Construction of Sheep PrP gene Standard plasmid DNA and curve using molecular beacon Real-time RT-PCR.

The aim of the present study was to prepare the plasmid and standard curve of real-time PCR for the quantitation of mRNA expression level of the digestive tract in sheep.Specific primer and molecular beacon were designed based on the conserved coding region from GenBank.Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR.Plasmid DNA was purified and identified by PCR amplification and sequencing after recycling and purification.The ARQ pattern plasmid was selected by PCR-SSCP,and was sequenced and analyzed.The positive plasmids were detected the real-time quantitative PCR after gradient dilution.The standard curve were generated automatically.The results showed that molecular beacons have high specificity and provide high sensitivity and accurate expression profiles.The correlation coefficients of the standard were 0.999,suggesting the strong linear relationship between the groups.Therefore,the construction of the standard plasmid and standard curve were established.