The Establishment Of Real-time Fluorescence Quantitative PCR Detection Method For CEV

Sheep Contagious Ecthyma is an acute,contagious disease of sheep and goats that is caused by the Contagious Ecthyma Virus (CEV).To diagnose the disease more quickly and accurately,two kinds of Real-time Fluorescence Quantitative PCR detection methods were established in this experiment by using F1L gene as a standard.These two methods were SYBR Green I Real-time Fluorescence Quantitative PCR and TaqMan Real-time Fluorescence Quantitative PCR.Two methods were established through two pairs of specific primers and a TaqMan probe that were synthesized using the Primer Express 3.0 based on the F1L gene sequence on CEV reference strains published by GenBank (AF097215).As standard templates,the recombining positive plasmids for two methods were established.The reaction system and response parameter of these two methods were optimized.Two standard curves were established.The correlation and amplification efficiencies were evaluated.The repeatability,specificity and sensitivity of these two Real-time Fluorescence Quantitative PCR methods were tested.Two kinds of Real-time Fluorescence Quantitative PCR were applied to detect 11 Sheep Contagious Ecthyma cases for demonstrating it’s practicability.The result showed that the correlation coefficiency of SYBR Green I Real-time Fluorescence Quantitative PCR was 0.99 for standard curves and it’s amplification efficiency was 1.18.The minimum detection limit was 65.47copies/μL.The coefficient of variation for intra-assay and inter-assay were 0.17%-0.59% and 0.74%～1.25% respectively.The result of TaqMan Real-time Fluorescence Quantitative PCR method showed that the correlation coefficiency was 0.997 for standard curves and the amplification efficiency was 1.08.The minimum detection limit was 13.40copies/μL.The coefficients of variation for intra-assay and inter-assay were 0.28%～0.55% and 0.45%～1.31% respectively.The repeatability of the two methods was good all.These two methods were both proved strong specificity,high sensitivity and no cross reactions with Goat Pox Vaccine (Living),Foot-and-Mouth Disease (type O and Asia 1) Bivalent Vaccine (Inactivated) and Peste des Petits Ruminants.Applying these two detection methods to detect 11 Sheep Contagious Ecthyma cases,all of the results were positive.