GENETIC DIVERSITY EEL Anguilla marmorata BY RAPD IN THUA THIEN HUE PROVINCE, VIETNAM

Anguilla marmorata is a species with high economic value and increasingly interested by organizations and scientists. So far, many of the eel's biological characteristics remain mysterious, and they are often classified according to morphological features such as pigmentation patches, number of vertebrae, ... It is even difficult to distinguish one individual from another in some species, especially in the larval stage. In this study, the Random amplification of polymorphic DNA (RAPD) molecular marker was used to evaluate the genetic diversity of 48 eels collected in Thua Thien Hue province. Results showed that the genetic diversity of individuals in the Eel population studied is quite high. With 8 random primers via PCR, 77 DNA tapes with 76 polymorphic tapes were obtained, the tape size ranged from 170-2,500 bp, in which primer S10 showed the highest diversity with an average Ho value of 0.563, followed by primer S8 (Ho = 0.558). The lowest diversity was in the OPD5 primer (Ho = 0.300). The OPG17 primer is the primer that produces the most polymorphic tapes (13/13 tapes) and the S3 primer for the least amplified tapes polymorphism (9/10 DNA tapes). The diversity coefficient in each random primer ranged from about 0.300 to 0.563, with an average of 0.433. The genetic variation in the Eel population is random. Genetic variation can be attributed mainly to different eel breeding conditions and origins. Genetic similarity coefficients among the Eels varied from 0.660 to 0.910 and were divided into two main groups in genetic similarity coefficient 0.660.

DNA Damage in Human Amniotic Cells: Antigenotoxic Potential of Curcumin and α-Lipoic Acid

Oxidative imbalances in the gestational phase are responsible for certain complications during pregnancy and for foetal and neonatal genetic disorders. In this work, using human amniocytes, we aimed to evaluate the protection provided to foetal DNA by two concentrations of antioxidant molecules, α-lipoic acid (LA) and curcumin (Cur), against hydrogen peroxide (H2O2)-induced damage. Genotoxicity tests, performed by the random amplification of polymorphic DNA (RAPD-PCR) technique and TUNEL tests, showed that the lowest concentration of LA-protected cells and DNA from H2O2 insults. However, a greater ability to protect the amniocytes’ DNA against H2O2 was observed following co-treatment with the highest concentration of Cur with H2O2. In fact, a genomic template stability (GTS%) similar to that of the negative control and a statistically significant reduction in the DNA fragmentation index (DFI) were revealed. Moreover, following a combined treatment with both antioxidants and H2O2, no statistical difference from controls was observed, in terms of both induced mutations and DNA breaks. Furthermore, no effect on morphology or cell viability was observed. The results demonstrate the ability of LA and Cur to protect the genetic material of amniocytes against genotoxic insults, suggesting their beneficial effects in pathologies related to oxidative stress.

Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis

Objective The aim of this study was to identify and isolate Trichosporon asahii (T. asahii) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii-specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon-specific PCR, 65 were found to be T. asahii as identified by T. asahii-specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

PERFIL MOLECULAR DE CANDIDA PARAPSILOSIS ISOLADAS DO AMBIENTE DE UMA UNIDADE DE TERAPIA INTENSIVA NEONATAL

Introdução: Candida spp. é o principal patógeno oportunista do reino Fungi que acomete indivíduos internados em serviços de saúde, sendo Candida parapsilosis a segunda espécie mais comum em amostras clínicas de recém-nascidos. Infecções desencadeadas pelo gênero Candida colocam em risco a saúde e desenvolvimento de neonatos críticos em função de sua maior susceptibilidade. Existem poucos relatos na literatura que avaliam a semelhança genética entre os isolados provenientes do ambiente hospitalar. Objetivo: Analisar o perfil molecular de isolados de C. parapsilosis provenientes do ambiente da Unidade de Terapia Intensiva Neonatal de um hospital universitário. Material e métodos: Foram incluídos no estudo isolados do complexo C. parapsilosis provenientes do ambiente da unidade. Através da técnica Random Amplification of Polymorphic DNA (RAPD-PCR) e utilizando quatro primers (OPA09, OPB11, OPA18 e OPG17) buscou-se avaliar a similaridade genética entre esses isolados. Para análise do RAPD-PCR, os produtos de amplificação do DNA foram submetidos à eletroforese em gel de agarose (2%). As bandas de amplificação observadas nos géis foram transformadas em matrizes de valores binários utilizando o programa estatístico Mult-Variate Statistical Package versão 3.21. As relações genéticas foram calculadas através do coeficiente de Jaccard (Sj), onde valores de Sj igual a 1,00 indicam o mesmo genótipo, valores entre 0,80 a 0,99 representam alta similaridade e valores inferior a 0,80 sugerem amostras distintas. Resultados: Dos cinco isolados analisados, quatro (80%) apresentaram alta similaridade entre si (Sj > 0,90) sendo que três desses (60%) apresentaram o mesmo genótipo (Sj = 1,00), os quais foram obtidos de diferentes leitos (2), da mesa de medicação e gaveta de armário, com um intervalo de 97 dias entre as coletas. Conclusão: O estudo sugere que o ambiente nosocomial pode ser um importante reservatório de C. parapsilosis, favorecendo sua permanência e disseminação pela unidade. Assim faz-se necessário reforçar a limpeza do ambiente hospitalar e estabelecer medidas de controle e prevenção a fim de evitar disseminação e consequentemente infecções invasivas.

Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

Human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.

Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

Polymorphism of Sakini Chicken Population From Different Locations/Altitudes of Nepal Using Randomly Amplified Polymorphic Dna Markers

A study was conducted to evaluate genetic polymorphism in three Sakini chicken populations using random amplification of polymorphic DNA (RAPD) with seven highly polymorphic primers. All populations showed polymorphism with these primers that generates 59 different bands with an average of 8.4 bands per primer with 78.6% polymorphism nature. Primer OPA-16 produced the highest number of polymorphism bands 47 % and the lowest number of bands was produced by the OPA-05 primer 24 %. Differences for genetic distance (D) among populations were significant (P<0.05). A consensus dendogram was therefore developed to show the phylogenetic relationship among the populations. The cluster pattern is well supported by the principle component analysis that also separates all three populations of Sakini chicken into six major groups. The results provide evidence of the applicability of RAPD to determining genetic relatedness within and among different poultry populations and in developing reproducible markers useful in evaluating individual variation in poultry. SAARC J. Agri., 18(2): 115-124 (2020)

Characteristic of Pseudomonas syringae pv. atrofaciens Isolated from Weeds of Wheat Field

The aim of this study was the identification of the causative agent of the basal glume rot of wheat Pseudomonas syringae pv. atrofaciens from the affected weeds in wheat crops, and determination of its virulent properties. Isolation of P. syringae pv. atrofaciens from weeds of wheat crops was carried out by classical microbiological methods. To identify isolated bacteria, their morphological, cultural, biochemical, and serological properties as well as fatty acids and Random Amplification of Polymorphic DNA (RAPD)-PCR (Polymerase chain reaction) profiles with the OPA-13 primer were studied. Pathogenic properties were investigated by artificial inoculation of wheat plants and weed plants, from which bacteria were isolated. For the first time, bacteria that are virulent both for weeds and wheat were isolated from weeds growing in wheat crops. It was shown that the fatty acids profiles of the bacteria isolated from the weeds contained typical for P. syringae pv. atrofaciens fatty acids, in particular, hydroxy acids: 3-hydroxydecanoic, 2-hydroxydodecanoic, and 3-hydroxydodecanoic. RAPD-PCR profiles of the newly isolated strains were identical to those of the collection strains P. syringae pv. atrofaciens UCM B-1011 and P. syringae pv. atrofaciens UCM B-1014 and contained a dominant fragment of 700 bp. The isolated strains, according to their phenotypic and genotypic properties, were identified as P. syringae pv. atrofaciens. It was established that the causative agent of basal glume rot of wheat P. syringae pv. atrofaciens is polyphagous and capable of infecting a wide range of plants. The main control measure for cereals diseases caused by P. syringae pv. Atrofaciens—crop rotations with nonhost species, should be revised, and alternative control methods must be proposed.