The Experts below are selected from a list of 17811 Experts worldwide ranked by ideXlab platform
Fernando Tavares - One of the best experts on this subject based on the ideXlab platform.
-
application of a Dot Blot hybridization assay for genotyping streptococcus uberis from brazilian dairy herds
Journal of Dairy Science, 2021Co-Authors: Bruna Gomes Alves, Leticia Wilke Franco Martins, Joana Ribeiro, Fernando Tavares, M Dos V SantosAbstract:Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot Blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to Dot Blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical Dot Blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.
-
application of a Dot Blot hybridization platform to assess streptococcus uberis population structure in dairy herds
Frontiers in Microbiology, 2017Co-Authors: Pedro Albuquerque, Niza Ribeiro, Alexandre Almeida, Irena Panschin, Afonso Porfirio, Marta Vales, Francisca Diniz, Helena Madeira, Fernando TavaresAbstract:Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a Dot Blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a six-month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the Dot Blots and increase the technique’s consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0,5 being considered positive results. These data allowed to confirm the isolates’ identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious transmission pattern depending on the herd. Overall, this work showed the utility of Dot Blot and MLSA to characterize population structure and epidemiological patterns of mastitis-causing S. uberis. This approach allowed to disclose prevalent virulence patterns and clonal lineages of S. uberis in two distinct herds, and gain insights on the impact of herd management practices on pathogen population structure.
Takeshi Nishio - One of the best experts on this subject based on the ideXlab platform.
-
prediction of the optimum hybridization conditions of Dot Blot snp analysis using estimated melting temperature of oligonucleotide probes
Plant Cell Reports, 2010Co-Authors: Sachiko Shiokai, Hiroyasu Kitashiba, Takeshi NishioAbstract:Although the Dot-Blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared Tm values estimated from nucleotide sequences using the DINAMelt web server, measured Tm values, and hybridization conditions yielding allele-specific signals. The estimated Tm values were comparable to the measured Tm values with small differences of less than 3°C for most of the probes. There were differences of approximately 14°C between the specific signal detection conditions and estimated Tm values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0× SSC corresponded to a difference of approximately 5°C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced Dot-Blot-SNP markers will become predictable.
-
improvement of the Dot Blot snp technique for efficient and cost effective genotyping
Molecular Breeding, 2010Co-Authors: Sachiko Shiokai, Kenta Shirasawa, Yutaka Sato, Takeshi NishioAbstract:Although the Dot-Blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.
-
s genotyping and s screening utilizing sfb gene polymorphism in japanese plum and sweet cherry by Dot Blot analysis
Molecular Breeding, 2008Co-Authors: Hiroyasu Kitashiba, Shao Ling Zhang, Jun Wu, Kenta Shirasawa, Takeshi NishioAbstract:Most Rosaceae fruit trees such as Japanese plum and sweet cherry have a gametophytic self-incompatibility (GSI) system controlled by a single S locus containing at least two linked genes with multiple alleles, i.e., S-RNase as a pistil determinant and SFB (S-haplotype-specific F-box gene) as a candidate for the pollen S determinant. For identification of S genotypes, many methods based on polymerase chain reaction (PCR) utilizing polymorphism in length of the S-RNase and SFB gene have been developed. In this study, we developed two Dot-Blot analysis methods for S-haplotype identification utilizing allele-specific oligonucleotides based on the SFB-HVa region, which has high sequence polymorphism. Dot-Blotting of allele-specific oligonucleotides hybridized with digoxigenin-labeled PCR products allowed S genotyping of plants with nine S haplotypes (S-a, S-b, S-c, S-e, S-f, S-h, S-k, S-7 and S-10) in Japanese plum and ten S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) in sweet cherry (Dot-Blot-S-genotyping). In addition, Dot-Blotting of PCR products of SFB probed with the allele-specific oligonucleotides, occasionally utilizing competitive hybridization, was successful in screening for a desirable S haplotype in sweet cherry (Dot-Blot-S-screening).
M Dos V Santos - One of the best experts on this subject based on the ideXlab platform.
-
application of a Dot Blot hybridization assay for genotyping streptococcus uberis from brazilian dairy herds
Journal of Dairy Science, 2021Co-Authors: Bruna Gomes Alves, Leticia Wilke Franco Martins, Joana Ribeiro, Fernando Tavares, M Dos V SantosAbstract:Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot Blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to Dot Blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical Dot Blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.
J R Bonami - One of the best experts on this subject based on the ideXlab platform.
-
Dot Blot hybridization and rt pcr detection of extra small virus xsv associated with white tail disease of prawn macrobrachium rosenbergii
Diseases of Aquatic Organisms, 2004Co-Authors: Joannes Sri Widada, D Qian, V Richard, J R BonamiAbstract:: The availability of specific and reliable detection methods is essential for monitoring the health status of farmed species, particularly for viral diseases. Extra small virus (XSV), a virus-like particle, is associated with Macrobrachium rosenbergii Noda virus (MrNV) in white tail disease (WTD) of M. rosenbergii. We developed 2 genome-based detection methods for the identification of XSV, namely Dot-Blot hybridization and a single-step RT-PCR. Detection limits were established and are ca. 2.5 pg and 5 fg of viral RNA for Dot-Blot hybridization and RT-PCR, respectively. Application of the methods to field samples indicated that some animals positively diagnosed with MrNV did not contain XSV, at least within the detection limit of the methodology. This raises the question of the actual role of XSV and its interactions with MrNV in WTD of M. rosenbergii.
-
genome based detection methods of macrobrachium rosenbergii nodavirus a pathogen of the giant freshwater prawn macrobrachium rosenbergii Dot Blot in situ hybridization and rt pcr
Journal of Fish Diseases, 2003Co-Authors: Sri J Widada, I Cambournac, D Qian, E Dejonghe, V Richard, J R BonamiAbstract:The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome-based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. Dot-Blot hybridization, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Detection limits were established for Dot-Blot hybridization and RT-PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT-PCR can be used for in-depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT-PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.
Pedro Albuquerque - One of the best experts on this subject based on the ideXlab platform.
-
application of a Dot Blot hybridization platform to assess streptococcus uberis population structure in dairy herds
Frontiers in Microbiology, 2017Co-Authors: Pedro Albuquerque, Niza Ribeiro, Alexandre Almeida, Irena Panschin, Afonso Porfirio, Marta Vales, Francisca Diniz, Helena Madeira, Fernando TavaresAbstract:Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a Dot Blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a six-month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the Dot Blots and increase the technique’s consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0,5 being considered positive results. These data allowed to confirm the isolates’ identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious transmission pattern depending on the herd. Overall, this work showed the utility of Dot Blot and MLSA to characterize population structure and epidemiological patterns of mastitis-causing S. uberis. This approach allowed to disclose prevalent virulence patterns and clonal lineages of S. uberis in two distinct herds, and gain insights on the impact of herd management practices on pathogen population structure.