Improvement of soil testing techniques for detecting spores of potato wart disease Synchytrium endobioticum using molecular methods

Synchytrium endobioticum (Schilb.) Percival. is a pathogen of potato wart disease and has a restricted distribution on the territory of the Russian Federation. Its main pathways are infected potato tubers and different planting material containing soil particles infected with spores of the fungus. One of the main problems is the use of toxic chemicals during detecting the disease in laboratory methods of direct soil testing to identify resting spores. This paper presents the assessment of molecular methods of soil diagnosis for detection of S. endobioticum by direct extraction of fungal DNA from soil samples using the MetaGen reagent kit. Identification was performed using the Fitoskrin. Synchytrium endobioticum-RT kit. The kit was pre-tested using DNA isolated from potato warts by various commercial kits. It was found that the optimal method of DNA isolation from the warts was using the FitoSorb-Avtomat 48 kit at the Tecan robotic station. Studies have shown that the sensitivity of the direct DNA extraction method from soil samples with various infection levels is the same as that of flotation method using carbon tetrachloride. Moreover, this method makes it possible to work with soil samples of different types, including peaty soils.

Prevalence of Epstain–Bar Virus among Patients with Periodontitis in Al–Najaf Al–Ashraf / Iraq

Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans which characterized by periodontal damage, alveolar bone resorption, pain, and eventual tooth loss. Epstein–Barr Virus (EBV) has widely infected >90% adults in the world and is associated with many human diseases, so that this study aimed to investigate the association between EBV and periodontitis. Patients and samples: Subgingival paper point samples were collected from 100 patients with periodontitis and 30 healthy people. All samples undergo direct DNA extraction to amplified EBVs DNA using PCR technique. The results indicated a high percentage of EBV infections (18%) in patient suffering from periodontitis while there was no EBV infection were detected in healthy persons. A high percentage of EBV was detected in female (56%) in comparison with male (44%). The results improved the association between EBV and periodontitis suggesting that EBV may serve as a pathogenic factor leading to periodontitis among patients.

An Improved Procedure of the Metagenomic DNA Extraction from Saline Soil, Sediment and Salt

A new modified protocol has been developed for extracting pure community inhibitors-free DNA from saline soils, sediments and salts. Amplification of DNA from soil and sediment is often inhibited by copurified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to saline samples. Using a widely used a newly modified direct DNA extraction method proposed in this report, DNA was extracted from samples of Urmia Lake in diverse geological location in Iran and quantity of the DNA were examined. We developed an improved method to extract DNA include the combination of physical, chemical and mechanical lysis methods from saline samples. In the earlier reports, skim milk as an adsorption competitor was added to buffer DNA extract. In current study, we added skim milk to buffer DNA extraction. The results showed that skim milk was useful as an additive for extract DNA from saline samples. This method is applicable to molecular community analysis of saline samples which strongly adsorb DNA. The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA. The extracted DNA was used to successfully amplify 16SrRNA region and functional genes. The amplicons were suitable for further applications such as diversity based analysis by denaturing gradient gel electrophoresis (DGGE) and cloning library.

The melioidosis agentBurkholderia pseudomalleiand related opportunistic pathogens detected in faecal matter of wildlife and livestock in northern Australia

SUMMARYThe Darwin region in northern Australia has experienced rapid population growth in recent years, and with it, an increased incidence of melioidosis. Previous studies in Darwin have associated the environmental presence ofBurkholderia pseudomallei, the causative agent of melioidosis, with anthropogenic land usage and proximity to animals. In our study, we estimated the occurrence ofB. pseudomalleiandBurkholderiaspp. relatives in faecal matter of wildlife, livestock and domestic animals in the Darwin region. A total of 357 faecal samples were collected and bacteria isolated through culture and direct DNA extraction after enrichment in selective media. Identification ofB. pseudomallei, B. ubonensis, and otherBurkholderiaspp. was carried out using TTS1, Bu550, andrecABUR3–BUR4 quantitative PCR assays, respectively.B. pseudomalleiwas detected in seven faecal samples from wallabies and a chicken.B. cepaciacomplex spp. andPandoraeaspp. were cultured from wallaby faecal samples, andB. cenocepaciaandB. cepaciawere also isolated from livestock animals. Various bacteria isolated in this study represent opportunistic human pathogens, raising the possibility that faecal shedding contributes to the expanding geographical distribution of not justB. pseudomalleibut other Burkholderiaceae that can cause human disease.

Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such asMycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.