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.

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.

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.

Background: Prenatal diagnosis and prevention of live-born children with Down syndrome is a principle priority of Iran’s ministry of health. The aim of this study was the rapid diagnosis of Down syndrome by quantitative Real-time PCR technique as a new method for prenatal diagnosis. Material and methods: In this experimental study, two milliliters of peripheral blood was obtained from each patient and normal control. Then genomic DNA was extracted using salting out method. DYRK1A2 gene as target gene and PMP22 gene as reference gene were analyzed by quantitative Real-time PCR technique. Results: DYRK1A2/PMP22 gene ratio was 1.68±0.13 and 1.00±0.09 in Down syndrome and normal samples, respectively (p<0.001), demonstrating 3 copies of target (DYRK1A2) gene in trisomy 21 syndrome and 2 copies in normal individuals.Conclusion: DYRK1A2/PMP22 gene ratio is significantly higher in patients with Down syndrome compared with normal individuals. So, quantitative Real-time PCR technique can be used as a sensitive, accurate and reliable technique for rapid diagnosis of trisomy 21 syndrome. 

Objective Calpastatin has been found in skeletal muscle tissue of animals and could prevent unrestrained cellular growth by suppressing calpain activity. The involvement of calpains in apoptosis has been a subject of debate, although their involvement is limited to certain cell types and to specific stimuli. Various studies have shown that this gene is involved in growth performance and meat quality, therefore the aim of this study was to investigate Calpastatin gene expression in different tissues of Raini cashmere goat using Real time PCR. Materials and Methods Totally 21 tissue samples including muscle, adipose, kidney, spleen, liver, lung and heart tissues were taken from 3 Raini cashmere goats. RNA was extracted and cDNA was synthesized. Real time PCR was performed using SYBR Green method to study relative gene expression. GAPDH gene was used as housekeeping gene. Pfaffl method was used to analyze achieved data. Results For calpastatin gene 89bp fragment and for GAPDH 101bp fragment were observed in all studied tissues. Real time PCR results of this study showed that Calpastatin gene is expressed in all studied (muscle, adipose, kidney, spleen, liver, lung and heart) tissues and the highest level of expression was observed in heart, spleen and liver tissues and the lowest level was seen in adipose tissue. Conclusions Results showed that Calpastatin are widespread in different tissues of goat. This study would also lay a foundation for further Calpastatin research in Raini cashmere goat. It is suggested that this study be conducted with greater number of livestock, different sexes, different ages and different physiological stages in different breeds of goats in order to reach a comprehensive conclusion.

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.