Direct DNA Extraction Research Articles

A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Loop-mediated isothermal amplification (LAMP) assays for rapid on-site detection of Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus in probiotic products

We developed the colorimetric loop-mediated isothermal amplification (LAMP) and a real-time LAMP with direct DNA extraction for the on-site detection of the three closely related Lacticaseibacillus species, such as Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus in probiotic products. Species-specific LAMP primers were designed targeting a unique gene in each species through comparative genomic analysis. Each LAMP primer set amplified target species specifically, distinguishing between the closely related species within the Lacticaseibacillus species. Based on the designed LAMP primers, two LAMP-based assays with direct DNA extraction were compared; their limit of detection was 103 CFU/mL for the Lacticaseibacillus species. In addition, both methods were applied to 44 commercial probiotic products and obtained identical results; it took about 45 min from sample preparation to confirming the presence or absence of the Lacticaseibacillus species. Thus, these methods can be rapid and simple in-field tests to detect L. casei, L. paracasei, and L. rhamnosus in probiotic products.

Cryptosporidium species diagnosis in handlers of domestic pigeons in Baghdad City: Molecular and microscopic approaches.

The aim of this study is to detect the prevalence and diagnosis of molecular characteristics of Cryptosporidium infection in handlers of domestic pigeons in Baghdad City. Traditional and molecular diagnostic methods were employed to detect and identify Cryptosporidium species. Sixty stool samples were collected from the handlers of domestic pigeons, and various techniques, including direct smear, flotation concentration, staining methods, and DNA extraction coupled with nested polymerase chain reaction (PCR), were utilized. The results obtained from traditional methods indicated an overall infection rate of 55% in handlers of domestic pigeons, while significant variations were noted between male and female handlers. Age group 21-40 years were found to have higher infection rates were found to have higher infection rates. The prevalence of Cryptosporidium also displayed temporal variability throughout the study period. Molecular analysis using nested PCR revealed higher infection rates of 86% in handlers of domestic pigeon samples. Genotyping and phylogenetic analysis identified the presence of Cryptosporidium parvam species in handlers of domestic pigeons, indicating zoonotic transmission potential. These findings underscore the high prevalence of Cryptosporidium infection among handlers of domestic pigeons in Baghdad City.

Identification of Microbial Populations Present in Agricultural and Nonarable Soils in the Talas Valley, Northern Kyrgyzstan, in Autumn

Soil bacterial and fungal communities were investigated in relation to soil type and farm management practices after vegetation harvesting in autumn. Soils from fields cultivated with Phaseolus vulgaris (bean) and Pyrus comminus (pear) and nonarable, natural areas were studied. Microbial diversity was analysed using cultivation-dependent methods (isolation of pure cultures) and cultivation-independent methods (direct extraction of DNA from soil, followed by PCR amplification of the 16S rRNA and 18S rRNA genes). The use of cultivation-dependent methods revealed that there were no differences in the biodiversity of the soil bacterial and fungal communities between fields cultivated with bean plants and pear trees. However, the use of cultivation-independent methods showed that there were clear soil and crop type-specific effects on the composition of the soil bacterial and fungal communities. The density of the bacterial population was two times higher in northern mountain-valley serozem (NMVS) soil samples than in light chestnut (LC) soil samples. In contrast, the densities of the fungal communities were almost equal in the studied soil types. The density of the actinomycetes community was almost two times higher in LC soil than in NMVS soil under bean plants. The Shannon index values showed that the bacterial biodiversity in the NMVS soil samples was greater than that in the LC soil samples. Soils under fallow appeared to have diverse bacterial communities that mainly consisted of local autochthonous microflora and a small amount of zymogenic microflora (since fresh plant residue does not enter the soil). The Shannon index results revealed two interesting facts: (1) the soil bacterial community was highly diverse in soils that supported bean plants and (2) the soil fungal biodiversity was high under pear trees in both soil types.

Detection of Clostridium difficile among diarrheic children using cultural and polymerase chain reaction technique

Introduction: Clostridium difficile is the most common cause of antibiotic-associated diarrhea and colitis. Several methods are available for the detection of C. difficile in stool samples. This study aimed to use glutamate dehydrogenase (GDH), toxin detection, culture and polymerase chain reaction (PCR) techniques for the diagnosis of this pathogen. Methodology: A total of 300 stool samples were collected from children with hospital acquired diarrhea (HA-D), community acquired diarrhea (CA-D), and hospitalized non-diarrheic children as control with ages ranging from 6 months to 6 years (mean 3.7 ± 1.7). Each stool sample was divided into two parts; one part was tested for the enzyme GDH, toxin A and B and then cultured on selective media; and the other part for direct DNA extraction. Results: From a total of 300 stool samples, 9 (3.0%) were positive for C. difficile by the PCR technique, 7 (7%) samples of which were from HA-D cases and 2 (2.0%) from CA-D cases; the control group samples were negative. The enzyme GDH was detected in 12 (12%) samples and toxins A and B in 8 (8%) samples from HA-D cases compared to 5 (5%) and 2 (2%), respectively from CA-D cases. Both GDH and the toxins were negative in control samples. Only 19 (19.0%) samples from HA-D cases gave suspected growth and all of these were negative by PCR. Conclusions: Based on the results of this study, we conclude that the PCR technique is the only reliable method for the diagnosis of this pathogen.

Assessing the diversity of plankton-associated prokaryotes along a size-fraction gradient: A methodological evaluation

Marine free-living (FL) and plankton-associated prokaryotes (plankton-microbiota) are at the basis of trophic webs and play crucial roles in the transfer and cycling of nutrients, organic matter, and contaminants. Different ecological niches exist along the plankton size fraction gradient. Despite its relevant ecological role, the plankton-microbiota has rarely been investigated with a sufficient level of size-fraction resolution, and it can be challenging to study because of overwhelming eukaryotic DNA. Here we compared the prokaryotic diversity obtained by 16S rRNA gene sequencing from six plankton size fractions (from FL to mesoplankton), through three DNA recovery methods: direct extraction, desorption pretreatment, enrichment post-treatment. The plankton microbiota differed strongly according to the plankton size-fraction and methodological approach. Prokaryotic taxa specific to each size fraction, and methodology used, were identified. Vibrionaceae were over-represented by cell desorption pretreatment, while prokaryotic DNA enrichment had taxon-specific effects, indicating that direct DNA extraction was the most appropriate method.

Prevalence of three Mycoplasma sp. by multiplex PCR in cattle with and without respiratory disease in central Mexico.

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ < 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.

Cecal Reduction of Brachyspira and Lesion Severity in Laying Hens Supplemented with Fermented Defatted ‘Alperujo’

Antimicrobial resistance demands the development of therapeutic alternatives such as prebiotics, probiotics, and nutraceuticals. The aim of this study was to assess the antimicrobial proprieties of the nutraceutical fermented defatted “alperujo”, derived from olive oil production, in a laying hen farm (n = 122,250) endemic with avian intestinal spirochetosis (Brachyspira spp.). Part of the batch (n = 1440) was divided into six groups of 240 hens each that included 80 or 108-week-old laying hens, supplemented with 0%, 2%, or 6% fermented defatted ‘alperujo’ for a month. At the end of the experiment, eight hens from each group were autopsied and cecal content was subjected to (i) Brachyspira culture and species identification by PCRs, and (ii) direct DNA extraction and Brachyspira qPCR. Furthermore, the ceca were processed for histopathology. Microbiological isolation revealed B. pilosicoli and B. hyodysenteriae co-infection in all groups. The 80-week-old hen group 2% supplemented showed a reduction in the cecal Brachyspira content (qPCR) compared with non-supplemented hens. Cecal histopathology showed a diffuse mild infiltration of lymphocytes, plasma cells, and heterophils; and hyperplasia of the gut-associated lymphoid tissue hyperplasia which decreased in severity in 80-week-old supplemented hens. The reduction in Brachyspira colonization and the severity of the lesions observed in supplemented hens highlights a potential protective function against avian intestinal spirochetosis.

Establishment of a rapid direct DNA preparation procedure for the detection of Clostridium botulinum serotype B from honey

Honey is at risk of contamination with Clostridium botulinum that causes botulism.&amp;nbsp;Rapid detection of the pathogen in honey is important in food safety assurance and food&amp;nbsp;poisoning surveillance. C. botulinum is capable of forming spores at low concentration; thus,&amp;nbsp;regular detection methods must begin with an enrichment step before the main procedure.&amp;nbsp;This additional step requires long time, complicated preparation, and might fail due to&amp;nbsp;improper selective media. Therefore, this study aimed to establish a rapid detection&amp;nbsp;procedure for C. botulinum serotype B directly from honey without enrichment culture by obtaining total DNA to use as template for the amplification of a fragment from botulinum&amp;nbsp;neurotoxin type B (BoNT/B) encoding gene. Using 6 honey samples provided by the&amp;nbsp;National Institute of Hygiene and Epidemiology with positive PCR results after enrichment,&amp;nbsp;all three DNA extraction methods including Exgene extraction kit, freeze-thaw cycling and&amp;nbsp;bead beating, showed amplification signals. Furthermore, when combined with the&amp;nbsp;isothermal loop-mediated amplification, the total time could be shortened to less than two&amp;nbsp;hours since collection. In addition, we observed PCR inhibition by the honey matrix and&amp;nbsp;recommended dilution of the sample 5-10 times for the reaction. With the advantages of&amp;nbsp;rapid and simple procedure, direct DNA extraction from honey could be used in field. &amp;nbsp;

Microbial communities of vegetable seeds and biocontrol microbes for seed treatment

Thirty-one species of vegetable seeds from Kyrgyzstan's State Register were analysed for mycological and bacterial composition using various methods, from simple visual to molecular. The modified roll-towel method simultaneously assesses the degree of contamination and seed germination. S creening in agar medium allows analysis of species on the outer and inner surfaces of vegetable seeds that are not detected using the roll-towel method. As a result of the phytopathological examination, 15 fungal and three bacterial species were found. Direct DNA extraction and amplification of the universal 16S rRNA gene of diseased seeds was used to identify the plant pathogenic bacteria, which included Pseudomonas syringae, Pseudomonas savastanoi, Pseudomonas cerasi, Pectobacterium carotovorum, Xanthomonas oryzae, Erwinia sp. and others from the Proteobacteria phylum. For the coating of vegetable seeds, S. alfalfae CI-4 was selected. This strain had a beneficial double effect on the tested vegetable seeds at 1 × 103 spores mL -1.

Diversity of Lysis-Resistant Bacteria and Archaea in the Polyextreme Environment of Salar de Huasco.

The production of specialized resting cells is a remarkable strategy developed by several organisms to survive unfavorable environmental conditions. Spores are specialized resting cells that are characterized by low to absent metabolic activity and higher resistance. Spore-like cells are known from multiple groups of bacteria, which can form spores under suboptimal growth conditions (e.g., starvation). In contrast, little is known about the production of specialized resting cells in archaea. In this study, we applied a culture-independent method that uses physical and chemical lysis, to assess the diversity of lysis-resistant bacteria and archaea and compare it to the overall prokaryotic diversity (direct DNA extraction). The diversity of lysis-resistant cells was studied in the polyextreme environment of the Salar de Huasco. The Salar de Huasco is a high-altitude athalassohaline wetland in the Chilean Altiplano. Previous studies have shown a high diversity of bacteria and archaea in the Salar de Huasco, but the diversity of lysis-resistant microorganisms has never been investigated. The underlying hypothesis was that the combination of extreme abiotic conditions might favor the production of specialized resting cells. Samples were collected from sediment cores along a saline gradient and microbial mats were collected in small surrounding ponds. A significantly different diversity and composition were found in the sediment cores or microbial mats. Furthermore, our results show a high diversity of lysis-resistant cells not only in bacteria but also in archaea. The bacterial lysis-resistant fraction was distinct in comparison to the overall community. Also, the ability to survive the lysis-resistant treatment was restricted to a few groups, including known spore-forming phyla such as Firmicutes and Actinobacteria. In contrast to bacteria, lysis resistance was widely spread in archaea, hinting at a generalized resistance to lysis, which is at least comparable to the resistance of dormant cells in bacteria. The enrichment of Natrinema and Halarchaeum in the lysis-resistant fraction could hint at the production of cyst-like cells or other resistant cells. These results can guide future studies aiming to isolate and broaden the characterization of lysis-resistant archaea.

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.

Simple direct DNA extraction protocols for efficient routine detection of plant pathogenic bacteria via conventional PCR

ABSTRACT Rapid, easy and direct DNA extraction protocols are required for use in routine analyses to detect plant pathogenic bacteria. For this purpose, five protocols for extracting bacterial DNA, which do not use toxic chemicals such as phenol and chloroform, were optimized for the detection of plant pathogenic bacteria quickly and directly from plant material using conventional polymerase chain reaction (PCR). The protocols are based on the use of different maceration and extraction buffers to ensure quality bacterial DNA extraction and to remove the PCR inhibitors. Results showed that the protocol #1 based on the use of Tris and TNPEE buffers and protocol #3 based on the use of 0.4% bovine serum albumin and 0.05% Tween 20 were able to detect Erwinia amylovora from apple and pear samples. Protocol #2 that used 2% polyvinyl pyrrolidone in maceration and extraction buffers was found to be efficient for detecting Erwinia amylovora, Agrobacterium tumefaciens, and Allorhizobium vitis directly from plant tissues. However, protocols #4 and #5 based on the use of 1% Triton X-100 and 0.1% Tween 20, respectively, were unable to detect the studied bacteria. Accordingly, the protocol #2 is proposed for efficient, direct and rapid detection of plant pathogenic bacteria in plant materials via PCR.

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 &gt;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.&#x0D; 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.&#x0D; 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%).&#x0D; The results improved the association between EBV and periodontitis suggesting that EBV may serve as a pathogenic factor leading to periodontitis among patients.

Direct next-generation sequencing analysis using endometrial liquid-based cytology specimens for rapid cancer genomic profiling.

Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid-based cytology (LBC) specimens. Since LBC specimens preserve high-quality DNA, cancer genome profiling using next-generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time. Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling. NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin-fixed paraffin-embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days. The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis)

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4–400 parasites/g, DNA-RR: 19–80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10–1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

An improved method for extraction of microbial DNA from alkaline-saline soil

A modified method for the direct extraction of DNA from alkaline-saline soils with minimum DNA fragmentation and a possible reduction in chimera formation during polymerase chain reaction (PCR) was developed. The commercial extraction kit Power Soil DNA (Mo Bio™ Laboratories, Inc.) was used as a reference technique. The method reported here was based on cell lysis employing ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and cell disruption with mechanical force with FastPrep-24™ equip followed by one cycle of freezing at -40 °C for 60 min and thawing at 65 °C for 20 min. The extraction method was tested for allophonic soils with large concentrations of organic matter, fulvic and humic acids, electrolytic conductivity (EC) ranging between 2.6 dS m-1 and 39.9 dS m-1, and pH between 8.8 and 10.9. The yield of DNA extracted depended on soil type, i.e., DNA extracted from soil varied between 2.35 (Texcoco-2) to 3.66 (Texcoco-1) μg DNA g-1 soil. The proposed method in this study produced enough DNA with yield and quality for PCR amplification of 16S rDNA when bovine serum albumin (BSA) was added to the reaction buffer. The DNA obtained had sufficient quality and yield for later use for 16S sequencing or possible use in other sequencing technologies, e.g. whole metagenome shotgun sequencing.

Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles

Abstract. Junaid M, Purwantara A, Guest G. 2021. Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles. Biodiversitas 22: 2838-2843. Understanding the biology of the fastidious Basidiomycete Ceratobasidium theobromae occupying host-tissue is essential for plant disease management. Direct pathogen DNA extraction from infected plant tissue avoids the need for isolation in artificial media. We report a modified DNA isolation protocol to obtain total plant DNA designed to overcome DNA extraction and isolation problems caused by infected petioles rich in polysaccharides and phenolic substances as a primary source of gummosis. This study examined and compared total plant DNA isolated from petioles high in polysaccharides and polyphenolic compounds using two methods: conventional CTAB lysis buffer and Kits (the standard method), and a new modified CTAB protocol to address these PCR inhibitors. The modified method resulted in higher quality and quantity of C. theobromae crude DNA and amplified PCR product. The modified method produced large quantities of clear, transparent, aqueous DNA-containing lysate (crude DNA) with a clear separation between the upper crude DNA and organic waste layers. Mean DNA absorbance was 1.80, and the lowest DNA yield was 836.6 ng/µl. With the standard method, the blurred, viscous lysate obtained showed signs of gummosis, with poor separation between layers of crude DNA, polysaccharides, protein, and organic waste layers and low yield. Gel electrophoresis indicated poor quality DNA extract. We conclude that this modified method will be valuable for genetic diversity and disease studies in a range of previously challenging plant tissues and their pathogens.

Detection of quantitative and qualitative for p.aeruginosa in contaminated and non-contaminated soil with oil residues targeting gyrb gene by QPCR

Comparison of P. aeruginosa counts is found in contaminated and non- contaminated soil with oil residue using a real-time quantitative PCR by Direct DNA extraction from soil samples as well as indirect DNA extraction from bacterial isolates. Sampling were collected randomly from ten different regions of the Ministry of Science &Technology during the period from October to December 2019. Twenty soil samples were investigated for hydrocarbon tolerance in selective broth containing hydrocarbon source, and then isolates diagnosed based on phenotype, microscopic, and biochemical tests, were used. Direct DNA extraction from soil samples using DNeasy Power Soil Pro Kit for the rapid detection of P. aeruginosa from soil, as well as wizard DNA extraction method from bacterial isolation. Quantitative and Qualitative PCR was performed with the Magnetic Induction Cycler (Mic), DNA extraction from soil and bacterial isolates amplified and detected using fluorescent reporter dye probes specific for Pseudomonas aeruginosa DNA and Internal Control IC. A total of twenty bacterial isolates were obtained from the different soil samples according to phenotyping tests, and also, we obtain 20 DNA samples from direct DNA extraction from soil, the results of purity were (1.70- 1.95) for wizard method and (1.46- 2.35) for direct extraction from soil. Pseudomonas aeruginosa counts in contaminated soil samples (8 x 107 Copies/μL) showed higher compared with the non- contaminated soil samples (2 x 102 Copies/μL) at maximum use of qPCR and soil DNA. Rapid and specific test (less than 4 hours) using Rt-PCR by targeting gyrB gene for Pseudomonas aeruginosa detection in contaminated and non-contaminated soil samples, combined with correctly adjusted samples processing methods, as well as soil DNA extraction were made which were applied within the sensitivity required for methods.

Effect of a Topical Formulation on Infective Viral Load in Lambs Naturally Infected with Orf Virus.

IntroductionOrf is a highly contagious eruptive viral disease of the skin and mucosa of sheep and goats. Although vaccination with live or attenuated orf virus is the preferred option for disease control, the vaccine is unavailable in many countries. Treatment of orf lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was developed for pain relief in sheep undergoing surgical husbandry procedures in Australia. Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control.MethodsOne-month-old lambs (n=14), naturally infected with orf, were recruited from a farm during a natural outbreak of the disease. The animals were selected at the early stages of the infection and randomly divided into two cohorts: Group A (n=11) treated with the topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat embryonic fibroblasts (TIGEF).ResultsAlthough no significant differences were found in the clinical progression of the lesions and PCR quantification (p=0.722) between these small cohorts, there was a significant difference (p<0.05) in reduction in infective viral load between the groups when assessed in OSF cell cultures between T0 and T3.ConclusionThese preliminary findings suggest that treatment of early stage lesions with this TS may reduce the infective viral load present in orf lesions.