scholarly journals Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology ofBartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California

2000 ◽  
Vol 38 (11) ◽  
pp. 4193-4200 ◽  
Author(s):  
Chao-Chin Chang ◽  
Rickie W. Kasten ◽  
Bruno B. Chomel ◽  
Darren C. Simpson ◽  
Carrie M. Hew ◽  
...  
Keyword(s):  
16S Rrna ◽  
Canis Latrans ◽  
Bacterial Genome ◽  
Clinical Signs ◽  
Rflp Analysis ◽  
Domestic Dog ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp.berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp.berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp.berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, threeBartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

PCR-RFLP analysis of fliC, fimH and 16S rRNA genes in Salmonella Typhimurium isolates of varied origin

2013 ◽  
Vol 64 (1) ◽  
pp. 177-183 ◽  
Author(s):  
Thangalazhy Gopakumar Sumithra ◽  
Vinod Kumar Chaturvedi ◽  
Praveen Kumar Gupta ◽  
Ajay Rai ◽  
Sunita Chougule ◽  
...  
Keyword(s):  
16S Rrna ◽  
Salmonella Typhimurium ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Rflp

scholarly journals PCR-RFLP Analysis of 12s and 16s Mitochondrial rRNA Genes from Brackishwater Finfish and Shellfish Species

2005 ◽  
Vol 18 (1) ◽  
Author(s):  
M.S. SHEKHAR ◽  
G. GOPIKRISHNA ◽  
I.S. AZAD
Keyword(s):  
Genetic Variation ◽  
16S Rrna ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
White Shrimp ◽  
Rrna Genes ◽  
Scylla Serrata ◽  
Aquatic Species ◽  
Asian Sea Bass ◽  
Pcr Rflp

The use of traditional genetic characterization techniques for detection of genetic variation in aquatic species based on morphological characters has its own limitations. One of the modern approaches to study genetic variation in aquatic species is by Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA). Amplification of mitochondrial 12s and 16s rRNA genes from tiger shrimp (Penaeus monodon, Penaeidae), white shrimp (Fenneropenaeus indicus, Penaeidae), grey mullet (Mugil cephalus, Mugilidae), tilapia (Oreochromis mossambicus, Cichlidae), Asian sea bass (Lates calcarifer, Centropomidae) and mud crabs (Scylla serrata and Scylla tranquebarica, Portunidae) and the characterization of the amplified PCR products by RFLP have been carried out in the present study. The use of the primers in the present study was found to be universal for amplification of mitochondrial 12s and 16s rRNA genes across the taxonomically different brackishwater species examined in this investigation. The amplified products of 12s and 16s rRNA mitochondrial gene segments obtained with these primers can be used for restriction digestion as an approach to obtain species-specific markers by PCR-RFLP analysis.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

672 IDENTIFICATION OF POLYBACTERIAL 16S RRNA GENES FROM ASCITIC FLUIDS WITH T-RFLP ANALYSIS AND DIRECT SEQUENCING

2012 ◽  
Vol 56 ◽  
pp. S266
Author(s):  
S. Krohn ◽  
J. Hartmann ◽  
A. Brodzinski ◽  
A. Chatzinotas ◽  
S. Bohm ◽  
...  
Keyword(s):  
16S Rrna ◽  
Direct Sequencing ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes

scholarly journals 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

2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length

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.

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil

2007 ◽  
Vol 53 (2) ◽  
pp. 277-283 ◽  
Author(s):  
Y. Badri ◽  
K. Zribi ◽  
M. Badri ◽  
T. Huguet ◽  
P. van Berkum ◽  
...  
Keyword(s):  
16S Rrna ◽  
Medicago Truncatula ◽  
Host Species ◽  
Sinorhizobium Meliloti ◽  
Fragment Length Polymorphism ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Rflp ◽  
Apparent Relationship ◽  
Polymerase Chain

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR–RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

scholarly journals Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

2003 ◽  
Vol 69 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Markus Egert ◽  
Michael W. Friedrich
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragments ◽  
Restriction Fragment Length

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.

scholarly journals 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

2001 ◽  
Vol 67 (4) ◽  
pp. 1893-1901 ◽  
Author(s):  
Gesche Braker ◽  
Héctor L. Ayala-del-Rı́o ◽  
Allan H. Devol ◽  
Andreas Fesefeldt ◽  
James M. Tiedje
Keyword(s):  
Community Structure ◽  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Fragment Length Polymorphism ◽  
Puget Sound ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length ◽  
Redox Gradients

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.

scholarly journals Methanogenic Community Dynamics during Anaerobic Utilization of Agricultural Wastes

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 91-97 ◽  
Author(s):  
A. M. Ziganshin ◽  
E. E. Ziganshina ◽  
S. Kleinsteuber ◽  
J. Pröter ◽  
O. N. Ilinskaya
Keyword(s):  
16S Rrna ◽  
Anaerobic Degradation ◽  
Community Dynamics ◽  
Biological Diversity ◽  
Biogas Production ◽  
Rflp Analysis ◽  
Methanogenic Archaea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene

This work is devoted to the investigation of the methanogenic archaea involved in anaerobic digestion of cattle manure and maize straw on the basis of terminal restriction fragment length polymorphism (TRFLP) analysis of archaeal 16S rRNA genes. The biological diversity and dynamics of methanogenic communities leading to anaerobic degradation of agricultural organic wastes with biogas production were evaluated in laboratory-scale digesters. T-RFLP analysis, along with the establishment of archaeal 16S rRNA gene clone libraries, showed that the methanogenic consortium consisted mainly of members of the genera Methanosarcina and Methanoculleus, with a predominance of Methanosarcina spp. throughout the experiment.

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