Use of single-enzyme PCR-restriction digestion barcode targeting the internal transcribed spacers [ITs rDNA] to identify dermatophyte species

Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum [A. obtusum] and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings

Preliminary identification and typing of pathogenic and toxigenic fusarium species using restriction digestion of ITS1-5.8S rDNA-ITS2 region

Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 universal primers followed by sequencing and restriction enzyme digestion of PCR products. The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS1-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species

Molecular characterization of Fasciola hepatica isolates by RAPD-PCR and ribosomal ITS1 sequencing

Understanding genetic structure and status of genetic variation of the Fasciola hepatica populations has important implications for epidemiology and effective control of fasciolosis. The aim of the present study was to genetically characterize F. hepatica isolates from different hosts, using sequence analysis of ribosomal ITS1 and RAPD-PCR. Fifty three adult F. hepaticas were isolated from naturally infected cattle, sheep, buffalo and goat from two regions in Iran. Genomic DNA was extracted from 70% ethanol preserved flukes. RAPD-PCR with a set of arbitrary primers [UBC90 and R151] was used to estimate genetic variation within the species. Ribosomal ITS1 region of the isolates was amplified, using primers specifically designed for this study. Ten samples [4 sheep, 2 cattle, 3 buffaloes and one goat isolate] were sequenced at ITS1 and analyzed, using DNASIS and ClustalW softwares. F. hepatica ITS1 region was amplified successfully for all samples and a band of 470 bp was shown in all cases. Different isolates did not show any significant genetic variations in rDNA-ITS1 as all the sequences showed to be 100% identical. RAPD results of 52 samples, in particular those with UBC90, showed different patterns within F. hepatica isolates of each host. RAPD data for this primer showed three different patterns for each of sheep and cattle isolates and two patterns in buffalo isolates. All the 14 cattle isolates come up with an identical pattern, using primer R151. The study showed the variability of F. hepatica isolates in Iran, using RAPD markers. No intraspecies variation was seen in the Iranian F. hepatica isolates at ITS1 rRNA gene, indicating highly conserved nature of this region

Comparison of five simple methods for DNA extraction from Echinococcus granulosus protoscoleces for PCR-amplification of ribosomal DNA

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. 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. 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. Boiling and crushing methods were the most suitable methods regarding their amplicon quality, easiness, quickness and cost effectiveness

Single strand conformation polymorphism analysis of PCR-amplified rDNA to differentiate medically important Aspergillus species

Aspergillus species are associated with allergic bronchopulmonary disease, mycotic keratitis, otomycosis, nasal sinusitis and invasive infection. In this study, we developed a PCR-Single Strand Conformational Polymorphism method to identify the most common Aspergillus species and we showed some advantages of this method comparing a PCR-Restriction Fragment Length Polymorphism with our designed restriction enzyme. We selected ITS2, as a short fragment within the rDNA region [length size: 330 bp] to be amplified as small size PCR product. We mixed 5 ml of the PCR product with an equal volume of loading buffer and followed by incubation for 5 min at 95°C and quenching in an ice bath. The mixture was applied to a 6%-12% Gradient Poly acryl amide gel to run in a vertical electrophoresis, then gel was stained with ethidium bromide and silver nitrate which followed by an ethidium bromide staining. Our results of restriction digestion showed a fine identification of 7 tested Aspergillus species during 5-6 hours after an overnight mycelial growth. As our results some of tested Aspergillus species: A. nidulans, A.fisheri, A. quadricincta, [A. fumigatus and A. niger] as a group and [A. flavus, A. tereus and A. ochraceus] as another group, can be discriminated. Moreover SSCP analysis enabled us to identify above Aspergillus species within 8-12 h after an over night growth without using an expensive restriction enzyme. It is concluded that Single Strand Conformational Polymorphism is a simple and rapid method for identification of some medically important Aspergillus

Colony-PCR is a rapid and sensitive method for DNA amplification in yeasts

Yeast infections are increasing cause of morbidity and mortality in immunocompromised patients. In order to perform a DNA-based diagnostic test, availability of a rapid and easy-to-perform DNA extraction protocol is essential. In the present study we evaluated colony-PCR as the easiest way to amplification of target DNA. Instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures. Serial cell dilution of three reference yeast strains including Candida albicans, Cryptococcus neoformans and Saccharomyces cerevisiae were used for determining the sensitivity of the colony-PCR. A total of one hundred yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4 complementary to the rDNA region. The colony-PCR resulted in a single band [with different sizes] for 106 cells or more for all reference species. Furthermore 98 out of 100 [98%] of samples showed a relevant single band after PCR. Directly application of the yeast cells obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests

new primer pair in ITS1 region for molecular studies on echinococcus granulosus

Echinocuccus granulosus, the causative agent of cystic echinococcosis has long been recognized as having a high degree of genetic divergence. The strains characterization seems to be essential for the establishment of a preventive and control strategy in every endemic area. Using DNA based methods for strain /genotype characterizations of E. granulosus have some difficulties, especially access to an efficient and pure concentration of DNA and proper primers. Using grinder method, a pure and high concentration DNA was extracted from 10 human hydatid cysts collected from Isfahan [central Iran] hospitals, and processed for PCR reaction. Using DNASIS, the primers were designed in internal transcribed spacer 1 [ITS1] region, following analysis of 30 E. granulosus nucleotide sequences, extracted from gene bank. This new and specific E. granulosus primer which amplified DNA thoroughly can be applied for molecular studies on echinococcosis