| Canine Infectious Hepatitis(CIH) is an acute and contagious canine infectious disease,due to high morbidity and mortality,which was caused by canine adenovirus type 1(CAV-I).There is not an perfect detect method so far. The routine detection of CAV-I is HA,HI,IHA,AGP,ELISA,RT-PCR et al. These methods have some disadvantages,for example,low speciality, time-consuming,low sensitivity,fake positiveity et al.Therefore,it is important for us to test a rapid and exact detection method.The real-time PCR is a new detection of nucleic acid,which is used to bacterial,virus,and genictic disease in the clinical detection.Based on the fluorescence quantitative PCR technology,we established the detection method of SYBR Green I real time PCR technique for Canine Infectious hepatitis virus.A pair of primers were designed based on reserved nucleotide sequence.Sequencing results of cloned plasmid and electrophore of the PCR production in 1%agarose indicated that the amplifictor is 100bp.And cloned plasmid was used as positive control in the following study.Then the reaction conditions were optimized to obtain high amplification efficiency and sensitive detection.Tm values of the special amplified products were always at 79±0.5℃,which is read-out criteria of the assay.Specificity experiment indicated that only CAV-I can be amplified and other virus amplification without classical amplification curve and dissociation curve.Sensitivity experiment showed that the method is more sensitive than common PCR and can replace its application.Subsequently,46 entry-exit samples were tested with the assay and ELISA and HA,indicating that it is more suitable for entry-exit quarantine.This assay is more sensitive and rapid than common PCR and without contamination on environment and needn't to do electrophoresis,which can be popularized as a rapid detection method.After trial and error,kits for CAV-I detection were assembled which can be popularized through veterinary labs. |
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