Accurate identification of the host species is critically important for disease detection and informing appropriate disease management decisions. Paecilomyces formosus, a causal agent of dieback and decline of oak, is an emerging threat that may cause severe risk to the Zagros forests of Iran in the future. In this study, a nested PCR assay for the identification of P. formosus was developed with the species-Specific primer pairs PaMF and PaMR designed from the comparisons of nucleotide sequences of the nuclear ribosomal DNA internal transcribed space regions (ITS) from P. formosus isolates and other closely related taxa. To accomplish this, we sampled forest trees with dieback symptoms in Kermanshah and Ilam provinces. The Paecilomyces isolates were identified based on morphological characteristics, acid production on keratin sucrose agar medium, and sequencing of the ITS-rDNA region and part of the beta tubulin gene. Nested PCR was successfully amplified a 441 bp product exclusively from P. formosus genomic DNA, and no cross-reactions were observed with any other species, and also P. variotii. The nested PCR method can detect 100 fg of P. formosus genomic DNA. Sixty of symptomatic forest trees from seven locations in Zagros foreste were assayed, resulting in the discovery of Amygdalus lycioides, Cerasus avium, Cerasus microcarpa, Quercus libani, Acer spp., Acer monspessulanum, Ficus carica, Ziziphus spina-christi, Tamarix ramosissima and Ziziphus spina-christi as new host species, and all seven infested areas. To fulfill Koch’s postulates, the experiments were carried out on detached branches and attached healthy branches of trees at the forests in Kermanshah and Ilam provinces, Iran. The PCR-based method developed here can be used for a fast and reliable diagnosis of P. formosus, monitoring the epidemics, and assessing management strategies in Zagros forests.

Phytophthora erythroseptica, the causal agent of potato pink rot, is one of the oomycete plant pathogens that causes significant losses in field and storage. In order to develop a sensitive and rapid method for detection and identification of P. erythroseptica, six nuclear and mitochondrial gene regions were investigated to design species-Specific Primers. Due to the high similarity of P. erythroseptica sequences to its closely related species, only one nuclear region, TigA, was appropriate to design Specific Primers. Using Specific Primers, a simple as well as a nested-PCR based method was developed for the identification and detection of P. erythroseptica. The Specificity of designed Primers was examined using a collection of Phytophthora species from different phylogenetic clades as well as close relatives of P. erythroseptica. In addition to pure DNA, designed Primers detected P. erythroseptica in infected plant tissues including potato, tomato and spinach. Specific Primers detected 10 pg of P. erythroseptica pure DNA, however, nested PCR increased Primers sensitivity at least 100 times. Moreover, Specific Primers designed in this study were able to detect P. erythroseptica as the maternal or paternal parent species in hybrid isolates that would make a significant help to recognize one of the parental species in hybrids of P. erythroseptica.

Objective: Evaluation of Primers designed due to the serovar Specific IS200 copy for detecting Salmonella abortusovis strains isolated in Iran.Design: Observational study.Samples: Ninety seven Salmonella abortusovis strains. Procedure: PCR amplification was carried out by serovar Specific Primers and different strains according to PCR results were studied by IS200 fingerprinting analysis. Results: All strains could be classified in 2 distinct genotypes by 2 kb and 900 bp amplicons in PCR amplification. These two genotypes were related to two different profiles with 11 and 9 kb band respectively in IS200 fingerprinting.Conclusion: PCR amplification by serovar Specific Primers was capable of grouping the strains in 2 major genotypic patterns.

Background: Designing the primer pairs is one of the most important factors in the amplification and quantitative analysis of the nucleic acid sequences of interest. Using in silico methods, the present study intends to design highly Specific Primers for quantitative analysis of the genes with minimum expression. To achieve this aim, we selected two candidate genes with little expression, namely, G-protein coupled receptor 120 (GPR120) and peroxisome proliferator-activated receptor-γ (PPARγ), in peripheral blood leukocytes of healthy volunteers. Methods: Peripheral blood was collected from 30 healthy volunteers. Primers for GPR120 and PPARγ were designed using online websites (UCSC, OligoCalc, and OligoAnalyzer) and the primer designing tool (NCBI). Total RNA extraction and cDNA synthesis were done using commercially available kits based on manufacturer instructions. Finally, the melting curve analysis of GPR120 and PPARγ was assessed using the quantitative real-time PCR method. Results: The in silico gene expression investigation revealed that GPR120 and PPARγ have minimal leukocyte expression. Besides, the melting curves analysis for both genes in the studied individuals showed only one melting peak, confirming the Specific amplification of the desired genes. Conclusion: Altogether, the study findings indicated that we could utilize the peripheral blood sample for assessing the gene expression and amplification of omega-3 fatty acids receptors, i. e. GPR120 and PPARγ as two candidate genes with very low expression in leukocytes.

A rapid and sensitive polymerase chain reaction (PCR) method is described for the determination of latent infections of Ralstonia solanacearum, the causal agent of bacterial wilt of solanaceous plants. The PCR tests with Specific Primers successfully detected R. solanacearum biovar 2 in all naturally and artificially infected potato samples with or without visible symptoms, and gave a characteristic 1019 bp band with division 2 Specific DlV2F / DlV2R Primers. As expected the PCR test with division I Specific DIVIF / DIVIR Primers did not produce any PCR product with any infected potato and tomato sample. None of the healthy control potato and tomato plants gave visible PCR products with the Specific Primers.

Mycoplasma gallisepticum (MG) as one the major pathogens of birds, causes significant economic losses in poultry industry. The main purpose of the present study was to detect Mycoplasma gallisepticum in clinical samples using the 16S rRNA PCR method. For serological screaming test, 18 commercial laying farms and 8 broiler breeder farms were selected and rapid serum agglutination test (RSAT) was performed. For polymerase chain reaction sampling, 10 of the 17 farms that were positive in RSAT were selected and 109 sterile swab samples were collected from the palatine cleft, trachea, air sacs and lungs in each farm. Three swabs from three birds were placed in test tube containing 1 ml of phosphate buffered saline and transferred to laboratory form PCR testing. In this study, Specific Primers for 16S rRNA gene were used. The aforementioned Primers are totally Specific for MG and can be differentiated from other Mycoplasma and bacteria present in the trachea of poultry of the 26 farms examined, 17 farms were positive in RSAT serologic test. The 530 bp PCR product produced by Specific Primers of all field strains appeared on electrophoresis gel in 46 samples from 10 farms accounting to 42.2%.The 16S rRNA PCR with very high sensitivity can be employed in definitive diagnosis of Mycoplasma gallisepticum infection in vitro.

Mutual relationship between endophytic fungi and some gramineuos plants is one of the recently known host-microbe interactions. Endophytic fungi are classified in class Ascomycetes, order Hypocreales, tribe Balansieae and mostly genus Neotyphodium. They are transferred into the progeny via maternal stock and have many beneficial effects to their host plants, such as resistance to pests and diseases, environmental stresses such as pH fluctuation, drought, salinity and grazing because Iran has a wide range of plant genomic resources and is known as one of the origins of gramineous plants, it is necessary to know more about endophytic fungi and their presence by using classical and molecular methods. Twenty isolates of endophytic fungi were obtained from seed and leaf sheaths of Bromus tomentellus, Lolium prenne, Festuca arundinacea and Festuca pratensis. Mycelia of endophytic fungi were observed red or pink in seed and leaf sheath when stained by Rose Bengal. The DNA of endophytic fungi was. extracted by Murry and Thompson method with minor modification. In order to confirm the identity of endophytes, three universal and Specific primer e. g. ITS1, ITS4, IS1, IS3, and 11-1, 11-2 were used. All isolates showed a 550-750 bp band with ITS1, ITS4 and eighteen of them showed a 444 bp band with IS1, IS3 Primers, which is an indicator of Neotyphodium endophytes. When Primers 11-1,11-2 were used, a 1000bp band was not observed in all isolates. Isolates FaSm and FpGon3 that did not shown a 444 bp band, may probably differ from Neotyphodium endophytic fungi.