Genetic diversity analysis of Morchella sp.by RAPD.

This study investigated the genetic diversity of morchella species using randomly amplified polymorphic DNA markers. Morchella species are an important group of edible mushrooms belonging to the family Helveliaceae with medicinal and economical significance. In this study we have developed an efficient method of genomic DNA isolation which was amplified by eight RAPD primers to test the polymorphism in three species of morchella. Out of all eight primers tested in current study, one of them (B8) resulted 100% polymorphism among the three studied species. Based on the RAPD profile a similarity matrix was generated to construct a dendrogram for phylogenetic analysis revealing the relationship among the three species of morchella.

Genomic variability of Helicobacter pylori isolates of gastric regions from two Colombian populations.

AIM: To compare the genomic variability and the multiple colonization of Helicobacter pylori (H. pylori) in patients with chronic gastritis from two Colombian populations with contrast in the risk of developing gastric cancer (GC): Túquerres-Nariño (High risk) and Tumaco-Nariño (Low risk). METHODS: Four hundred and nine patients from both genders with dyspeptic symptoms were studied. Seventy-two patients were included in whom H. pylori was isolated from three anatomic regions of the gastric mucosa, (31/206) of the high risk population of GC (Túquerres) and (41/203) of the low risk population of GC (Tumaco). The isolates were genotyped by PCR-RAPD. Genetic diversity between the isolates was evaluated by conglomerates analysis and multiple correspondence analyses. RESULTS: The proportion of virulent genotypes of H. pylori was 99% in Túquerres and 94% in Tumaco. The coefficient of similarity of Nei-Li showed greater genetic diversity among isolates of Túquerres (0.13) than those of Tumaco (0.07). After adjusting by age, gender and type of gastritis, the multiple colonization was 1.7 times more frequent in Túquerres than in Tumaco (P = 0.05). CONCLUSION: In Túquerres, high risk of GC there was a greater probability of multiple colonization by H. pylori. From the analysis of the results of the PCR-RAPD, it was found higher genetic variability in the isolates of H. pylori in the population of high risk for the development of GC.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

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.

Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

Correlation of Oxacillinase Gene Carriage With the Genetic Fingerprints of Imipenem-Resistant Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: Emergence of multidrug-resistant Acinetobacter baumannii has resulted in the treatment failure of related infections and an increase in patient mortality. The presence of class D ß-lactamases (oxacillinases) in this organism is an important mechanism underlying resistance to beta-lactam antibiotics. OBJECTIVES: The aim of this work was to investigate the correlation between oxacillinase gene carriage and genetic fingerprints in imipenem-resistant burn and non-burn isolates of A. baumannii. MATERIALS AND METHODS: Fifty-eight A. baumannii isolates were collected from October 2011 to April 2012, which included 28 burn isolates from Shahid Motahari Hospital and 30 non-burn isolates from Imam Hossein Hospital. The minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) of imipenem were measured by the broth microdilution method. The presence of oxacillinase genes (OXA-23-, OXA-24-, OXA-51-, and OXA-58-like genes) was shown using type-specific primers and PCR. Genetic profiles were generated by RAPD-PCR fingerprinting. RESULTS: OXA-23 was observed in 81% of the isolates and its distribution was similar within the two groups. The presence of OXA-51 was shown in 58.6% of the isolates, of which most were burn isolates (67.6%). OXA-24 was present in 20.7% of the isolates, all belonging to the burn group; OXA-58 was not observed in any of the isolates. RAPD-PCR fingerprints revealed two clusters at a similarity level of 70% (A, B). At a similarity level of 85%, nine different groups were observed for burn and non-bun isolates. CONCLUSIONS: Our results showed that bla OXA-23 was the most prevalent gene, followed by bla OXA-51 , among the burn and non-burn clinical isolates of A. baumannii. Bla OXA-24 -like genes were detected at a lower level and were only found among the burn isolates, which also showed higher heterogeneity compared to the non-burn group.

Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol.

In Present work, the main objective is to develop less time consuming protocol for genomic DNA isolation from leaves of Passiflora foetida. Optimized protocol is cost effective, as it avoided use of expensive liquid nitrogen. The important parameters of CTAB buffer composition such as Polyvinylpyrrolidone PVP40000 (without PVP, 1%, 2%, 3.5%, 4.0%, 4.5%, 5.0%), CTAB (w, 1%, 2%, 3%, 4%, 5%), water bath temperature (30°C to 70°C) and duration on water bath for half hr and one and half hr has been optimized. CTAB (2%), PVP (1%), water bath temperature (70%), duration on water bath (1 hr) has efficiently yielded DNA quality of 200-1782 µg/0.5gm from leaf, stem, root, tendril and flower. However, 168 µg - 1782 µg of DNA has been obtained from 0.5 g of leaf of Passiflora foetida. Polyphenol contamination has been overcome using 5M NaCl and PVP. Acetate has been used for obtaining double-stranded DNA in stabilized form. Current DNA extraction protocol takes maximum of four hours for completion, which is many time savings. RAPD-PCR reaction parameters such as DNA concentration (100ng), Primer concentration (2 µM), Dream Taq polymerase (2 U), annealing temperature (29°C) and number of cycles for amplification of DNA has been optimized. Primer fragment Akansha 7 shows high polymorphism of 7 fragments ranges from 200bp - 2500 bp. Current optimized protocol of DNA isolation is specifically for Passiflora foetida, which can be used for downstream molecular techniques.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

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.

Molecular epidemiology of cystic echinococcosis: genotypic characterization in humans and different livestock / Epidemiología molecular de la equinococosis quística: caracterización genotípica en humanos y diferentes animales

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.

Genetic relatedness of Candida albicans bloodstream infection clinical isolates in Malaysia

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

POPULATION GENETICS OF Atta sexdens rubropilosa (HYMENOPTERA: FORMICIDAE) / Genética de poplaciones de Atta sexdens rubropilosa (Hymenoptera: Formicidae)

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.

Interactions of Newly Isolated Orchid Mycorrhizal Fungi with Korean Cymbidium kanran Hybrid 'Chungsu'

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.