Genotyping of hydatid cyst isolated from human and domestic animals in ilam province, western Iran Using PCR-RFLP

Hydatidosis or cystic hydatid disease is one of the most important diseases in human and animals. Identification of strains is important for improvement of control and prevention of disease. The aim of this study was to determine the strains isolated from human and domestic animals in Ilam Province, Iran, using PCR-RFLP method. Respectively, 30 and 4 animal and human hydatid cysts were collected from different slaughterhouses and hospitals of the province. Protoscolices were separated and their DNA genome was extracted by extraction kit. rDNA-ITS1 of each isolated samples was duplicated by BD1 [Forward] and 4s [Reverse] Primers. PCR products were studied by electrophoresis and then were digested using TaqI, HpaII, Rsal and Alul restriction enzymes. RFLP products were studied using electrophoresis on 1% agar gel. A fragment of l000bp was produced from amplification of rDNA-ITS1 of protoscolices using PCR method. After digestion of PCR product by Alul enzyme, 200bp and 800bp, by Rsal, 655bp and 345bp and by HpaII 700bp and 300bp sizes were obtained. TaqI enzyme had no change in fragment size and it remained l000bp. Considering the method. Ham strains was specified as E. granulosus sensu stricto [G1-G3]. Although sheep strain [G1] is dominated in human and different animal in Iran and the world, but more efforts should be done to clarify the true genotype of Ilam strains specified as E. granulosus sensu stricto [G1-G3]

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

Identification of aspergillus species using morphological characteristics

Although molecular methods continue to improve and become more rapidly available, microscopy and culture remain commonly used and essential tools for identification of Aspergillus spp. In this study we emphasize on morphological methods including; macroscopic and microscopic characteristics for identification of Aspergillus species isolated from environmental and clinical specimens. We used four differential media: czapek dox agar [CZ], czapek yeast agar [CYA], malt extract agar [MEA], and czapek yeast 20% sucrose agar. Morphological features of colonies on above culture media as well as microscopically characteristics for the major strains were studied and then compared with those of standard Aspergillus strains. Our major subjects were Iranian Aspergillus strains isolated from clinical and environmental specimens. Standard Aspergillus strains for study development included; A. fumigatus, [JCM 10253], A. flavus [JCM 2061], A. niger [JCM 10254], A. nidulans [JCM 02728], A. tereus [JCM 10227]. Morphological features of Aspergillus cultures were studied, the major and remarkable macroscopic features in species identification were the colony diameter, color [conidia and reverse], exudates and colony texture. Microscopic characteristics for the identification were conidial heads, stipes, color and length vesicles shape and seriation, metula covering, conidia size, shape and roughness also colony features including diameter after 7 days, color of conidia, mycelia, exudates and reverse, colony texture and shape. Finally we compared the morphological characteristics of tested Aspergillus isolates with those of the standard species Aspergillus isolates were identified in the level of species using the differential culture media. A total of 205 Aspergillus isolates studied included: 153[75%] environmental Aspergilli and 52 [25%] clinical isolates. Within 11 Aspergillus species identified, A.flavus [55%], A.niger [31.7%] and A. fumigatus [8.7%] were the most common Aspergillus isolates from all of the specimens. In our view morphological method using the differential media is the most reliable and sensitive assay to identify more medically important Aspergillus species

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