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Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.
Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.
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Animales , Bagres/genética , ADN/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , GenómicaRESUMEN
Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.
Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.
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Echinococcus Granulosus (EG) is the major cause of cystic echinococcosis in humans and livestock in the world. In Chile is a zoonosis of great importance. The most frequently affected geographic areas are the Regions of Aysén, Los Rios, Los Lagos, Coquimbo and the Araucanía. Hence, it was discovered that in endemic areas of hydatidosis there could be several strains and genotypes of EG. In addition, there is evidence that some strains and genotypes are more infectious for human beings than others. This interesting phenomenon of the biology of EG has been studied using molecular biology techniques based on polymerase chain reaction (PCR) and DNA sequence analysis, which has made it possible to characterize the cestode species complex called EG sensu lato (s l) as being comprised of EG sensu stricto (s.s.) (Genotypes G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10), which present an important phenotypic variation detectable in characteristics of the biological cycle, specificity of the intermediate host, pattern of development, pathogenicity, antigenicity, transmission dynamics and, consequently, in the measures needed to control the disease. The aim of this manuscript is to describe the different genotypes of EG described in humans and different livestock host reported in the literature.
Echinococcus granulosus (EG) es la principal causa de equinococosis quística en humanos y ganado en el mundo. En Chile hay una zoonosis de gran importancia. Las zonas geográficas más afectadas son las Regiones de Aysén, Los Ríos, Los Lagos, Coquimbo y la Araucanía. Por lo tanto, se descubrió que en áreas endémicas de hidatidosis podría haber varias cepas y genotipos de EG. Además, hay pruebas de que algunas cepas y genotipos son más infecciosos para los seres humanos que otros. Este interesante fenómeno de la biología del EG ha sido estudiado utilizando técnicas de biología molecular basadas en la reacción en cadena de la polimerasa (PCR) y análisis de secuencias de ADN, lo que ha permitido caracterizar el complejo de cestode llamado EG sensu lato (sl) EG (G3) y E. canadensis (G6-G10), que presentan una importante variación fenotípica detectable en las características del ciclo biológico, especificidad del huésped intermedio, patrón de desarrollo, patogenicidad, antigenicidad, dinámica de transmisión y, por consiguiente, en las medidas necesarias para el control de la enfermedad. El objetivo de este manuscrito fue describir los diferentes genotipos de EG descritos en humanos y diferentes animales de ganado reportados en la literatura.
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Humanos , Animales , Echinococcus granulosus/genética , Equinococosis/parasitología , Equinococosis/veterinaria , Echinococcus granulosus/clasificación , Complejo IV de Transporte de Electrones/genética , Genotipo , Ganado/parasitología , Tipificación Molecular , Técnica del ADN Polimorfo Amplificado Aleatorio , Especificidad de la EspecieRESUMEN
Aims: The aim of this study was to investigate the genetic relatedness of the most prevalent Candida bloodstream infection (BSI) species in in a Malaysian population via Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) fingerprinting. Methodology and results: The genomic DNA of 43 Candida BSI blood culture samples obtained from Universiti Malaya Medical Centre (UMMC) was isolated, after which species identification was carried out using PCR with ITS-1 and ITS-4 pan-fungal primers in conjunction with CHROMagar™ Candida. The predominant Candida species in the BSI samples is Candida albicans (14 out of 43 isolates). RAPD-PCR on these 14 C. albicans clinical isolates was performed using PST as the arbitrary primer. Data analysis using MEGA found an overall non-relatedness of these 14 clinical isolates [average similarity coefficient (SAB) value 0.733±0.172]. Following in-depth analysis, five of the 14 isolates were observed to be identical (SAB values of 1.00 each), four isolates had SAB values of 0.80-0.99, indicating that they are highly similar, but are non-identical, while five isolates are unrelated (SAB lower than 0.80). This suggests that microevolution might have occurred and that these clinical isolates may possibly belong to different strains. Conclusion, significance and impact of study: A fair degree of genetic heterogeneity was found among the 14 C. albicans isolates from UMMC. To our knowledge, this is the first report on the genetic profiles of C. albicans bloodstream infection isolates from Malaysia, warranting further studies in the possible evolutionary trends within this Candida species in Malaysia. Keywords: Randomly Amplified Polymorphic DNA PCR (RAPD-PCR), Candida albicans, Candida bloodstream infections, Genetic relatedness, DNA fingerprinting
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Candida albicansRESUMEN
The genetic variability of Atta sexdens rubropilosa leaf-cutting ants collected from five brazilian localities was evaluated with PCR-RAPD technique. We used 15 primers producing 148 fragments of which 123 (83.11 %) contained polymorphisms. The estimated Shannon index was 0.3836 ± 0.2335 showing that these ants possess high genetic diversity. The G ST value was 0.2372 and ΦPT = 0.184, indicating that the analyzed populations are moderately differentiated and 82 % of the variation obtained occur within populations. Although Mantel's test had shown correlation between genetic distances and geographic was observed that Ivatuba and Itambé (33.8 km) have the small geographical distance and the largest genetic distance. The lower genetic distance was estimated for Maringá and Ivatuba but this localities have a small geographic distance (42.3 km), indicating that there are no barriers for mating among reproducers in these populations. The high degree of polymorphism (83.11 %) and the ability to cross among the populations in the studied regions indicate that this species of leaf-cutting ant is well adapted to the region; therefore, integrated control programs can be developed.
La variabilidad genética de las hormigas Atta sexdens rubropilosa colectadas en cinco lugares distintos de Brasil fueron evaluados por la técnica PCR-RAPD. Un total de 15 primers produjeron 148 fragmentos, de los cuales 123 fueron polimórficos, lo que corresponden al 83,11 %. La estimación de la diversidad genética por el índice de Shannon fue 0,3836 y el desviación estándar fue de ± 0,2335. Estos valores demuestran una alta diversidad genética. El valor de G ST fue 0,2372 y ΦPT = 0,184 lo que indica que las poblaciones están moderadamente diferenciadas y que el 82 % de la variación obtenida se produce dentro de las poblaciones. Aunque la prueba de Mantel ha demostrado una correlación entre la distancia genética y geográfica se observó que Ivatuba e Itambé (33,8 km) tiene una pequeña distancia geográfica y la mayor distancia genética. La distancia inferior genética fue estimada para Maringá e Ivatuba pero estas localidades cuentan con una distancia geográfica pequeña (42,3 km), lo que indica que no hay barreras para el apareamiento entre los reproductores en estas poblaciones. El alto valor de polimorfismo (83,11 %) y la capacidad de emparejamiento entre las poblaciones presentes en las regiones estudiadas, indican que esta especie de hormiga cortadora está bien adaptada a la región, y deben ser desarrollados programas integrados de control si se convierten en plagas.
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Introduction: Nosocomial Citrobacter spp. is emerging as a successful nosocomial pathogen in neonates in Nepal. The important risk factor being poor infection prevention and control practices. The objective of this study was to investigate the clonal relatedness of Citrobacter freundii isolated from clinical and nonclinical sources in Neonatal Intensive Care Unit (NICU) and to determine the presence of Extended Spectrum Beta-Lactamase (ESBL) genes and class 1 integron element. Materials and Methods: Polymerase chain Reaction (PCR) and PCR-Randomly Amplified Polymorphic DNA typing of the isolates were performed in three isolates to amplify class 1 integron element integrase gene, ESBL genes, and to study the clonal relatedness, respectively. Results: Two isolates harbored class 1 integron element. The blaCTX-M was present in all isolates and blaTEM-1 was present in one isolate. An isolate carried blaCTX-M and blaTEM-1 genes. All of these isolates were not clonally related. Conclusion: The study for the first time documented the emergence and spread of ESBL genes and class 1 integron element in multidrug resistant C. freundii in Nepal and urge for monitoring and surveillance of these strains.
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Two fungal isolates obtained from roots of Cymbidium goeriingii in Jeju island were confirmed to be symbiotic with orchid plantlets, and were compared with other orchid mycorrhizal (OM) fungi previously isolated. The two isolates differed in their peloton structures formed in the roots of Cymbidium kanran hybrid 'Chungsu' and in responses of orchid plant. These two isolates differed from the additionally tested OM fungi in some features, and from root damaging species of Rhizoctonia and Fusarium as based on cluster analysis after PCR-RAPD with the primers, Bioneer-28 and OPO-2. With this simple and fast technique, it was possible to distinguish OM fungi from the plant root pathogenic fungi based on calculation of their polymorphic bands. This technique can therefore be helpful to distinguish the OM fungi from the root pathogens. Particularly, the new isolates are considered as new resource of symbiotic fungi for horticultural industries.