基于novel⁃miR1检测家兔豆状囊尾蚴感染的滚环扩增方法的建立和应用  

Establishment and application of a rolling circle amplification method based on novel⁃miR1 for detection of Cysticercus pisiformis infection in rabbit

作　　者：陈国梁 王立群 李艳萍 刘婷丽 李红 张少华[1] 骆学农[1] 强文军 CHEN Guoliang;WANG Liqun;LI Yanping;LIU Tingli;LI Hong;ZHANG Shaohua;LUO Xuenong;QIANG Wenjun(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,Gansu,China;Animal Husbandry and Veterinary Technology Extension Center,Liangzhou District,Wuwei 733000,Gansu,China)

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,兰州730046 [2]甘肃省武威市凉州区畜牧兽医技术推广中心,武威733000

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第3期294-299,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(32072889)。

摘　　要：目的建立检测家兔豆状囊尾蚴感染的滚环扩增(RCA)方法。方法以家兔血清中豆状囊尾蚴来源的novel⁃miR1为诊断靶标,设计连接序列和锁式探针,并对连接序列和锁式探针的比例、反应时间、酶用量、dNTP用量以及扩增反应时间等5个重要条件进行优化,建立检测豆状囊尾蚴感染的RCA方法。将novel⁃miR1标准品倍比稀释为1 fmol/L至100 nmol/L的9个不同浓度的样品,评价RCA方法的灵敏度。用优化后的RCA方法分别检测健康家兔和豆状囊尾蚴感染家兔血清miRNA各20份(实验室保存样品),并通过受试者工作特征(ROC)曲线分析其敏感性和特异性。20只雌性家兔每只经口感染1000个豆状带绦虫虫卵,采集感染前和感染后每个月的家兔血样制备血清miRNA,用优化后的RCA方法进行检测,评价其应用效果。结果电泳结果显示,RCA扩增产物的DNA停留在加样孔中,形成一条明亮条带。反应条件优化试验结果显示,连接序列浓度为2μmol/L、锁式探针浓度为1μmol/L(连接序列和锁式探针的比为2∶1)、T4 DNA连接酶为350 U、连接180 min时连接效率最高。在100μl的扩增体系中,dNTP浓度为0.5μmol/L,扩增240 min时,RCA扩增效果最佳。优化后RCA的最低检测浓度为10 pmol/L。RCA检测健康家兔和豆状囊尾蚴感染家兔血清miRNA样品的平均荧光值分别为53.298±1.707和97.498±5.892,差异有统计学意义(t=7.206,P<0.01)。ROC分析结果显示,当阳性临界值为61.69时,RCA方法的敏感性和特异性均为95%,AUC为0.9550,似然比为19.00。RCA检测豆状囊尾蚴感染后不同时间的家兔血清miRNA结果显示,20份感染前的血清中19份检测结果为阴性,1份为阳性;感染后1个月的血清中,17份为阳性,2份为疑似,1份为阴性;感染后2个月的血清,1份为阴性,其余为阳性;感染后3个月的血清,3份为阴性,其余为阳性。结论建立了检测家兔豆状囊尾蚴感染的RCA方法,该方法在豆状囊尾蚴感染后3�Objective To establish a rolling circle amplification(RCA)method for detection of Cysticercus pisiformis infection in rabbits.Methods The novel⁃miR1 derived from C.pisiformis in rabbit serum served as the diagnostic target to establish an RCA diagnostic method for cysticercosis pisiformis fection in rabbit.To establish the RCA method,ligation sequence and the locking probe sequence were designed,and five important reaction con⁃ditions were optimized,including the ratio of ligation sequence and padlock probe,ligation reaction time,ligase dos⁃age,amplification reaction time,and dNTP dosage.The sensitivity of the RCA method was assessed,the novel⁃miR1 standard was serially diluted into 9 samples of different concentrations ranging from 1 fmol/L to 100 nmol/L.The sensitivity and specificity of the optimized RCA methods were assayed using the serum miRNA from 20 healthy rabbits and 20 C.pisiformis infected rabbits(Laboratory preserved samples),and analyzed by the receiver operating characteristic curve(ROC curve)method.To evaluate the application effectiveness of the RCA,20 female rabbits were infected by gavage with 1000 eggs of T.pisiformis for collection of serum every month post⁃infection,which were used to prepare miRNA for application in RCA method under optimized condition.Results The agarose gel electrophoresis results showed that the RCA amplification products remained in the sampling wells of agarose gel,forming a bright band.The tests to optimize RCA condition indicated that the concentration of ligation se⁃quence was 2μmol/L,padlock probe was 1μmol/L(ratio of ligation sequence and padlock probe was 2∶1),T4 DNA ligase was 350 U,and the efficacy of ligation reaction was found the highest when ligating for 180 min.The optimal amplification reaction system was 0.5μmol/L dNTP and amplification reacted 240 min in 100μl amplifica⁃tion reaction system.Lastly,the RCA method limit of detection was proved to be 10 pmol/L.The RCA method de⁃tected showed that the average serum miRNA fluorescence i

关 键 词：豆状囊尾蚴病 novel⁃miR1 滚环扩增 

分 类 号：R532.39[医药卫生—内科学]