摘要
目的建立一种新的敏感、特异且能快速检测细粒棘球绦虫G1的重组酶介导扩增技术(Recombinase aided isothermal amplification,RAA)。方法以细粒棘球绦虫G1(E.granulosus G1,EgG1)线粒体基因序列(GenBank No.AF297617)作为靶序列设计合成引物,以EgG1 DNA为模板进行RAA扩增,反应产物预期大小为284 bp。以聚合酶链式反应(PCR)作为平行对照,应用RAA扩增不同稀释浓度的基因组DNA及梯度稀释的含目的基因片段不同拷贝数的pMD19-T(Simple)克隆质粒,以评价RAA检测方法的检测灵敏度;应用此方法检测多房棘球绦虫、牛带绦虫、亚洲带绦虫、多头带绦虫、犬复孔绦虫、犬弓首蛔虫、毛首鞭形线虫、贾第鞭毛虫、肝片吸虫、卫氏并殖吸虫、大片吸虫以及华支睾吸虫基因组DNA,以评价方法的特异性。方法优化后用于检测感染EgG1的动物组织标本(10份)、模拟现场阳性犬粪标本(3份)以及现场阳性犬粪标本(5份),以验证该项检测技术的可靠性和实用性。结果建立的RAA法可在40 min内特异性扩增EgG1的目的基因片段,最低可检测量为10 pg;以重组质粒为模板,RAA法最低检出质粒拷贝数为104个。用建立的RAA法检测其他寄生虫(包括多房棘球绦虫)结果均为阴性。用优化的RAA方法检测感染EgG1的动物组织标本以及阳性粪便标本(包括模拟现场粪便标本),结果均为阳性,且与PCR检测结果一致。结论建立的针对EgG1的RAA方法,其具有简便快捷、检测灵敏度与特异性均较好的特点,可望用于细粒棘球绦虫虫种的鉴定以及棘球蚴病基因诊断。
Objective To establish a sensitive,specific,and quick tool for the identification of Echinococcus granulosus G1 based on recombinase-aided isothermal amplification(RAA).Methods In accordance with the principle of an RAA reaction,primers were designed and synthesized based on the gene sequences of mitochondrial DNA(mtDNA)of E.granulosus G1(GenBank No.AF297617).E.granulosus G1 DNA served as a template for amplification using RAA;the expected size of the RAA reaction product was 284 bp.A polymerase chain reaction(PCR)was used as a parallel control.E.granulosus G1 genomic DNA at various concentrations and a pMD19 T(Simple)vector containing various copies of the target gene fragment were amplified to evaluate the sensitivity of RAA at detecting E.granulosus G1.RAA was also used to evaluate specificity by amplifying the genomic DNA of E.multilocularis,Taenia saginata,T.asiatica,T.multiceps,Dipylidium caninum,Toxocara canis,Trichuris trichiura Linnaeus,Giardia lamblia,Fasciola hepatica,Paragonimus westermani,Fasciola gigantica,and Clonorchis sinensis.Optimized RAA was performed to identify specimens with E.granulosus G1,including animal tissue(no.=10),simulated positive canine fecal specimens from the field(no.=3),and positive canine specimens from the field(No.=5),in order to verify its reliability and feasibility.Results The RAA assay established in this study specifically amplified the genomic DNA of E.granulosus G1 within 40 min,and the minimum detection limit was 10 pg.With a recombinant plasmid as a template,the minimum detectable copy number of the plasmid according to RAA was 104.The established method did not amplify the genomic DNA of other parasites(including E.multilocularis)tested in this study.Optimized RAA was used to detect E.granulosus G1 in animal tissue and positive fecal specimens(including simulated fecal specimens from the field).The positive specimens were all detected.The results were consistent with those of conventional PCR.Conclusion A method of RAA to identify E.granulosus G1 has been established and preliminarily used in tests involving real samples,and it was simple,quick,sensitive,and specific.This method could be used to differentiate E.granulosus G1 by species in the field.
作者
周鸿让
王晓玲
许秋利
艾琳
陈木新
余晴
肖宁
ZHOU Hong-rang;WANG Xiao-ling;XU Qiu-li;AI Ling;CHEN Mu-xin;YU Qing;XIAO Ning(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Disease Research,WHO Collaborating Centre for Tropical Diseases,National Center for International Researchon Tropical Diseases,Ministry of Science and Technology,Key Laboratory of Parasite and Vector Biology,National Health Commission of China,Shanghai,China 200025)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第6期655-660,共6页
Journal of Pathogen Biology
基金
国家重点研发计划项目(No.2016YFC1200500)
中国疾病预防控制中心包虫病综合示范项目(No.201901)。
关键词
细粒棘球绦虫G1
重组酶核酸等温扩增技术
虫种检测
Echinococcus granulosus G1
recombinase-aided isothermal amplification(RAA)
identification
