discrimination using rDNA T-RFLP for identification of wood decay fungi." Holzforschung 59, no. 6 (November 1, 2005): 696–702. http://dx.doi.org/10.1515/hf.2005.111.

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AbstractIn the present work PCR technology was used as a tool to detect the early stages of wood decay and was compared with microscopic evaluation. The wood decay fungiPostia placentaandConiophora puteanawere detectable in interior wood samples by terminal restriction fragment length polymorphism (T-RFLP) after 2weeks of incubation with monocultures, while microscopic detection of hyphae was not possible until after 7 weeks. A potential problem when fungal communities are studied with T-RFLPs of rDNA is that intra-specific variation complicates data analysis. In this work, we show that intra-specific sequence variation in the internal transcribed spacer of the rDNA inConiophora puteanaallows T-RFLP identification of this species. This is due to intra-specific variations in fragment length, in combination with the absence of point mutations in the selected restriction sites.

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Lueders, Tillmann, and Michael W. Friedrich. "Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts." Applied and Environmental Microbiology 69, no. 1 (January 2003): 320–26. http://dx.doi.org/10.1128/aem.69.1.320-326.2003.

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ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.

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Seldin, M. F., H. C. Morse, J. P. Reeves, C. L. Scribner, R. C. LeBoeuf, and A. D. Steinberg. "Genetic analysis of autoimmune gld mice. I. Identification of a restriction fragment length polymorphism closely linked to the gld mutation within a conserved linkage group." Journal of Experimental Medicine 167, no. 2 (February 1, 1988): 688–93. http://dx.doi.org/10.1084/jem.167.2.688.

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A linkage map of distal mouse chromosome 1 was generated using restriction fragment length polymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease.

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Danovaro, R., G. M. Luna, A. Dell'Anno, and B. Pietrangeli. "Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments." Applied and Environmental Microbiology 72, no. 9 (September 2006): 5982–89. http://dx.doi.org/10.1128/aem.01361-06.

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ABSTRACT We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.

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Jackson, Colin J., Richard C. Barton, and E. Glyn V. Evans. "Species Identification and Strain Differentiation of Dermatophyte Fungi by Analysis of Ribosomal-DNA Intergenic Spacer Regions." Journal of Clinical Microbiology 37, no. 4 (1999): 931–36. http://dx.doi.org/10.1128/jcm.37.4.931-936.1999.

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Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization ofEcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates ofT. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonucleaseMvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense andT. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

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Penny, Christian, Thierry Nadalig, Malek Alioua, Christelle Gruffaz, Stéphane Vuilleumier, and Françoise Bringel. "Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization." Applied and Environmental Microbiology 76, no. 3 (November 30, 2009): 648–51. http://dx.doi.org/10.1128/aem.01556-09.

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ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.

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Egert, Markus, and Michael W. Friedrich. "Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure." Applied and Environmental Microbiology 69, no. 5 (May 2003): 2555–62. http://dx.doi.org/10.1128/aem.69.5.2555-2562.2003.

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ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

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Jernberg, Cecilia, Ã¯Â¿Â½sa Sullivan, Charlotta Edlund, and Janet K. Jansson. "Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism." Applied and Environmental Microbiology 71, no. 1 (January 2005): 501–6. http://dx.doi.org/10.1128/aem.71.1.501-506.2005.

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ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

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Yu, Chang-Ping, Rajiv Ahuja, Gary Sayler, and Kung-Hui Chu. "Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia." Applied and Environmental Microbiology 71, no. 3 (March 2005): 1433–44. http://dx.doi.org/10.1128/aem.71.3.1433-1444.2005.

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ABSTRACT A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.

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Braker, Gesche, Héctor L. Ayala-del-Rı́o, Allan H. Devol, Andreas Fesefeldt, and James M. Tiedje. "Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes." Applied and Environmental Microbiology 67, no. 4 (April 1, 2001): 1893–901. http://dx.doi.org/10.1128/aem.67.4.1893-1901.2001.

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ABSTRACT Steep vertical gradients of oxidants (O2 and NO3 −) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers,Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs ofnirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis ofnirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity ofArchaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

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Junier, Pilar, Thomas Junier, and Karl-Paul Witzel. "TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets." Applied and Environmental Microbiology 74, no. 20 (August 29, 2008): 6452–56. http://dx.doi.org/10.1128/aem.01394-08.

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ABSTRACT We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.

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Awaluddin, Awaluddin, Rizalinda Sjahrir, and Farida Ilyas. "PENGGUNAAN METODE PCR – RFLP (POLYMERASE CHAIN REACTION – RESTRICTION FRAGMENT LENGTH POLYMORFISM) DALAM MENDETEKSI JAMUR DERMATOFIT." JURNAL MEDIA KESEHATAN 14, no. 1 (June 30, 2021): 96–102. http://dx.doi.org/10.33088/jmk.v14i1.615.

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ABSTRAK Dermatofitosis adalah salah satu jamur yang terdiri dari tiga genus : Epidermophyton, Trichophyton dan Microsporum. Penelitian ini bertujuan untuk mengidentifikasi jenis jamur dermatofit dengan metode PCR-RFLP. Penelitian ini merupakan penelitian observasional laboratorium dengan dengan menguji 23 sampel yang diperoleh dari beberapa klinik dan sekolah dasar di Makassar. Hasil penelitian menunjukkan bahwa semua sampel teridentifikasi yakni M. canis 26,1%, Trichophyton rubrum 13,1%,T. mentagrophytes 21,6%, T. tonsurans 8,7%, T. verrucosum 4,2% dan spesies unclassified 26,1%. Kami menyarankan Teknik PCR-RFLP dapat digunakan untuk konfirmasi jenis jamur dermatofit. Kata Kunci : Dermatofitosis, Jamur Dermatofit, PCR – RFLP

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Engebretson, Jeff J., and Craig L. Moyer. "Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4823–29. http://dx.doi.org/10.1128/aem.69.8.4823-4829.2003.

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ABSTRACT An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

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Hartmann, Martin, and Franco Widmer. "Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices." Applied and Environmental Microbiology 72, no. 12 (October 13, 2006): 7804–12. http://dx.doi.org/10.1128/aem.01464-06.

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ABSTRACT Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.

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Blackwood, Christopher B., Deborah Hudleston, Donald R. Zak, and Jeffrey S. Buyer. "Interpreting Ecological Diversity Indices Applied to Terminal Restriction Fragment Length Polymorphism Data: Insights from Simulated Microbial Communities." Applied and Environmental Microbiology 73, no. 16 (June 29, 2007): 5276–83. http://dx.doi.org/10.1128/aem.00514-07.

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ABSTRACT Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E var) and true Shannon diversity (H′), with correlations between 0.71 and 0.81. TRF-H′ and true H′ were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H′ cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E var). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

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Stankovic, Srdjan, Ivana Moric, Aleksandar Pavic, Branka Vasiljevic, Barrie Johnson, and Vladica Cvetkovic. "Investigation of the microbial diversity of an extremely acidic, metal-rich water body (Lake Robule, Bor, Serbia)." Journal of the Serbian Chemical Society 79, no. 6 (2014): 729–41. http://dx.doi.org/10.2298/jsc130605071s.

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An investigation of the microbial diversity of the extremely acidic, metal-rich Lake Robule was carried out using culture-dependant and culture-independent (T-RFLP) methods, and the ability of indigenous bacteria from the lake water to leach copper from a mineral concentrate was tested. T-RFLP analysis revealed that the dominant bacteria in lake water samples were the obligate heterotroph Acidiphilium cryptum (~50% of total bacteria) and the iron-oxidizing autotroph Leptospirillum ferrooxidans (~40%) The iron/sulfur-oxidizing autotroph Acidithiobacillus ferrooxidans had been reported to be the most abundant bacteria in the lake in an earlier study by other authors, but it was not detected in the present study using T-RFLP. Although it was isolated on solid media and detected in enrichment (bioleaching) cultures. The presence of the two bacterial species detected by T-RFLP (L. ferrooxidans and A. cryptum) was also confirmed by cultivation on solid media. The presence and relative abundance of bacteria inhabiting Lake Robule was explained by the physiological characteristics of the bacteria and the physico-chemical characteristics of the lake water.

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Moeseneder, Markus M., Jesús M. Arrieta, Gerard Muyzer, Christian Winter, and Gerhard J. Herndl. "Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis." Applied and Environmental Microbiology 65, no. 8 (August 1, 1999): 3518–25. http://dx.doi.org/10.1128/aem.65.8.3518-3525.1999.

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ABSTRACT The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.

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Székely, Anna J., Rita Sipos, Brigitta Berta, Balázs Vajna, Csaba Hajdú, and Károly Márialigeti. "DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost." Microbial Ecology 57, no. 3 (July 25, 2008): 522–33. http://dx.doi.org/10.1007/s00248-008-9424-5.

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Frey, Julie C., Jessica M. Rothman, Alice N. Pell, John Bosco Nizeyi, Michael R. Cranfield, and Esther R. Angert. "Fecal Bacterial Diversity in a Wild Gorilla." Applied and Environmental Microbiology 72, no. 5 (May 2006): 3788–92. http://dx.doi.org/10.1128/aem.72.5.3788-3792.2006.

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ABSTRACT We describe the bacterial diversity in fecal samples of a wild gorilla by use of a 16S rRNA gene clone library and terminal-restriction fragment length polymorphism (T-RFLP). Clones were classified as Firmicutes, Verrucomicrobia, Actinobacteria, Lentisphaerae, Bacteroidetes, Spirochetes, and Planctomycetes. Our data suggest that fecal populations did not change temporally, as determined by T-RFLP.

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Tsao, Kimberly, Stephen J. Bent, and Durland Fish. "Identification ofBorrelia burgdorferi ospCGenotypes in Host Tissue and Feeding Ticks by Terminal Restriction Fragment Length Polymorphisms." Applied and Environmental Microbiology 79, no. 3 (November 26, 2012): 958–64. http://dx.doi.org/10.1128/aem.03106-12.

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ABSTRACTWe developed a high-throughput method based on terminal restriction fragment length polymorphisms (T-RFLP) to identifyospCgenotypes from field-collected samples ofBorrelia burgdorferi. We first validated the method by analyzingB. burgdorferi ospCpreviously identified by sequencing. We then analyzed and comparedospCgenotypes detected from ear biopsy tissue from natural populations of the white-footed mouse, a majorB. burgdorferireservoir host species in the eastern United States, and larval ticks feeding on those individual mice. The T-RFLP method enabled us to distinguish all 17ospCgenotypes tested, as well as mixed samples containing more than one genotype. Analysis costs compare favorably to those of alternativeospCidentification methods. The T-RFLP method will facilitate large-scale field studies to advance our understanding of genotype-specific transmission patterns.

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Wang, Jia Jia, Yu Mei Li, Zhi Wen Zhao, Qiang Li, Ying Zi Liu, Chun Jiang Ye, Da Ke Hao, Yan Hong Qu, Qing Qing Zhang, and Jun Fa Li. "T-RFLP Analysis of Soil Microbial Community from Shandong Province for Forensic Science." Advanced Materials Research 599 (November 2012): 250–53. http://dx.doi.org/10.4028/www.scientific.net/amr.599.250.

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Soil microbial community very little had been found as trace evidence， however， it could be vital for forensic science. In this paper, soil microbial community diversities from seventeen different regions of Shandong province were investigated by Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results of diversity index analysis show the highest Margalef index and Eveness index are 4.609 for sample D and 0.970 for sample E, respectively. In T-RFLP profile, the absolute advantage peak are 488bp (62.6% ) from sample O and 496.5bp (20.8%) from Sample H. The analysed terminal restriction fragments (T-RFs) are from Firmicutes and Proteobacteria. These results reveal that there are the obvious regional characteristic for microbial community of all the detected samples, which suggests T-RFLP analysis of microbial community diversity will be a rapid effective fingerprinting method for forensic detection.

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Savichtcheva, O., and S. Okabe. "Qualitative and quantitative estimation of host-specific fecal pollution using Bacteroides–Prevotella 16S rRNA genetic markers by T-RFLP and real-time PCR analyses." Water Science and Technology 59, no. 9 (May 1, 2009): 1831–40. http://dx.doi.org/10.2166/wst.2009.213.

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Rapid and reliable determination of the non-point sources of fecal pollution is a critical issue for the environmental microbiologists all over the world. In this work we evaluated the use of anaerobic bacterial group Bacteroides–Prevotella as an alternative fecal pollution indicator. Terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (RT-PCR) analyses were used to monitor and quantify human-, cow- and pig-specific fecal contamination in natural river waters. We also included DNA sequence analysis of the identified fecal markers revealed by T-RFLP in order to clarify the specificity of each marker. It was suggested that the most influent peaks for each fecal source could be used to identify the source of fecal pollution. Development of specific probes based on these markers permit to quantify source of contamination by quantitative RT-PCR. Therefore, we combined the T-RFLP results and RT-PCR assay to quantify fecal contamination by certain host. We can conclude that T-RFLP and RT-PCR analyses showed high reproducibility and sensitivity during analyzing real water samples and can be used to identify, track and quantify host-specific bacterial genetic markers in complex natural water environments.

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Kasuga, I., D. Shimazaki, and S. Kunikane. "Influence of backwashing on the microbial community in a biofilm developed on biological activated carbon used in a drinking water treatment plant." Water Science and Technology 55, no. 8-9 (April 1, 2007): 173–80. http://dx.doi.org/10.2166/wst.2007.256.

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The influence of backwashing on the biofilm community developed on biological activated carbon (BAC) used in a drinking water treatment plant was investigated by means of bacterial cell enumeration and terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting analysis of bacterial and eukaryotic ribosomal RNA genes (rDNA). After backwashing, the attached bacterial abundance in the top layer of the BAC bed decreased to 64% of that before backwashing. The community level changes caused by backwashing were examined through the T-RFLP profiles. In the bacterial 16S rDNA analysis, the relative abundances of some terminal-restriction fragments (T-RFs) including the Planctomycetes-derived fragment increased; however, the relative abundances of some T-RFs including the Betaproteobacteria-derived fragments decreased. In the eukaryotic 18S rDNA analysis, the relative abundances of some T-RFs including the protozoan Cercozoa-derived fragments increased; however, the relative abundances of some T-RFs including the metazoan Chaetonotus- and Paratripyla-derived fragments decreased. The T-RFLP analysis suggests that backwashing can cause changes in the relative compositions of microorganisms in a BAC biofilm in the top layer of the bed.

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Mengoni, Alessio, Eva Grassi, and Marco Bazzicalupo. "Cloning Method for Taxonomic Interpretation of T-RFLP Patterns." BioTechniques 33, no. 5 (November 2002): 990–92. http://dx.doi.org/10.2144/02335bm04.

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Coolen, Marco J. L., Eduard Post, Catherine C. Davis, and Larry J. Forney. "Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes." Applied and Environmental Microbiology 71, no. 12 (December 2005): 8729–37. http://dx.doi.org/10.1128/aem.71.12.8729-8737.2005.

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ABSTRACT To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.

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Zerba, K. E., A. M. Kessling, J. Davignon, and C. F. Sing. "Genetic structure and the search for genotype-phenotype relationships: an example from disequilibrium in the Apo B gene region." Genetics 129, no. 2 (October 1, 1991): 525–33. http://dx.doi.org/10.1093/genetics/129.2.525.

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Abstract We analyzed allelic associations (disequilibria) for four restriction fragment length polymorphisms (RFLPs) in the region of the 43-kb Apo B gene in a sample of 233 unrelated individuals from Montreal, Canada, sampled for health. This total sample (T) included 160 individuals of known French Canadian (FC) ancestry. We present a rigorous application of current methodology to these samples, including estimation of type II error probabilities and correlations between markers for estimates of disequilibria. We then consider the utility of these estimates of allelic disequilibria for the interpretation of genotype-phenotype relations. Significant deviations from Hardy-Weinberg equilibrium were not predicted by proximity to other markers in disequilibrium. We found significant quadri-allelic disequilibrium for two marker pairs despite absence of significant deviations from Hardy-Weinberg equilibrium for either marker or tri-allelic disequilibrium, respectively. Altogether these results underscore the complexity of the genotypic structure of the data. A combination of nonevolutionary factors, including sampling for health, small sample size and data exclusion due to methodological constraints of not successfully typing all members of the sample for every RFLP, is a likely explanation for this complexity. These types of factors are common to many RFLP studies. Patterns of composite di-allelic disequilibrium indicated that some RFLP allele pairs may have a longer shared evolutionary history than others and that disequilibrium is not predicted by distance between RFLPs. Type II error probabilities were generally much higher than those for type I errors. Correlations between marker pairs for disequilibria were generally not high. We show from a review of 14 published studies of association between the XbaI RFLP and variation in a total of 15 lipid traits that deviations from Hardy-Weinberg equilibrium can cause substantial differences in the estimation of variability associated with phenotypic differences among marker genotypes relative to Hardy-Weinberg conditions.

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Fogarty, Lisa R., and Mary A. Voytek. "Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species." Applied and Environmental Microbiology 71, no. 10 (October 2005): 5999–6007. http://dx.doi.org/10.1128/aem.71.10.5999-6007.2005.

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ABSTRACT To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

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Villanueva da Fonseca, Luisa Andrea, Maria Anilda Santos Araújo, Denise Maria Wanderlei Silva, and Fernanda Cristina De Albuquerque Maranhão. "ITS-RFLP optimization for dermatophyte identification from clinical sources in Alagoas (Brazil) versus phenotypic methods." Journal of Infection in Developing Countries 16, no. 11 (November 29, 2022): 1773–77. http://dx.doi.org/10.3855/jidc.17077.

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Introduction: Dermatophytoses are superficial mycoses, and the identification of their etiological agents is routinely performed by culture and microscopic features, which is time-consuming and relies on personnel expertise. Molecular approaches have been developed to provide faster and reliable results; therefore, this study aimed to identify dermatophytes isolated from Alagoas state patients, employing phenotypical and molecular methods. Methodology: Clinical samples for morphological identification were collected from private and public laboratories and cultivated on Sabouraud dextrose agar. DNA extraction was followed by ITS amplicon analysis after restriction enzyme digestion DdeI (ITS-RFLP). Results: Out of fourteen representative strains, ITS-RFLP with DdeI efficiently identified Microsporum canis, Nannizzia gypsea, and Trichophyton rubrum, while species of the complex T. tonsurans/T. mentagrophytes presented the same restriction pattern. After genotyping, 2 T. tonsurans and 1 Microsporum sp. strain were reclassified as T. rubrum. Conclusions: RFLP of ITS-region followed by DdeI digestion produced faster and relatively reliable results than classic methods; however, this method has not been as efficient for closely related dermatophytes cryptic species.

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Dinoto, Achmad, Akarat Suksomcheep, Satoshi Ishizuka, Hanae Kimura, Satoshi Hanada, Yoichi Kamagata, Kozo Asano, Fusao Tomita, and Atsushi Yokota. "Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve." Applied and Environmental Microbiology 72, no. 1 (January 2006): 784–92. http://dx.doi.org/10.1128/aem.72.1.784-792.2006.

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ABSTRACT To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

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Campbell, M. A., D. Chen, and P. C. Ronald. "Development of Co-Dominant Amplified Polymorphic Sequence Markers in Rice that Flank the Magnaporthe grisea Resistance Gene Pi7(t) in Recombinant Inbred Line 29." Phytopathology® 94, no. 3 (March 2004): 302–7. http://dx.doi.org/10.1094/phyto.2004.94.3.302.

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Pi7(t), a dominant blast resistance gene derived from the rice cultivar Moroberekan, confers complete resistance against the fungal pathogen Magnaporthe grisea. Pi7(t) previously was positioned on chromosome 11 by restriction fragment length polymorphism (RFLP) mapping of a recombinant inbred line population. One derivative of this population, recombinant inbred line (RIL)29, was designated as the representative line for Pi7(t). A segregating F2 population was created from RIL29 in order to determine the location of Pi7(t). The new mapping data indicate a position for Pi7(t) 30 centimorgans distal to the original location. Pi7(t) shares a common position with the previously mapped Pi1 M. grisea resistance gene. RIL29 carries DNA not derived from either parent used to create the RIL population at the newly assigned Pi7(t) locus. RFLP analysis has identified a possible donor source.

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Järve, K., H. O. Peusha, J. Tsymbalova, S. Tamm, K. M. Devos, and T. M. Enno. "Chromosomal location of a Triticum timopheevii - derived powdery mildew resistance gene transferred to common wheat." Genome 43, no. 2 (March 15, 2000): 377–81. http://dx.doi.org/10.1139/g99-141.

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A dominant powdery mildew resistance gene introduced from Triticum timopheevii in line 146-155-T of common wheat, Triticum aestivum, was located on chromosome 6B by monosomic analysis. Restriction fragment length polymorphism (RFLP) and microsatellite analyses detected the presence of a T. timopheevii segment, translocated to chromosome 6B, with breakpoints between the loci Xpsr8/Xpsr964 on 6BS and Xpsr154/Xpsr546 on 6BL. The novel powdery mildew resistance gene, which has been designated Pm27, was shown to cosegregate with the microsatellite locus Xpsp3131, which is located on the introgressed T. timopheevii segment. The molecular data confirm the location of Pm27 on the translocated 6B chromosome. Key words: monosomic analysis, RFLP, microsatellites, Pm27.

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Ondreičková, Katarína, Andrej Ficek, Daniel Mihálik, Marcela Gubišová, Martina Hudcovicová, Hana Drahovská, and Ján Kraic. "Bacterial Communities in Rhizosphere of Maize Studied by T-RFLP." Agriculture (Pol'nohospodárstvo) 60, no. 3 (December 10, 2014): 98–104. http://dx.doi.org/10.2478/agri-2014-0011.

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Abstract The terminal restriction fragment length polymorphism munities from different collecting places was evaluated was used to determine the bacterial diversity in rhizo- by principal component analysis. Results showed that sphere of maize (Zea mays L.) collected from four sites the most different bacterial community originated from of experimental field plot in two dates of the vegetation marginal part of the experimental field plot collected in season (July and September). The 16S rRNA gene was September was caused probably by combination of the amplified from metagenomic DNA using universal eubac- marginal effect and drought before sampling date in Sep- terial primers and PCR products were digested separately tember. Other rhizosphere samples showed from moderate with three restriction enzymes. Significant differences in to small differences in the structure of the bacterial com- the number of terminal restriction fragments among rhi- munity. Nevertheless, significant differences among all zosphere samples and between sampling dates were not collected bacterial communities were not observed. detected (P < 0.05). Variation within the bacterial communities from different collecting places was evaluated by principal component analysis. Results showed that the most different bacterial community originated from marginal part of the experimental field plot collected in September was caused probably by combination of the marginal effect and drought before sampling date in September. Other rhizosphere samples showed from moderate to small differences in the structure of the bacterial community. Nevertheless, significant differences among all collected bacterial communities were not observed.

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Rüger, M., and U. Reichl. "Charakterisierung medizinisch relevanter bakterieller Mischkulturen mittels t-RFLP und Durchflusszytometrie." Chemie Ingenieur Technik 84, no. 8 (July 25, 2012): 1292. http://dx.doi.org/10.1002/cite.201250246.

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Vaccino, P., M. Accerbi, and M. Corbellini. "Cultivar identification in T. aestivum using highly polymorphic RFLP probes." Theoretical and Applied Genetics 86, no. 7 (August 1993): 833–36. http://dx.doi.org/10.1007/bf00212609.

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Galal-Khallaf, Asmaa, Khaled Mohammed-Geba, Alaa G. M. Osman, Khaled Y. AbouelFadl, Yaisel J. Borrell, and Eva Garcia-Vazquez. "SNP-based PCR-RFLP, T-RFLP and FINS methodologies for the identification of commercial fish species in Egypt." Fisheries Research 185 (January 2017): 34–42. http://dx.doi.org/10.1016/j.fishres.2016.09.031.

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Waldron, L. S., B. C. Ferrari, M. R. Gillings, and M. L. Power. "Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces." Applied and Environmental Microbiology 75, no. 1 (October 31, 2008): 108–12. http://dx.doi.org/10.1128/aem.01341-08.

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ABSTRACT Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

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Dagar, Sumit S., Sanjay Kumar, Priti Mudgil, Rameshwar Singh, and Anil K. Puniya. "D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp." Applied and Environmental Microbiology 77, no. 18 (July 22, 2011): 6722–25. http://dx.doi.org/10.1128/aem.05441-11.

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ABSTRACTThis study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation ofOrpinomyces joyoniiandOrpinomyces intercalarisbased on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T inO. intercalariscreated an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

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Conn, Vanessa M., and Christopher M. M. Franco. "Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones." Applied and Environmental Microbiology 70, no. 3 (March 2004): 1787–94. http://dx.doi.org/10.1128/aem.70.3.1787-1794.2004.

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ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

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Kent, Angela D., Dan J. Smith, Barbara J. Benson, and Eric W. Triplett. "Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities." Applied and Environmental Microbiology 69, no. 11 (November 2003): 6768–76. http://dx.doi.org/10.1128/aem.69.11.6768-6776.2003.

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ABSTRACT Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.

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Bae, H., Y. C. Chung, and J. Y. Jung. "Microbial community structure and occurrence of diverse autotrophic ammonium oxidizing microorganisms in the anammox process." Water Science and Technology 61, no. 11 (June 1, 2010): 2723–32. http://dx.doi.org/10.2166/wst.2010.075.

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The enrichment of anaerobic ammonium oxidizing (anammox) bacteria using an upflow anaerobic sludge bioreactor was successfully conducted for 400 days of continuous operation. The bacterial community structure of anammox bioreactor included Proteobacteria (42%), Chloroflexi (22%), Planctomycetes (20%), Chlorobi (7%), Bacteroidetes (5%), Acidobacteria (2%), and Actinobacteria (2%). All clones of Planctomycetes were affiliated with the anammox bacteria, Planctomycete KSU-1 (AB057453). The presence and diversity of ammonia oxidizing bacteria (AOB) and archaea (AOA) were identified by terminal restriction fragment length polymorphism (T-RFLP) based on the amoA gene sequences. The AOB in anammox bioreactor were affiliated with the Nitrosomonas europaea cluster. The T-RFLP result of AOA showed the diverse microbial community structure of AOA with three terminal restriction fragments (T-RFs).

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Hayes, Dawn C., Rebecca R. Anderson, and Richard L. Walker. "Identification of Trichomonadid Protozoa from the Bovine Preputial Cavity by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Typing." Journal of Veterinary Diagnostic Investigation 15, no. 4 (July 2003): 390–94. http://dx.doi.org/10.1177/104063870301500417.

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Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.8S rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.8S rRNA gene and ITSR sequence results and PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.

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Dopheide, Andrew, Gavin Lear, Rebecca Stott, and Gillian Lewis. "Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods." Applied and Environmental Microbiology 75, no. 16 (June 26, 2009): 5261–72. http://dx.doi.org/10.1128/aem.00412-09.

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ABSTRACT Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3′ and 5′ terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.

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Matsuda, M., D. Inoue, Y. Anami, H. Tsutsui, K. Sei, S. Soda, and M. Ike. "Comparative analysis of DNA-based microbial community composition and substrate utilisation patterns of activated sludge microorganisms from wastewater treatment plants operated under different conditions." Water Science and Technology 61, no. 11 (June 1, 2010): 2843–51. http://dx.doi.org/10.2166/wst.2010.212.

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In this study, the microbial community structure and carbon source utilisation profile of activated sludge samples collected from full-scale municipal wastewater treatment plants (WWTPs) operated under different conditions were characterised and compared, respectively, using terminal-restriction fragment length polymorphism (T-RFLP) analysis and Biolog assay. Samples were collected from each biological treatment tank of six conventional activated sludge, two anaerobic–oxic, two anaerobic–anoxic–oxic, and one step-aeration processes in eight full-scale WWTPs in Osaka, Japan. Results of the T-RFLP analysis of eubacterial 16S rDNA showed that microbial communities of activated sludge differed greatly among samples, and that they were affected by process-based operational conditions. In contrast, the carbon source utilisation profiles of activated sludge samples were mutually similar, but appeared to be influenced slightly by aerated conditions at each reaction tank. Similar carbon source utilisation profiles among all samples suggest that the activated sludge community possesses functions that are necessary for wastewater treatment even if the phylogenetic composition is different. Different results from the T-RFLP analysis and Biolog assay suggest that the phylogenetic composition of microbial community might not necessarily reflect the wastewater treatment functions of the activated sludge.

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Thies, Frank L., Wolfgang König, and Brigitte König. "Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting." Journal of Medical Microbiology 56, no. 6 (June 1, 2007): 755–61. http://dx.doi.org/10.1099/jmm.0.46562-0.

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Bacterial vaginosis (BV) is a prevalent infection in women of reproductive age associated with numerous sequelae, including preterm delivery, amniotic fluid infections and an increased risk of acquiring sexually transmitted diseases. The vaginal microbiota in BV patients is characterized by a shift from lactobacilli to a diverse spectrum of mostly anaerobic bacteria. In this study, terminal restriction fragment length polymorphism (T-RFLP) was used to characterize the vaginal bacterial communities from 50 women with BV and 20 healthy subjects. In the BV samples, 23 species or phylotypes from 17 genera could be identified, including Atopobium vaginae, Megasphaera sp., Lactobacillus iners, Gardnerella vaginalis and three recently described phylotypes from the order Clostridiales. The number of detected species or phylotypes was on average 6.3 per sample (range 2–14). In contrast, in normal samples, only Lactobacillus species could be identified. In conclusion, T-RFLP provides a rapid and reliable technique to investigate the diversity of the predominant vaginal microbiota and allows differentiation of the flora of BV and healthy women. As such, T-RFLP may be helpful both in the diagnosis of BV from vaginal fluids and in a better understanding of the bacterial succession involved in the aetiology of BV.

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Anderson, J. A., and S. S. Maan. "Interspecific nuclear–cytoplasmic compatibility controlled by genes on group 1 chromosomes in durum wheat." Genome 38, no. 4 (August 1, 1995): 803–8. http://dx.doi.org/10.1139/g95-102.

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Triticum longissimum cytoplasm is incompatible with the T. turgidum nuclear genome. Two nuclear genes, scs and Vi, derived from the nuclear genome of T. timopheevii and by a spontaneous mutation, respectively, restore nuclear–cytoplasmic compatibility, normal plant vigor, and male fertility in these alloplasmic genotypes. The objectives of this study were (i) to determine the chromosomal locations of scs and Vi; (ii) to identify DNA markers for scs and Vi; and (iii) to determine the interactions involving the dosage of scs and Vi. Two populations segregating for scs and Vi were produced and scored for seedling vigor (indicating presence of scs) and degree of self-fertility (indicating presence of Vi). Four RFLP markers were mapped near scs. Aneuploid analysis revealed that these markers, and hence the scs gene, are located on the long arm of chromosome 1A. Four RFLP markers were mapped near Vi on 1BS. Results indicated that other factors may be inhibiting the expression of Vi. We determined the dosage of scs and Vi in both populations with the aid of the linked RFLP markers. Individuals with two versus one dose of scs had reduced self-fertility, while individuals with two versus one dose of Vi had similar self-fertility.Key words: scs, Vi, Triticum, nucleocytoplasmic compatibility, RFLP.

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Segonds, Christine, Thierry Heulin, Nicole Marty, and Gerard Chabanon. "Differentiation of Burkholderia Species by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene and Application to Cystic Fibrosis Isolates." Journal of Clinical Microbiology 37, no. 7 (1999): 2201–8. http://dx.doi.org/10.1128/jcm.37.7.2201-2208.1999.

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Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiateBurkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderiaand to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrocinia T were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includesB. multivorans T) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepaciagenomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.

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Pandey, J., S. S. Sood, and R. K. Jain. "Terminal restriction fragment length polymorphism (T-RFLP) analysis: Characterizing the unseen." Indian Journal of Microbiology 47, no. 1 (March 2007): 90–91. http://dx.doi.org/10.1007/s12088-007-0017-7.

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Journal articles on the topic 'T-RFLP'

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