Analysis Of Genes Sequence Of CEV Dunhua Strains And Detection Of PCR

Sheep Contagious Ecthyma is a kind of acute and contagious disease,which is caused by Contagious Ecthyma virus(CEV)and infects ruminants such as goats and sheep.The symptoms of CEV,are extremely similar to those of viruses including the sheep pox virus,goat pox virus and foot-and-mouth disease.At present,the sensitivity and accuracy of the detection methods used by CEV,is actually low.In order to diagnose the virus quickly,efficiently and accurately,a method called SYBR Green I real-time fluorescence quantitative PCR,with high specificity and sensitivity,is put forward and applied.In text 1,culturing MDBK cells in vitro,CEV Dunhua strain was isolated from the skin of diseased goats in Dunhua.The isolated strain was identified as the sheep Contagious Ecthyma virus,after the research of the cultured characteristics,the observation of electron microscopy,and a series of tests including ether sensitivity,chloroform sensitivity,acid resistance,resistance Alkaline,heat resistance,ultraviolet irradiation and animal regression.In text 2,the F1 L gene sequences of the isolated CEV Dunhua strain,were compared with those of the amino acid sequences corresponding to different strains,which have been published at home and abroad.The results showed that the homologies between the isolates and the published ranged from 97% to 99%,and the phylogenetic relationships between the isolates and the Jilin strain were the closest,which had the farthest ones with the OV/Torino.The results show that F1 L gene is genetically highly conserved,but still has some variations.According to the IL-10-LIKE PROTEIN gene sequence of the CEV,published by Genbank,in order to verify the practicability of the proposed method,measures can be listed as follows.A pair of specific fluorescent primers was designed and synthesized;the recombinant plasmid was constructed as a standard template;the dyestuff method was used for PCR amplification;the standard curve was drawn;the correlation coefficient of the standard product was evaluated;the reproducibility and specificity tests were performed;clinical materials of 14 clinical cases of suspected infections were tested.The results showed that the minimum detection concentration of newly established method ran up to 82.9 copies/?L,and the melting curve had a single specific peak,featured by specific amplification,excellent repeatability,obvious sensitivity stability.In addition,the method had no cross reaction with the sheep pox vaccine,the goat pox vaccine and the foot-and-mouth disease vaccine.The positive rate of virus materials of 14 clinically suspected sheep infectious impetigo diseases was 100%.The above results indicated that the SYBR Green I real-time fluorescence quantitative PCR established in this study,was valid and practicable.