scholarly journals High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

2011 ◽  
Vol 78 (1) ◽  
pp. 163-169 ◽  
Author(s):  
Peter Andeer ◽  
Stuart E. Strand ◽  
David A. Stahl
Keyword(s):  
Stable Isotope ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Microbial Populations ◽  
Bacterial Isolates ◽  
Content Type ◽  
Restriction Fragment Length

ABSTRACTStable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)–TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli,Rhodococcus,Variovorax, andMicrobacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

scholarly journals Identification of Critical Members in a Sulfidogenic Benzene-Degrading Consortium by DNA Stable Isotope Probing

2008 ◽  
Vol 74 (20) ◽  
pp. 6476-6480 ◽  
Author(s):  
A. R. Oka ◽  
C. D. Phelps ◽  
L. M. McGuinness ◽  
A. Mumford ◽  
L. Y. Young ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Stable Isotope Probing ◽  
Polymorphism Analysis ◽  
Benzene Degradation ◽  
Anaerobic Benzene Degradation ◽  
Restriction Fragment Length

ABSTRACT Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the 13C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

scholarly journals Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation

2010 ◽  
Vol 76 (5) ◽  
pp. 1695-1698 ◽  
Author(s):  
Erin M. Gallagher ◽  
Lily Y. Young ◽  
Lora M. McGuinness ◽  
Lee J. Kerkhof
Keyword(s):  
Stable Isotope ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Anaerobic Bacteria ◽  
Stable Isotope Probing ◽  
Restriction Fragment Length ◽  
Harbor Sediments

ABSTRACT 2,4,6-Trinitrotoluene (15N or 13C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [15N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [13C]TNT cultures and was related to Lysobacter taiwanensis.

scholarly journals An Update on Clostridium difficile Toxinotyping

2015 ◽  
Vol 54 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Maja Rupnik ◽  
Sandra Janezic
Keyword(s):  
Clostridium Difficile ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Diagnostic Tests ◽  
Fragment Length Polymorphism ◽  
Molecular Diagnostic ◽  
Content Type ◽  
Phylogenetic Studies ◽  
Heterogenous Group ◽  
Restriction Fragment Length

Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation ofClostridium difficilestrains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis forC. difficilephylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature.

scholarly journals Development of a 16S rRNA Gene Primer and PCR-Restriction Fragment Length Polymorphism Method for Rapid Detection of Members of the Genus Megasphaera and Species-Level Identification

2011 ◽  
Vol 77 (15) ◽  
pp. 5533-5535 ◽  
Author(s):  
Akihiro Ohnishi ◽  
Shinko Abe ◽  
Shiho Nashirozawa ◽  
Sayaka Shimada ◽  
Naoshi Fujimoto ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Direct Detection ◽  
Rrna Gene ◽  
Content Type ◽  
Detection And Identification ◽  
Restriction Fragment Length

ABSTRACTThe genusMegasphaerais relevant to the environment, human health and food, and renewable energy for the future. In this study, a primer set was designed for PCR-restriction fragment length polymorphism (RFLP) analyses to detect and identify the members ofMegasphaera. Direct detection and identification were achieved for environmental samples and isolates.

scholarly journals Influence of Salmonella enterica Serovar Enteritidis Infection on the Development of the Cecum Microbiota in Newly Hatched Chicks

2012 ◽  
Vol 79 (2) ◽  
pp. 745-747 ◽  
Author(s):  
H. Juricova ◽  
P. Videnska ◽  
M. Lukac ◽  
M. Faldynova ◽  
V. Babak ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Gut Microbiota ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Salmonella Enterica ◽  
Fragment Length Polymorphism ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Content Type ◽  
Cecal Microbiota ◽  
Restriction Fragment Length

ABSTRACTTerminal restriction fragment length polymorphism and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of the ordersEnterobacteriales,Clostridiales, andLactobacillales.Salmonella entericaserovar Enteritidis infection caused the greatest changes in the gut microbiota when 1-day-old chicks were infected, compared with the infection of 4- and 16-day-old chicks.

scholarly journals Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

2011 ◽  
Vol 77 (12) ◽  
pp. 3998-4007 ◽  
Author(s):  
Norma J. Ruecker ◽  
Rebecca M. Hoffman ◽  
Rachel M. Chalmers ◽  
Norman F. Neumann
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Content Type ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACTMolecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene ofCryptosporidiumspecies were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis,C. parvum,C. felis,C. meleagridis,C. ubiquitum,C. muris, andC. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies forC. andersoniandC. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures ofCryptosporidiumat template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity ofCryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

scholarly journals An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification ofcry1-Type Genes

2013 ◽  
Vol 79 (21) ◽  
pp. 6706-6711 ◽  
Author(s):  
Changlong Shu ◽  
Dongming Liu ◽  
Zishan Zhou ◽  
Jilin Cai ◽  
Qi Peng ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Improved Method ◽  
Hyphantria Cunea ◽  
Content Type ◽  
Pcr Rflp ◽  
Restriction Fragment Length

ABSTRACTThecry1-type genes ofBacillus thuringiensisrepresent the largestcrygene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identifycry1-type genes using current methods because of the increasing number ofcry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes ofcry1-type genes was developed. This improved method was used to identifycry1-type genes in 20B. thuringiensisstrains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clusteredcry1-type genes and can be used to evaluatecry1-type genes in novel strain collections ofB. thuringiensis. Among the detectedcry1-type genes, we identified four novel genes,cry1Ai,cry1Bb,cry1Ja, andcry1La. The bioassay results from the expressed products of the four novelcrygenes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic againstPlutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity againstOstrinia furnacalis,Hyphantria cunea,Chilo suppressalis, andBombyx morilarvae and considerable weight loss activity againstHelicoverpa armigera.

scholarly journals Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting

2016 ◽  
Vol 54 (10) ◽  
pp. 2547-2552 ◽  
Author(s):  
Halima M. Said ◽  
Keshav Krishnamani ◽  
Shaheed V. Omar ◽  
Andries W. Dreyer ◽  
Bianca Sansom ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Data Exchange ◽  
Fragment Length Polymorphism ◽  
Content Type ◽  
Restriction Fragment Length ◽  
Rflp Typing

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing ofMycobacterium tuberculosisusing the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing ofM. tuberculosisisolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.

scholarly journals flaBGene as a Molecular Marker for Distinct Identification of Borrelia Species in Environmental Samples by the PCR-Restriction Fragment Length Polymorphism Method

2011 ◽  
Vol 77 (19) ◽  
pp. 7088-7092 ◽  
Author(s):  
Beata Wodecka
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Sequence Similarity ◽  
Restriction Enzymes ◽  
Borrelia Miyamotoi ◽  
Content Type ◽  
Restriction Fragment Length

ABSTRACTA new protocol employing nested PCR-restriction fragment length polymorphism (RFLP) based on theflaBgene and two restriction enzymes was worked out. This protocol allows the identification of allBorreliaspecies transmitted byIxodes ricinusin Europe, includingBorrelia miyamotoiand 3 genetic variants ofB. garinii. A dendrogram offlaBsequence similarity was in accordance with RFLP variants.

Sign in / Sign up

Export Citation Format

Share Document