Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP

The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP]. Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on modified Leeming and Notman agar medium. DNA extracted from the yeast colonies was amplified by PCR using primers specific for 26S rDNA. RFLP of the PCR products was performed using Hin6I enzyme, and PCR and RFLP products were visualized by agarose gel electrophoresis. Malassezia yeasts were detected at the following frequencies: 15.46% in horses, 12.74% in cattle, 12.38% in sheep, 28.33% in dogs, 26.66% in cats and 26% in aquatic birds. Eighty colonies of 6 species were isolated: Malassezia globosa 41.25%, Malassezia furfur 22.5%, Malassezia restricta 15%, Malassezia sympodialis 15%, Malassezia pachydermatis 5% and Malassezia slooffiae 1.25%. Therefore different lipophilic Malassezia species are found in a wide diversity of animals and aquatic birds. PCR-RFLP is a suitable technique for identification of different Malassezia species

Investigation of double-band electrophoretic pattern of ITS-rDNA region in Iranian isolates of leishmania tropica

Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer [ITS] region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis [ACL] in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs. Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products. Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis. Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.

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]

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

Sequence analysis of the second internal transcribed spacer [ITS2] region of rDNA for species identification of trichostrongylus nematodes isolated from domestic livestock in Iran

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification. Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism [RFLP] profile was designed to differentiate Trichostrongylus species. A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings. Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species

Mycological microscopic and culture examination of 400 bronchoalveolar lavage [BAL] samples

The frequency of invasive opportunistic mycoses has increased significantly over the past decades especially in immunocompromised patients. Invasive aspergillosis [IA] has become a major cause of morbidity and mortality among these patients. As bronchoalveolar lavage [BAL] fluid samples are generally useful specimens in the diagnosis of invasive pulmonary aspergillosis [IPA], this study was designed to evaluate the incidence of fungal elements in at-risk patients by direct microscopy and culture of BAL samples. In a 16-month period, 400 BAL samples were obtained from several groups of different patients with pulmonary and respiratory disorders and examined by using both direct microscopy and culture. Of the 400 samples, 16 [4%] were positive direct examination with branching septate hyphae and 46 [11.5%] were positive culture: 25 [54%] Aspergillus flavus, 6 [13%] A. fumigatus, 5 [10.9%] A. niger, 1 [2.2%] A. terreus, 3 [6.5%] Penicillium spp. and 6 [13%] mixed A. flavus/A. niger. A. flavus was the most common cause of Aspergillus infection or colonization. Bone marrow transplant [BMT] recipients were the most susceptible group to fungal infection and/or colonization. Among Aspergillus species, A. flavus was the most common isolate in both infections and colonization in Iran. More studies are needed to clarify the epidemiological aspect of aspergillosis in Iran

Morphological and morphometrical description of trichostrongylus species isolated from domestic ruminants in Khuzestan province, southwest Iran

Genus Trichostrongylus [Nematoda: Trichostrongylidae] is one of the most important zoonotic nematodes with wide geographic distribution in the world. The purpose of the present study was to describe morphological and morphometrical characteristics of male Trichostrongylus species, currently prevalent in domestic ruminants of Khuzestan Province, southwest Iran. Gastro-inetstinal organs of 1600 sheep, goats, cattle, and buffalos, slaughtered in Khuzestan Province, southwest Iran, were examined for infectivity with Trichostrongylus species. For examination and measurements of helminthes, Azo-carmine staining was performed, followed by camera lucida drawings of morphological characters and measurements of morphometrical criteria with a calibrated microscope. Using valid nematodes systematic keys, almost all the parasites were identified at the level of species. Overall, 114 animals were found infected with at least one species of Trichostrongylus. Considering morphlogifcal characteristics of male Trichostrongylus, six species were identified including T. colubriformis, T. vitrinus, T. probolorus, T. capricola, T. longispicularis and Trichostrongylus sp.. Although, compared to the previous decades, currently Trichostrongylus is much less prevalent in the domestic ruminants of the study area, but still different species occur in these animals

Molecular diagnosis of Strongyloides stercoralis infection by PCR detection of specific DNA in human stool samples

Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples. A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar's chi[2] test, with consideration of a P-value of <0.05 as indication of significant difference. In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture. Single PCR method amplifying a short [100bp] target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target

Species identification and strain typing of candida isolates by PCR-RFLP and RAPD-PCR analysis for determining the probable sources of nosocomial infections

Since the incidence of nosocomial Candida infections have been increasing in parallel with the raising in the number of patients involving in predisposing factors, determining the sources and the ways of acquisition of infection is necessary as an efficient strategy for controlling the diseases. The aim of this study is identification and strain typing of Candida strains isolated from hospitals to facilitate tracing the sources of infections in hospitalized patients in addition to assess the discriminatory power of some random primers by using RAPD analysis. Samples were collected from patients who were hospitalized in oncology, intensive care unit [ICU], and organ transplants wards of the Shohadaye-Tajrish Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran, and their environments. The yeasts were isolated on CHRO Magar Candida. Species identification was performed by PCR-amplification of ITS1-5.8SrDNA-ITS2 region followed by restriction digestion with the enzyme MspI. To determine the probable origin of Candidia infections, in case of each patient whose the clinical and relevant environmental isolates were identified as the same species, a set of eight primers namely number 1 to 8, were applied in RAPD-PCR to find out the possible homogeny or variation within the isolated strains. One hundred and four Candida strains were identified. The most common species was C. albicans [57.5%] followed by C. tropicalis [13.5%], C. glabrata [12.5%], C. parapsilosis [8.65%], C. famata [3.8%], C. krusei [1.9%], C. guilliermondii [0.96%]. and C. lusitaniae [0.96%]. While the source of infection for three patients were not determined by RAPD analysis, interpretable results from RAPD-typing of Candida species isolated from 8/18 of cases implied that the infections might originate from the exogenous sources. Moreover, according to the function of each primer, primer No. 1 was determined as a best primer for typing of Candida albicans strains. The species of yeast isolates were determined by PCR-RFLP. It was found that RAPD assay can point out the genomic variability within the Candida species. Besides, the method could show a probable relationship between acquired infections and their sources

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 epizootiology of rodent leishmaniasis in a hyperendemic area of Iran

Zoonotic cutaneous leishmaniasis [ZCL] is an expanding disease and public health problem in Iran. In the current study, natural Leishmania infection rate and seasonal fluctuation of the infection in Rhombomys opimus population of a hyperendemic focus of ZCL in Iran was investigated. The study was conducted from October 2006 to October 2008 in Esfahan Province, central part of Iran. An extensive sampling of rodents using Sherman traps was done in different seasons. Nested PCR assay was used for detection and identification of Leishmania species and the results were confirmed using PCR-RFLP. Leishmania infection rate was 58.6% [34 of 58] using nested PCR. 44.8% of the gerbils were infected only with L. turanica and 1.7% with L. gerbilli alone. A mixed natural infection with L. major and L. turanica was seen in 12.1% of the rodents. L. major infection alone was not seen in R. opimus population in the study area. The highest and lowest Leishmania infection rates were observed in fall and spring respectively. L. turanica infection was observed throughout the year whereas mixed infections with L. major and L. turanica was not seen in spring. It is concluded that in the study area, L. major, L. gerbilli and L. turanica circulate in the population of R. opimus. Leishmania major infection usually accompanied by L. turanica in naturally infected gerbils with the highest rate in fall. It is recommended that the role of L. turanica in the epidemiology and transmission of ZCL be revisited

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

Plasmodium vivax MSP-3 beta gene as a genetic marker for the parasite detection in comparison with SRNA gene

The importance of accurate diagnosis of all of major diseases cannot be underestimated and efficient laboratory testing is vital to identifying and treating life-threatening illnesses including malaria. In this study, we compared the potential of one of merozoite surface protein genes, PvMSP-3 beta, for detection of Plasmodium vivax in blood samples by PCR with routinely used marker, ssrRNA gene. One hundred P. vivax microscopy-positive blood samples were simultaneously tested with two genetic markers, including PvMSP-3 beta gene and ssrRNA gene by PCR and nestedPCR method, respectively, and their sensitivity and specificity in detection of P. vivax was compared. An important difference was seen in sensitivity between the 2 genetic markers, 100% in case of ssrRNA gene vs. 95% of PvMSP-3 beta gene. The specificity of the two markers was same [100%]. Microscopic diagnoses of thick and thin blood smears was used as "golden standard" method. Due to critical importance of accurate detection of the parasite in malarious area, the PvMSP-3 beta gene cannot be a suitable marker for detection of P. vivax in blood sample by PCR. More investigations are needed to find other valid markers

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

Partial sequence analysis of merozoite surface protein-3 alpha gene in plasmodium vivax isolates from malarious areas of Iran

Approximately 85-90% of malaria infections in Iran are attributed to plasmodium vivax, while little is known about the genetics of the parasite and its strain types in this region. This study was designed and performed for describing genetic characteristics of plasmodium vivax population of Iran based on the merzoite surface protein-3 alpha gene sequence. Through a descriptive study we analyzed partial P. viva merozoite surface protein-3 alpha gene sequences from 17 clinical P. vivax isolates collected from malarious areas of Iran. Genomic DNA was extracted by Q1 Aamp DNA blood mini kit, amplified through nested PCR for a partial nucleotide sequence of PvMSP-3alpha gene in P. vivax. PCR-amplified products were sequenced with an ABI Prism Perkin-Elmer 310 sequencer machine and data were analyzed with clustal W software. Analysis of PVMSP-3alpha gene sequence demonstrated extensive polymorphisms, but the sequence identity between isolates of same types was relatively high. We identified specific insertions and deletions for the types A, B and C variants of vivax in our isolates. In phylogenetic comparison of geographically separated isolates, there was not a significant geographical branching of the parasite populations. The highly polymorphic nature of isolates suggests that more investigations of the PvMSP-3alpha gene are needed to explore its vaccine potential

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

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

Diversity of merozoite surface protein-3beta gene of plasmodium vivax isolates from Iran

Plasmodium vivax malaria accounts for approximately 88% of malaria cases in Iran. There is limited information on genetic diversity of P. vivax in the country and a need to develop and apply an effective vaccine against the disease is necessary. Among many potential candidates, MSP -3beta gene is promising target. This study was designed and carried out to determine the variation of this gene as genetic marker in population of malarious areas of Iran. Blood sample of 85 P. vivax isolates from four southern and east-southern provinces of the country assessed for polymorphism of PvMSP-3beta gene by PCR/RFLP method. Based on the size of PCR product of the gene, 7 genetically different types of parasite has been distinguished. Two alleles were simultaneously visible in 19% of the cases. Results from PCR/RFLP analysis of PvMSP-3beta gene showed at least 15 allelic groups. Multiple infections have been found in 2.4% of the cases. PvMSP-3beta gene was highly diverse in P. vivax isolates of malarious areas of Iran, and can be a suitable marker for population genetic studies of P. vivax. More investigations on PvMSP-3beta genes are needed to reveal genetic structure of P. vivax in Iran