One of the challenges in interpreting the results obtained in qPCR is to make sure the amplicons are specific and that the melting curve analysis is used to examine it. Additional peaks in the melting curve is not always indicative of a problem, for this purpose, in this paper, a webbased tool called uMelt SM is suggested to researchers as a practical and simple way for the correct analysis of the melting curve which provides the possibility of predicting the DNA melting curve and denaturation profiles of the high-fluorescence resolution of PCR products. The results of this study showed that the melting curves were generated based on parameters were generated and an appropriate algorithm for working with this software was presented. Finally, actin and superoxide dismutase genes from Pyricularia oryzae were presented as a suitable model for determining the predicted curve using this software and Real-Time PCR curve was also drawn. Results of the uMelt SM predicted melting curves showed a high degree of compliance with the real-time PCR melting curves, which confirms the advantages of ease of use, saving time, cost, and effort in the experimentalist part when using uMelt SM software.

Newcastle disease is one of the most serious viral diseases in the poultry worldwide. Since the traditional strategies have been not control it well, the aim of this study was to introduce new methods for early and rapid diagnosis of Newcastle.In this study, to detect the virus, a Real-time PCR method was optimized, Viral RNA was extracted from strain B1 using the kit RNease mini (Qiagen, USA), according to the manufacturer's instructions. This sample had 68.23×109 copy numbers of viral RNA per each microliter. Then, a serial dilution as 100-fold was prepare from the initial sample. Then, these dilutions were simultaneously applied in reverse transcription and Realtime PCR using commercial kits (Genekam Biotechnology, Germany) according to the manufacture’s instruction. The sensitivity of Real-time PCR reaction was determined based on serial dilutions of 1×10-34 copy number per micro liter. Since, speed and accuracy in diagnosis of contagious of Newcastle disease virus plays an important role to control the disease, so adopting this method is recommended as a diagnostic test with high sensitivity.

During 2009-10, real time RT-PCR and conventional RT-PCR techniques Used for detecting BTVs RNA in 310 blood samples. For real time and gel based RT-PCR segment-1 and segment-10 selected as conserve genes to search any BTV strains. Using these methods, 58 (%18.7) and 14 (%4.5) positive samples were detected among the clinically suspected sheep. Sensitivity of both molecular techniques evaluated by log-10 serial dilutions of BTV16 RNA, and determined 101.8 and 103.8 TCID50/ml in rRT-PCR and conventional RT-PCR respectively. This report confirmed rRT-PCR assay could detect weak BTV positive samples even at end stage of infection. In this study Virus isolation from selected positive samples failed by inoculation to embryonated chicken egg, Vero and KC cell.

In the recent years, Real-time PCR technique introduced as a choice method for diagnosis of infectious diseases in many laboratories. During each cycle of the PCR reaction, this technique combines the polymerase chain reaction chemistry with the utilization of fluorescent reporter molecules for monitoring the production of amplification products. Therefore, the set of features including the high sensitivity and specificity, repeatable data and low contamination risk has made the Real-time PCR technology as an attractive alternative to conventional PCR. This technique is often used to quantify the level of gene expression. Since the whole Real-time PCR reaction is performed within a closed tube, the risk of contamination is reduced and eventually prevent false-positive results. The aim of present study was to provide a general overview on different types of Real-time PCR methods, their benefits and applications.

Objective Raini Cashmere goat is one of the most important goat breeds in Iran. These animals are breed both for meat production and cashmere production. One of the basic measures in the domestic animals is the study of genes and proteins associated with economic traits and their study at cellular or chromosomal levels. One of these important genes is the leptin. Leptin is produced by white adipose tissue and plays an important role in the regulation of feed intake, energy balance, fertility and immune functions. The aim of this research was to study leptin gene expression in adipose tissue, liver, kidney, lung and heart of Raini Cashmere goat using Real Time PCR technique. Materials and methods Tissue sampling from heart, lung, liver, kidney and adipose tissue (3 replicates from each tissue) of 6 animals was performed and RNA was extracted. Extracted RNA were immediately stored at-80° C. The Quality and quantity of RNA were evaluated and cDNA was synthesized and Real Time PCR was performed. PCR Products were electrophoresed on 1. 5% agarose gel. Melting curves from Real Time PCR were examined and were evaluated different levels of expression in the studied different tissues. Results The results of Real Time PCR curves and observation of electrophoresis of PCR products on agarose gel showed that the leptin gene was expressed in all the tested tissues and the highest level of expression was observed in adipose tissue (4. 5) and liver (3. 7) and the lowest level was detected in heart (1. 4). Conclusions These results may show that leptin plays a particular role in fat metabolism. Further studies are needed to clarify role of leptin in the physiology of fat metabolism and other materials. This would help us to better understand the mechanisms for the known effect of nutritional factors and body fatness on various functions.

Background: Despite advances in cancer studies, colorectal cancer, as the third most common cancer, has the highest mortality rate worldwide. Due to its high prevalence in the younger ages and advanced stages, screening of this cancer with molecular methods is necessary. Studies have shown that HOTAIR gene plays an important role in cancers. Our aim in the present study was to determine the expression of HOTAIR gene in tumor and tumor margins of patients with colorectal cancer using Real-Time PCR. Materials and method: In the present case-control study, 47 colorectal cancer patients who had referred to Imam Reza Hospital in Tabriz from May to March 2011 were evaluated. Samples were subjected to extraction of RNA after confirmation by the pathologist, and then the HOTAIR gene expression was measured and analyzed using Real Time-PCR and Graph Pad Prism, respectively. Findings: The expression level of HOTAIR in tumor samples was about seven times higher than that of marginal samples (p = 0. 0009). Also, by increasing the degree of tumor differentiation, the expression of HOTAIR gene decreased (p = 0. 023). Conclusion: HOTAIR gene is involved in the pathogenesis of colorectal cancer, and the use of expression analysis of this gene as a biomarker can be helpful in diagnosis, treatment, and prognosis of patients, although more studies are needed to verify this claim.

In this study, Molecular method to measure the rhizopus oryzae Real Time PCR was used in tomato paste. Because of problems mold count HMC method such as Proofer error, low and non-repeatability precision, accuracy and repeatability developing a standard method that can enable high accuracy and repeatability and mold counts it seems necessary. Real Time PCR molecular methods can be used as a suitable method to identity and quantity the precise mold is considered that based on the measurement of absolute and relative genes is the existing molds. Tomato paste samples with different amounts of mold spores (101,103 and 105) were contaminated per gram. After incubation and growth of mold spores and mycelium extraction DNA from each sample was performed using al-sammari method. Then, using Real Time PCR reaction of DNA copy number of molds using SYBR Green reagent and primers of the rhizopus oryzae genus was determined. The results showed that increasing amount of DNA molds in controls and inoculated samples resulted Real Time curve in the lower Cr values are entered logarithmic phase. The correlation coefficient of linear calibration Real Time was R2 = 0.943. This indicate that the accuracy of this test is quantitative detection. The reaction specificity was determined using melting curve of the primers and indicated that the reaction has been completely dedicated.