mecA-Gene Detection and MDR Profile of S. Aureus Isolates From Patients Attending The Referral Hospitals of Amhara Reginal State, Ethiopia

Background: Staphylococcus aureus causes different types of human infections, and has an ability to develop resistance to many antibiotics. There is scarcity of data on mecA gene and MDR profiles of this organism in developing countries, like Ethiopia. The aim of the present study is therefore, to investigate MDR profiles and mecA-gene profile of S. aureus from Referral Hospitals of Amhara Reginal State. Methods: Of the total of 110 isolates collected from Amhara Region Referral Hospitals, 70 MDR isolates were further processed for isolation of S. aureus mecA gene. Genomic DNA was isolated using SIGMA ALDRICH genomic DNA isolation Kit for Gram positive bacteria. Amplification of S. aureus mecA gene was done using amplicon size of 533 bp. Agarose gel electrophoresis was prepared with 1.5% agarose by TAE solvent and 0.5μg/mL of ethidium bromide. Electrophoresis was carried out for 1 hour and a half (7 Volts/ cm²) in 1X tris acetate EDTA (TAE) buffer. Data were analysed by using descriptive statistics.Results: Majority of the isolates were recovered from patients age less than 5 years, 51 (36.7%) and least from age greater than 60 years, 6 (4.3%). Most of the isolates were from blood, 61 (43.9%), followed by wound, 45 (32.4%). High resistance rate was observed in penicillin 81 (73.6%), followed by cotrimoxazole 78 (70.9%), ceftriaxone 76 (69%), erythromycin 66 (60%) and tetracycline 65 (59.1%). Phenotypically, considering cefoxitin as a surrogate marker, 38 (34.5%) of the isolates were methicillin resistant. The overall MDR isolates were 92 (83.6%). The PCR amplification result of mecA gene was 14 (20%).Conclusions and recommendations: There are high rate of MDR and MRSA isolates of S. aureus. PCR amplification result indicates 20% of MRSA isolates are mecA gene producers. Largescale study for detection of MDR strains of S. aureus including MRSA using molecular techniques should be encouraged in Amhara region.

A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits

DNA isolation from formalin fixed paraffin embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. We developed a new protocol (IARCp) to improve better quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6 to 394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments. Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. Our proposed protocol does not require the use of any commercial kits for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries (LMICs).

Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells

Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to other organisms.

Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.HighlightA chromosome-level genome assembly of C. cinerea #326A fast and efficient high-molecular-weight fungal genomic DNA isolation protocolStructural variant and single nucleotide variant calling using Nanopore readsA series of solutions and reference parameters for fungal genomic analysis on MinION

Molecular Typing of Staphylococcus Aureus Isolated from Various Environmental Sources

RAPD-PCR was employed for the characterization of S. aureus isolates from different sources such as soil, water, milk, meat and skin swab etc.,. Isolated pure cultures of S.aureus strains were subjected to genomic DNA isolation, purification, separation and quantification. Isolated DNA samples were distinguished by using 4 different random primers. Genome profile analysis obtained from the S.aureus demonstrated that it was possible to differentiate the S. aureus strains from different sources by RAPD technique. Results indicate possible relationships between host origin and genetic variation among S. aureus isolates from various sources. This RAPD method was thus useful for epidemiological studies of the S. aureus flora.

A one-tube method for rapid and reliable plant genomic DNA isolation for PCR analysis

BackgroundConsistent isolation of high quality plant genomic DNA is a prerequisite for successful PCR analysis. Time consumption, ease of operation and procedure cost are important secondary considerations for selecting an effective DNA extraction method. The simple, reliable and rapid DNA extraction method developed by Edwards and colleagues in 1991 [1] has proven to be the gold standard.ResultsThrough modification of the Edwards method of extraction, we have developed a one-tube protocol that greatly improves the efficiency of plant DNA extraction and reduces the potential for sample contamination while simultaneously yielding high quality DNA suitable for PCR analysis. We further show that DNA extracts prepared with this method are stable at room temperature for at least three months.ConclusionThe one-tube extraction method yields high quality plant DNA with improved efficiency while greatly minimizing the potential for cross contamination. This low-cost and environment-friendly method is widely applicable for plant molecular biology research.

Genetic variation and molecular relationships taxa of Conringia heist. ex Fabr. (Brassicaceae) based on RAPD markers in Turkey

In this study, genetic variation and phylogenetic analysis of 13 populations of 6 species belonging to Conringia genus spreading in Turkey were performed using RAPD markers. Genomic DNA isolation from the leaves of the Conringia plant samples was performed via using a commercial kit. Seven RAPD primers were used to identify the genetic diversity between the populations. Polymerase Chain Reaction (PCR) was performed using DNA samples and primers. PCR products were resolved using agarose gel electrophoresis and visualized under UV light. All gel images were analyzed, and the absence and presence of polymorphic bands were scored. The total of 34 DNA bands were detected by seven RAPD primers. PAUP 4.0b10 analysis program was used to calculate phylogenetic tree and genetic distances between the species. The phylogenetic tree was obtained using the UPGMA algorithm and it was composed of two clades. According to the PAUP analysis, the species having the closest distance between each other are C. planisiliqua (Ankara-Aya?) and C. planisiliqua (Ankara-Nall?han) with the value of 0.000 and those having the longest distance are C. grandiflora (Akseki ?ukurk?y) and C. orientalis (Elaz??-Baskil) with the value of 0.6000. The results suggest that the RAPD markers are useful tools to demonstrate the genetic relationships between populations of the Conringia species.

Some aspects of the genomic DNA isolation from lavender plants of different origin.

The method of DNA extraction from lavender plants has some difficulties. Moreover, molecular genetic studies often use not only fresh materials but also microplants from an in vitro culture or frozen material. For all these types of samples, the DNA isolation technique was developed, which is based on the CTAB buffer, but without the use of liquid nitrogen, mercaptoethanol and sodium acetate, which, in turn, simplifies and reduces the cost of the method.