实时定量PCR测定爱德华氏菌隐蔽质粒的拷贝数  

Real time PCR quantification of the copy number of cryptic plasmids in Edwardsiella ictaluri

作　　者：付立霞 高雯[2] 韩先干[3] 高波[2] 张晓君[2] 刘晓丹[2] 曾令兵 FU Li-xia;GAO Wen;HAN Xian-gan;GAO Bo;ZHANG Xiao-jun;LIU Xiao-dan;ZENG Ling-bing(Key Lab of Freshwater Biodiversity Conservation,Ministry of Agriculture;Yangtze River Fisheries Research Institute,CAFS,Wuhan 430223,China;2.Jiangsu Key Laboratory of Zoonosis,College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,Jiangsu,Chin;3.Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)

机构地区：[1]农业部淡水生物多样性保护重点实验室,中国水产科学研究院长江水产研究所,武汉430223 [2]江苏省人畜共患病学重点实验室,扬州大学动物科学与技术学院,江苏扬州225009 [3]中国农业科学院上海兽医研究所,上海200241

出　　处：《淡水渔业》2018年第4期63-70,共8页Freshwater Fisheries

基　　金：农业部淡水生物多样性保护重点实验室开放课题(LFBC0910);江苏省人兽共患病学重点实验室项目(R1510);上海市科技兴农重点攻关项目(2015HNG1-9);国家自然科学基金项目(31671313)

摘　　要：为测定爱德华氏菌(Edwardsiella ictaluri)隐蔽质粒的拷贝数,并探究不同基因组制备方式及定量策略对质粒拷贝数测定的影响,分别采用试剂盒和水煮法制备爱德华氏菌基因组,通过绝对定量和相对定量PCR测定两种不同总DNA制备方式下的隐蔽质粒拷贝数,同时测定试剂盒对染色体DNA和质粒DNA的回收率。结果显示,以试剂盒提取的总DNA测得的pEI1和p EI2拷贝数绝对定量和相对定量分别为3.63±0.30、5.04±0.18和4.22±0.15、5.13±0.50,显著低于以水煮法测得的pEI1和p EI2拷贝数的绝对定量11.84±0.80、11.70±0.25和相对定量13.85±1.64、11.90±0.97。回收率结果显示,试剂盒对染色体DNA的回收率(45.8±4.1)%,显著高于对质粒pEII的回收率(25.1±0.5)%和pEI2的回收率(31.3±1.7)%。结果表明:通过实时定量PCR测定爱德华氏菌隐蔽质粒的拷贝数时,基因组的制备以水煮法为宜,爱德华氏菌隐蔽质粒pEI1和pEI2均属于中拷贝质粒。Edwardsiella ictaluri genome was prepared by commercial kit and water boiling method to determine the copy number of cryptic plasmids in it and explore the effects of different total DNA preparation and quantification strategy on plasmid copy number determination.Absolute and relative real time PCR were performed to measure the copy number of cryptic plasmids in E.ictaluri .Meanwhile,the recovery rates of chromosomal DNA and plasmids DNA recycled by kits were determined.The results showed that the absolute and relative quantification of plasmid pEI1 and pEI2 copy number with the commercial kit-extracted DNA were 3.63±0.30,5.04±0.18,4.22±0.15 and 5.13±0.50,respectively,which was significantly lower than that of corresponding plasmids with water-boiling method of the copy number of 11.84±0.80,11.70±0.25,13.85±1.64 and 11.90±0.97.The recovery assay showed that recovery rate of chromosomal DNA was （45.8±4.1）%,which was significantly higher than that of plasmid pEII of （25.1±0.5） % and pEI2 of （31.3±1.7）% by the DNA mini kit.In conclusion,the method of DNA preparation by water-boiling is better for the real time PCR quantification of the copy number for cryptic plasmids in E.ictaluri ,and cryptic plasmids pEI1 and pEI2 in E.ictaluri can be classified as medium copy number plasmids.

关 键 词：鮰爱德华氏菌(Edwardsiella ictaluri) 隐蔽质粒 质粒拷贝数 实时定量PCR 

分 类 号：S941.4[农业科学—水产养殖]