中国兽医科学2024，Vol.54Issue(5)：602-608,7.DOI:10.16656/j.issn.1673-4696.2024.0095
犬猫弓形虫病real-time荧光定量PCR检测方法的建立与应用
Establishment and application of real-time fluorescent quantitative PCR for detection of toxoplasmosis in dogs and cats
摘要
Abstract
To establish a highly sensitive,specific and automated molecular diagnostic method for detecting Toxoplasma gondii in dogs and cats,this study extracted the DNA of the RH strain of T.gondii and used the conserved region of the dense granular protein 7(GRA7)gene as the target.Specific primers were designed and a standard plasmid pMD18-GRA7 was constructed,A SYBR Green Ⅰ fluorescent quantitative PCR(real time PCR or qPCR)detection method was established using the standard plasmid pMD18-GRA7 as a template.The results showed that the specific primers of this method could only amplify DNA samples of T.gondii,and no amplification curve was found in the negative samples and other positive samples of dog and cat parasites.The detection limit of the established qPCR method is 3 copies,when the standard con-centration is 3 copies/μL,the Ct value detected is 29.4.The detection of clinical samples from dogs and cats showed a positive and negative coincidence rate of 100%compared to the results of commercial reagent kits.The above results indicate that the SYBR Green Ⅰ fluorescence quantitative PCR method established in this study has good specificity,strong sensitivity,and stable results,and can be used for clinical detection of canine and cat toxoplasmosis.关键词
弓形虫/犬/猫/GRA7/实时荧光定量PCRKey words
Toxoplasma gondii/dogs/cats/GRA7/real-time PCR分类
农业科技引用本文复制引用
施远国,胡潇予,柯艳坤,李颖鑫,文青元,杨子鹏,杨留禄,袁子国..犬猫弓形虫病real-time荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2024,54(5):602-608,7.基金项目
弓形虫检测技术研究项目(NJ-FW-20230068) （NJ-FW-20230068）
国家自然科学基金项目(31972707) （31972707）
广东省基础与应用基础研究基金项目(2023A1515011795) （2023A1515011795）
