Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes

Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. © 2013 Elsevier Inc

Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes

Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. © 2013 Elsevier Inc

Differentiating agents of dermatophytosis (Trichophyton rubrum and Trichophyton interdigitale) in human by dual polymerase chain reaction

Background: Dermatophytes create the most common fungal disease in humans, called dermatophytosis. The two species of Trichophyton rubrum and Trichophyton interdigital are responsible for over 80% of types of dermatophytosis. So far, several morphological and physiological methods have been used to differentiate these very similar species, but these methods are generally time-consuming and have low specificity. The purpose of this study was to introduce a simple and rapid duplex polymerase chain reaction (PCR) reaction to differentiate these two species from each other. Methods: This research was an analytical and experimental study that was carried out from 2017 to 2018 in the Medical Mycology Laboratory, School of Public Health, Tehran University of Medical Sciences, Iran. For this purpose, the nucleotide sequences of the 4 regions of internal transcribed spacer (ITS), beta-tubulin, elongation factor 1 alpha and calmodulin in the two considered species of fungi were conducted bioinformatics analysis. The differences and similarities of nucleotides between two species in each of these genes were studied for selecting the primer. The specificity of selected primers was tested for duplex PCR reaction against sequenced isolates of dermatophyte species. Results: According to the total data, the specific primers were selected from elongation factor 1 alpha gene. These primers produced a product of 173 and 384 bp, in Trichophyton rubrum and Trichophyton interdigital, respectively. They had high specificity in the face of various dermatophytes. The length of nucleotide sequences found in the genebank of this gene in the two species is between 700 and 770 bp. The similarity of the two species in this region is 94.6% and differs by 78 bp. Of the 107 extracted DNAs from clinical dermatophyte isolates, in duplex PCR 24 isolates were positive with Trichophyton interdigital primer and 71 isolates against Trichophyton rubrum. The remaining isolates, which included 6, were negative in this reaction, which included other dermatophyte species. Conclusion: This method is a specific and fast differential method compared to conventional methods for identifying Trichophyton rubrum and Trichophyton interdigital from each other

Comparison of Five Simple Methods for DNA Extraction from Echinococcus granulosus Protoscoleces for PCR-Amplification of Ribosomal DNA

"nBackground: Cystic hydatid disease is an important zoonosis, affecting humans and animals and is a significant public health and economic problem throughout the world and Iran. Since extraction of DNA from the parasite is a primary and crucial step which has a principal effect on PCR results, in the current study five simple methods for DNA extraction from protoscoleces of Echinococcus granulosus were ap­plied and compared with each other. "nMethods: After collecting hydatid cysts from an abattoir, DNA samples were extracted from two cyst isolates from sheep, two from goats and two from camels using five different methods involving the use of glass beads, mechanical grinder, freeze-thaw, boiling and crushing. For all DNA samples ex­tracted, one PCR assay based on amplifying rDNA-ITS1 region was performed and amplicons re­solved on 1.5% agarose gels. "nResults: The methods were compared regarding to DNA and PCR bands, time and cost effectiveness and laborious amount. The target DNA was successfully amplified from all samples using all methods produced an expected band size. All methods showed some advantages and disadvantages in PCR gels. The boiling method, which was the most time and cost effectiveness method, achieved the thick­est bands in the PCR following grinder, crushing, freeze-thaw and glass beads."nConclusion: Boiling and crushing methods were the most suitable methods regarding their amplicon quality, easiness, quickness and cost effectiveness

Identification andDifferentiation of Trichophyton Mentagrophytes and T.rubrum by Polymerase Chain Reaction and Enzymatic Digestion

Introduction & Objective: Trichophyton rubrum and T. mentagrophytes are most common causative agents of dermatophytosis in the world. Differentiation of these species is important from the epidemiological and pathological point of view. Conventional methods including macroscopic and microscopic morphology and biochemical tests are time-consuming (in some cases it takes 3-4 weeks), laborious and still sometimes insufficient to identify these agents. The aim of this study was to use polymerase chain reaction followed by enzymatic digestion for differentiation of these 2 species. Materials & Methods: In this descriptive–experimental study one hundred strains were isolated from patients with dermatophytosis. Preliminary identification was done by morphological methods. DNA was isolated and purified by glass-bead methods and ITS1- 5.8SrDNA-ITS2 region was amplified by PCR and the amplicon was digested by the restriction enzyme MvaI. The products were visualized after agarose gel electrophoresis and staining. Differentiation of the species was based on sequence analysis and the electrophoretic patterns. Results: Morphological tests were not able to definitely differentiate the two tested species, especially for isolates with intermediate features. Using molecular methods, it was found that 45 isolates are T. rubrum and 54 are T. mentagrophytes. One isolate was Fusarium spp. Physiological tests were confirmed the results except for 4 isolates. It was also found that hair perforation test is more reliable than urease test for differentiation of these two species. Conclusion: We found that DNA-based method, although expensive, is a fast and reliable method for differentiation of T. rubrum and T. mentagrophytes. The frequency of mentioned species was almost similar in the tested isolates. The method is recommended for differentiation of other dermatophytes

Frequency of Candida species isolated from patients in children’s medical center, Tehran, Iran

Background: Candida species originating from either endogenous or exogenous sources are one of the main causes of opportunistic infections. Colonization is an important independent risk factor for invasive candidiasis, and many patients admitted to neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU) are colonized with Candida species that may result in invasive candidiasis. Awareness among clinicians about various aspects of colonization is critical to optimal management. The aim of this study was to determine the frequency and species distribution of Candida strains isolated from predisposed patients hospitalized at Children’s Medical Center (CMC), Tehran, Iran. Methods: From June 2014 to June 2016, 347 Candida isolates were collected from 341 patients either hospitalized in different wards or referred as outpatients. The yeasts were identified by colony color characteristics using CHROMagar Candidamediumand by amplification of the ITS1-5.8S rDNA-ITS2 region in DNA extracted from each isolate followed by analysis of species specific electrophoretic patterns of PCR products digested with the restriction enzyme MspI. Results: Of the 341 patients, 213 were males and 128 were females. Most samples were obtained from the 1 – 12-month age group, and the majority of samples represented urine (n = 182), throat swabs (n = 57), and stool samples (n = 53), respectively. The samples were mostly from patients in general wards. The most commonly isolated species was C. albicans (77%), followed by C. tropicalis (8.4%), C. parapsilosis (7.5%), C. glabrata (2.3%), C. kefyr (1.7%), C. krusei (1.1%), C. lusitaniae (0.6%), C. guilliermondii (0.3%), C. albicans + C. parapsilosis (1.4%), and C. albicans + C. glabrata (0.3%). Conclusions: C. albicans is the most common species isolated from children in Iran, followed by C. tropicalis and C. parapsilosis, a prevalence pattern that is relatively different from studies in other countries. Neonates and infants 1 - 12 months of age hospitalized in ICU, were more colonized by Candida species than other group