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Cássio Do Nascimento - One of the best experts on this subject based on the ideXlab platform.
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Impact of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA–DNA Hybridization method
Archives of oral biology, 2013Co-Authors: Cássio Do Nascimento, Janine Navarro Dos Santos, Vinícius Pedrazzi, Murillo Sucena Pita, Nadia Monesi, Ricardo Faria Ribeiro, Rubens Ferreira De AlbuquerqueAbstract:Abstract Purpose Molecular diagnosis methods have been largely used in epidemiological or clinical studies to detect and quantify microbial species that may colonize the oral cavity in healthy or disease. The preservation of genetic material from samples remains the major challenge to ensure the feasibility of these methodologies. Long-term storage may compromise the final result. The aim of this study was to evaluate the effect of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA–DNA Hybridization. Methods Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted (n = 364) and processed according to proposed protocols: immediate processing and processed after 2 or 4 weeks, and 6 or 12 months of storage at 4 °C, −20 °C and −80 °C. Results Either total or individual microbial counts were recorded in lower values for samples processed after 12 months of storage, irrespective of temperatures tested. Samples stored up to 6 months at cold temperatures showed similar counts to those immediately processed. The microbial incidence was also significantly reduced in samples stored during 12 months in all temperatures. Conclusions Temperature and time of oral samples storage have relevant impact in the detection and quantification of bacterial and fungal species by Checkerboard DNA–DNA Hybridization method. Samples should be processed immediately after collection or up to 6 months if conserved at cold temperatures to avoid false-negative results.
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Effect of sample storage time on detection of Hybridization signals in Checkerboard DNA–DNA Hybridization
Canadian Journal of Microbiology, 2012Co-Authors: Cássio Do Nascimento, Katia Muller, Sandra Sato, Rubens Ferreira De Albuquerque JuniorAbstract:Long-term sample storage can affect the intensity of the Hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the Hybridization signals of 13 bacterial species detected by the Checkerboard DNA–DNA Hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of Hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at –20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of Hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p 0.05). The Checkerboard DNA–DNA Hybridization method was suitable to detect Hybridization signals from all groups evaluated, and the in...
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Use of the checkerboard DNA-DNA Hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.).
Parasites & vectors, 2011Co-Authors: Analiz De Oliveira Gaio, Cássio Do Nascimento, Rívea Cristina Custódio Rodrigues, Nágila Francinete Costa Secundino, Francisco Lemos, Paulo F. P. Pimenta, Nadia MonesiAbstract:Background: Bacteria associated with insects can have a substantial impact on the biology and life cycle of their host. The checkerboard DNA-DNA Hybridization technique is a semi-quantitative technique that has been previously employed in odontology to detect and quantify a variety of bacterial species in dental samples. Here we tested the applicability of the checkerboard DNA-DNA Hybridization technique to detect the presence of Aedes aegypti-associated bacterial species in larvae, pupae and adults of A. aegypti. Findings: Using the checkerboard DNA-DNA Hybridization technique we could detect and estimate the number of four bacterial species in total DNA samples extracted from A. aegypti single whole individuals and midguts. A. aegypti associated bacterial species were also detected in the midgut of four other insect species, Lutzomyia longipalpis, Drosophila melanogaster, Bradysia hygida and Apis mellifera. Conclusions: Our results demonstrate that the checkerboard DNA-DNA Hybridization technique can be employed to study the microbiota composition of mosquitoes. The method has the sensitivity to detect bacteria in single individuals, as well as in a single organ, and therefore can be employed to evaluate the differences in bacterial counts amongst individuals in a given mosquito population. We suggest that the checkerboard DNA-DNA Hybridization technique is a straightforward technique that can be widely used for the characterization of the microbiota in mosquito populations.
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Use of Checkerboard DNA-DNA Hybridization to Evaluate the Internal Contamination of Dental Implants and Comparison of Bacterial Leakage With Cast or Pre-Machined Abutments
Clinical oral implants research, 2009Co-Authors: Cássio Do Nascimento, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Rubens Ferreira De Albuquerque JuniorAbstract:Aims: To evaluate the checkerboard DNA–DNA Hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Materials and methods: Nine plastic abutments cast in a Ni–Cr alloy and nine pre-machined Co–Cr alloy abutments with plastic sleeves cast in Ni–Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 μl of a solution containing 108 cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant–abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA–DNA Hybridization method. Results: DNA–DNA Hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P
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The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA Hybridization method.
Microbiological research, 2007Co-Authors: Cássio Do Nascimento, Nadia Monesi, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Rubens Ferreira De Albuquerque JuniorAbstract:Summary Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA–DNA Hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA–DNA Hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard Hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA–DNA Hybridization technique.
Rubens Ferreira De Albuquerque Junior - One of the best experts on this subject based on the ideXlab platform.
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Effect of sample storage time on detection of Hybridization signals in Checkerboard DNA–DNA Hybridization
Canadian Journal of Microbiology, 2012Co-Authors: Cássio Do Nascimento, Katia Muller, Sandra Sato, Rubens Ferreira De Albuquerque JuniorAbstract:Long-term sample storage can affect the intensity of the Hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the Hybridization signals of 13 bacterial species detected by the Checkerboard DNA–DNA Hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of Hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at –20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of Hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p 0.05). The Checkerboard DNA–DNA Hybridization method was suitable to detect Hybridization signals from all groups evaluated, and the in...
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Use of Checkerboard DNA-DNA Hybridization to Evaluate the Internal Contamination of Dental Implants and Comparison of Bacterial Leakage With Cast or Pre-Machined Abutments
Clinical oral implants research, 2009Co-Authors: Cássio Do Nascimento, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Rubens Ferreira De Albuquerque JuniorAbstract:Aims: To evaluate the checkerboard DNA–DNA Hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Materials and methods: Nine plastic abutments cast in a Ni–Cr alloy and nine pre-machined Co–Cr alloy abutments with plastic sleeves cast in Ni–Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 μl of a solution containing 108 cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant–abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA–DNA Hybridization method. Results: DNA–DNA Hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P
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The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA Hybridization method.
Microbiological research, 2007Co-Authors: Cássio Do Nascimento, Nadia Monesi, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Rubens Ferreira De Albuquerque JuniorAbstract:Summary Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA–DNA Hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA–DNA Hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard Hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA–DNA Hybridization technique.
Nadia Monesi - One of the best experts on this subject based on the ideXlab platform.
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Impact of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA–DNA Hybridization method
Archives of oral biology, 2013Co-Authors: Cássio Do Nascimento, Janine Navarro Dos Santos, Vinícius Pedrazzi, Murillo Sucena Pita, Nadia Monesi, Ricardo Faria Ribeiro, Rubens Ferreira De AlbuquerqueAbstract:Abstract Purpose Molecular diagnosis methods have been largely used in epidemiological or clinical studies to detect and quantify microbial species that may colonize the oral cavity in healthy or disease. The preservation of genetic material from samples remains the major challenge to ensure the feasibility of these methodologies. Long-term storage may compromise the final result. The aim of this study was to evaluate the effect of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA–DNA Hybridization. Methods Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted (n = 364) and processed according to proposed protocols: immediate processing and processed after 2 or 4 weeks, and 6 or 12 months of storage at 4 °C, −20 °C and −80 °C. Results Either total or individual microbial counts were recorded in lower values for samples processed after 12 months of storage, irrespective of temperatures tested. Samples stored up to 6 months at cold temperatures showed similar counts to those immediately processed. The microbial incidence was also significantly reduced in samples stored during 12 months in all temperatures. Conclusions Temperature and time of oral samples storage have relevant impact in the detection and quantification of bacterial and fungal species by Checkerboard DNA–DNA Hybridization method. Samples should be processed immediately after collection or up to 6 months if conserved at cold temperatures to avoid false-negative results.
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Use of the checkerboard DNA-DNA Hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.).
Parasites & vectors, 2011Co-Authors: Analiz De Oliveira Gaio, Cássio Do Nascimento, Rívea Cristina Custódio Rodrigues, Nágila Francinete Costa Secundino, Francisco Lemos, Paulo F. P. Pimenta, Nadia MonesiAbstract:Background: Bacteria associated with insects can have a substantial impact on the biology and life cycle of their host. The checkerboard DNA-DNA Hybridization technique is a semi-quantitative technique that has been previously employed in odontology to detect and quantify a variety of bacterial species in dental samples. Here we tested the applicability of the checkerboard DNA-DNA Hybridization technique to detect the presence of Aedes aegypti-associated bacterial species in larvae, pupae and adults of A. aegypti. Findings: Using the checkerboard DNA-DNA Hybridization technique we could detect and estimate the number of four bacterial species in total DNA samples extracted from A. aegypti single whole individuals and midguts. A. aegypti associated bacterial species were also detected in the midgut of four other insect species, Lutzomyia longipalpis, Drosophila melanogaster, Bradysia hygida and Apis mellifera. Conclusions: Our results demonstrate that the checkerboard DNA-DNA Hybridization technique can be employed to study the microbiota composition of mosquitoes. The method has the sensitivity to detect bacteria in single individuals, as well as in a single organ, and therefore can be employed to evaluate the differences in bacterial counts amongst individuals in a given mosquito population. We suggest that the checkerboard DNA-DNA Hybridization technique is a straightforward technique that can be widely used for the characterization of the microbiota in mosquito populations.
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The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA Hybridization method.
Microbiological research, 2007Co-Authors: Cássio Do Nascimento, Nadia Monesi, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Rubens Ferreira De Albuquerque JuniorAbstract:Summary Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA–DNA Hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA–DNA Hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard Hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA–DNA Hybridization technique.
Balázs Kriszt - One of the best experts on this subject based on the ideXlab platform.
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Sequence analysis of 16S rRNA, gyrB and catA genes and DNA–DNA Hybridization reveal that Rhodococcus jialingiae is a later synonym of Rhodococcus qingshengii
International Journal of Systematic and Evolutionary Microbiology, 2014Co-Authors: András Táncsics, Tibor Benedek, Milán Farkas, István Máthé, Károly Márialigeti, Sándor Szoboszlay, József Kukolya, Balázs KrisztAbstract:The results of 16S rRNA, gyrB and catA gene sequence comparisons and reasserted DNA–DNA Hybridization unambiguously proved that Rhodococcus jialingiae Wang et al. 2010 and Rhodococcus qingshengii Xu et al. 2007 represent a single species. On the basis of priority R. jialingiae must be considered a later synonym of R. qingshengii .
James M Tiedje - One of the best experts on this subject based on the ideXlab platform.
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DNA–DNA Hybridization values and their relationship to whole-genome sequence similarities
International Journal of Systematic and Evolutionary Microbiology, 2007Co-Authors: Johan Goris, Konstantinos T Konstantinidis, Joel A Klappenbach, Tom Coenye, Peter Vandamme, James M TiedjeAbstract:DNA-DNA Hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of Hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA Hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.
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dna dna Hybridization values and their relationship to whole genome sequence similarities
International Journal of Systematic and Evolutionary Microbiology, 2007Co-Authors: Johan Goris, Konstantinos T Konstantinidis, Joel A Klappenbach, Tom Coenye, Peter Vandamme, James M TiedjeAbstract:DNA-DNA Hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of Hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA Hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.
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bacterial species determination from dna dna Hybridization by using genome fragments and dna microarrays
Applied and Environmental Microbiology, 2001Co-Authors: Jaechang Cho, James M TiedjeAbstract:Whole genomic DNA-DNA Hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA Hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the Hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-Hybridizations and can provide an open database of Hybridization profiles, avoiding the limitations of traditional DNA-DNA Hybridization.