جستجوی مقالات مرتبط با کلیدواژه « time pcr » در نشریات گروه « علوم دام »

The aim of the present study was to compare the expression of lactoferrin and TLR4 genes in cow milk somatic cells to investigate genetic differences effects and crossbreeding on the gene expression of the immune system as well as the effect of mastitis. Total RNA was extracted from fresh milk cells of 3 healthy Holstein¡ 3 Guilan Native and 3 F1 crossbred cattle belong to the National Animal Breeding Center of Iran and 3 Holstein with mild symptoms of clinical mastitis. After cDNA synthesis genes expression was measured using Real-time PCR relative to the reference gene GAPDH. The results showed while in crossbred cows the average expression of both genes was higher than parents (P0.05). This study indicated that while there was non-significance difference between Guilan native and Holstein¡ but the crossbreeding clearly affected on the expression of two genes associated with genetic resistance.

Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC) PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR) technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor), using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR.
Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2) was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve.
Results and Discussion The specific primers of Butyrivibrio fibrisolvens were successfully used for amplifying the specific fragments from this bacteria. The main and important factors for increasing the accuracy of Q-C PCR is the degree of similarity between competitor and target fragment. In this study the competitor fragment was highest homology to target sequences. In this regards it seems obtained results have considerable accuracy. The intensity of bands was evaluated and analyzed using Image J software. The results of band intensity analysis showed linear trend between competitor and target in different serial dilution of competitors. The specific fragment from 16S rDNA region of Butyrivibrio fibriolvents Bacteria was amplified using specific primers and cloned in pTZ57R/T plasmid. After amplifying the competitor fragment and target sequence simultaneously, the two bands were detectable in gel electrophoresis. The range of 10-1 to 10-6 serial dilution from competitor was selected for QC-PCR reaction. The results of this section showed that the considerable linear correlation was exist between competitor and target fragment in QC-PCR reaction (R2=0.985). In this study, Real-time PCR was also used for quantification of Butyrivibrio fibriolvents Bacteria strain. Melting analysis was showed that the reaction in Real-time PCR had appropriate condition for amplifying the target sequence. We used a standard in this study. This standard was designed as a vector contain of competitor fragment. This kind of standard was successfully used for QC-PCR and Real-time PCR analyses. Standard competitor could be used for absolute quantification for this bacterial strain. Overall, our results showed that the designed standard and optimized QC-PCR have considerable potential for Butyrivibrio fibriolvents Bacteria strain.
Conclusion In this study, the two methods of quantification of nucleotide acids in biological samples were optimized. These two methods were performed in order to optimize Butyrivibrio fibriolvents Bacteria strain quantification. Our results showed that both techniques have the capabilities to use as valuable research methodologies for enumerating the Butyrivibrio fibrisolvens strains.

IL8RB gene is one of the chemokine receptors presented on neutrophil surfaces whichare necessary for maximizing of neutrophils ability during infection. Different sequence and expression of IL8RB gene lead to different resistance to mastitis in cow populations. The aim of current study was to investigate expression changes of IL8RB gene resulted from crossbreeding of Guilan Native cows with Holstein cows. We collect 56 blood samples from 5 different cow populations in Guilan Native cattle breeding center. After RNA extraction and cDNA synthesis, realtime PCR reactions were performed in all samples with three repeats for each sample. We used GAPDH and RPLP0 as internal reference control genes. The maximum amount of IL8RB gene (0.74±0.77)average normalized expression was in Native cow populations and the minimum expression (0.12±0.22) was in 75 percent Holstein crossbred cow populations. Highest to lowest amount of IL8RB gene average normalized expression was in Native cow (0.74±0.77), 50 percent sire Native and dam Holstein crossbred cow (0.3±0.27), 50 percent sire Holstein and dam Native crossbred cow (0.26±0.3), Holstein cow (0.23±0.26), and 75 percent Holstein crossbred cow (0.12±0.22), respectively. With Consideration of highest gene expression in Native cow and decreasing in gene expression respect to increasing in blood contribution of Holstein cow which they are sensitive to mastitis, consequently changes in gene expression of IL8RB are related to resistance to mastitis. As this study showed the effect of crossbreeding on IL8RB gene expression changes, then it is necessary to consider the consequences of crossbreeding on native cattle breeding program.

Foot-and-mouth disease (FMD) is a severely contagious viral disease in cloven-hooved animals that it causes considerable economic losses in livestock productivity. Vaccination is one of the most effective procedures for control of FMD. One of vaccines performances is stimulating expression of some immune system genes، which called cytokines. In this study، expression changes of IFN-γ and TGFβ1 genes were evaluated in vaccinated guinea pigs with FMD type O inactivated vaccine. Blood samples were collected from vaccinated and control (no vaccinated) guinea pigs in three distinct times. After blood sampling، RNA was extracted and converted to cDNA. For measuring IFN-γ and TGFβ1 genes expression، relative Real-time PCR procedure was used. The results showed that expression of IFN-γ and TGFβ1 genes in the second and third blood sampling were significantly increased in comparison to the first blood sampling. Because increasing of cytokines expression is an indicative of the immune system response، these genes can be used as indicators for testing effects of the recombinant vaccines.