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A novel method for DNA extraction from small number of oocysts of eimeria species

A novel method for DNA extraction from small number of oocys...

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中国动物学会寄生虫学专业委员会第十届全国寄生虫学青年工作者学术研讨会

作者： Xiaoli Tang Guangping Huang Xianyong Liu Xun Suo National Animal Protozoal laboratory & College of Veterinary Medicine China Agricultural University

Introduction Coccidiosis caused by intracellular protozoan parasites of the genus eimeria is an economically important enteric disease *** identification of different species of eimeria genus is critical for prevention,surveillance,and control of *** diagnostic methods are labour intensive and time-consuming,and do not ***,DNA-based molecular biology approaches have been providing assistance in the species identification of the *** investigate whether it is possible to extract DNA from a small number of oocysts,200 oocysts were used in the present study as the lowest detection *** & methods Both the purified and unpurified oocyst suspension was divided into 16 equal portions of 1×10 oocysts *** sample is centrifuged at 1000 g for 10 min to sediment the *** supernatant is discarded completely and the pellet is re-suspended in 10μl sodium hypochlorite(10%available chlorine).Every 4 of 16 was treated at 4℃ for 0.5、1、1.5、2h,*** the same time treatment of the 4 portions,they were combined with 35 μl saturated salt solution and incubated at 55 ℃ for 0.5、1、1.5、2h,*** centrifuged at lOOOg for 5 min,the floating part、supernatant、precipitate are observed and pictured under a *** see the DNA directly,both the purified and unpurified oocyst suspension of *** was diluted into eight groups which include 2×10,5×10,1×10,5×10,1×10,5×10,1×10,and 5×10 sporulated oocysts,*** the treatment of sodium hypochlorite(4℃,1.5h) and saturated salt solution(55 ℃,1.5h),the oocyst suspension was prepared for protease digestion through the addition of 300 μl TE buffer(10 mM Tris-HCl,0.1 mM EDTA pH 8),3 μl of10%(w/v) SDS and 10 μl of proteinase K(20 mg/ml).After another one hour incubation at 37 ℃,oocyst DNA was extracted by using a commercially available nucleic acid purification kit following the manufacturer instructions(TIANGEN,China).PCR amplif

关键词： coccidiosis eimeria spp DNA extraction

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