Development of a New Semi-nested PCR Real-time Fluorescence Technology for Detection of Toxoplasma gondii
CHEN Meng-Tao1, ZHU Long-Jiao2,3, LIU Hai-Yan4,*, XU Wen-Tao2,3,*
1 School of Public Health, North China University of Science and Technology, Tangshan 063210, China; 2 Department of Nutrition and Health, Key Laboratory of Food Precision Nutrition and Quality Control, China Agricultural University, Beijing 100083, China; 3 College of Food Science & Nutritional engineering, Key Laboratory of Transgenic Biological Safety Evaluation (Food Safety)/Food Science and Nutrition Engineering, Beijing Laboratory of Food Quality and Safety, China Agricultural University, Beijing 100083, China; 4 Research Center for Sports Nutrition and Eudainomics, Tianjin University of Sport, Tianjin 301617, China
Abstract：Toxoplasma gondii is a worldwide zoonotic parasite with a high chronic infection rate in pigs (Sus scrofa) and small ruminants. It can cause great harm to individuals after being infected with immunocompromised people through food-borne transmission. In this study, a set of specific primers were designed according to the 529 bp gene sequence of T. gondii (DQ779191.1) published in GenBank, and a semi-nested PCR real-time fluorescence method was established based on SYBR GreenⅠ dye to detect T. gondii. In the first-round of amplification, the 529 bp repetitive sequence of T.gondii was amplified by external primers, and then the 529 bp gene fragment was amplified by real-time fluorescence amplification with the internal primer of the second-round of amplification combined with SYBR GreenⅠ dye, and the final product fragment length was 260 bp. Under the optimized reaction conditions, the detection limit of this method was 18.4 fg/μL with high sensitivity. There was no cross reaction with the genomes of Echinococcus granulosus and Taenia solium, and the specificity was sound. Therefore, this study successfully established a new semi-nested PCR real-time fluorescence detection method with high sensitivity and specificity, which provides an effective technical means for monitoring T. gondii infection in livestock and meat products.
陈梦陶, 朱龙佼, 刘海燕, 许文涛. 弓形虫半巢式PCR实时荧光检测方法的建立[J]. 农业生物技术学报, 2023, 31(12): 2665-2673.
CHEN Meng-Tao, ZHU Long-Jiao, LIU Hai-Yan, XU Wen-Tao. Development of a New Semi-nested PCR Real-time Fluorescence Technology for Detection of Toxoplasma gondii. 农业生物技术学报, 2023, 31(12): 2665-2673.
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