重组酶介导的多重核酸等温扩增法鉴别细粒棘球绦虫和多房棘球绦虫技术的建立与应用

Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis

目的建立一种快速检测细粒棘球绦虫G1(EgG1)与多房棘球绦虫(Em)DNA的重组酶介导的多重核酸等温扩增方法(mRAA)。方法以EgG1和Em线粒体基因作为靶序列,设计特异性引物,建立快速检测棘球绦虫的mRAA方法。分别扩增浓度为10.00、5.00、1.00、0.50、0.10、0.05、0.01 ng/μl棘球绦虫基因组DNA及10^5、10^4、10^3、10^2、10个拷贝/μl的pMD19-T(Simple)克隆质粒,评价mRAA方法的敏感性;分别以牛带绦虫、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、蓝氏贾第鞭毛虫、肝片形吸虫、卫氏并殖吸虫、大片形吸虫以及华支睾吸虫基因组DNA为模板,评价mRAA方法的特异性。采用优化后的m RAA方法,分别对3份EgG1和Em混合模拟犬粪样、19份现场采集的棘球绦虫感染动物肝脏组织(10份EgG1感染、9份Em感染)、7份现场采集的棘球绦虫阳性犬粪(5份EgG1感染、2份Em感染)进行检测,验证m RAA方法的可靠性和实用性。结果建立的mRAA方法可特异性扩增EgG1和Em线粒体基因片段,长度分别约为250 bp、500 bp。mRAA方法对EgG1和Em基因组DNA的最低检测限为2 pg/μl,对EgG1和Em重组质粒的最低检测限为200个拷贝/μl。mRAA方法对牛带绦虫、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、蓝氏贾第鞭毛虫、肝片形吸虫、卫氏并殖吸虫、大片形吸虫以及华支睾吸虫基因组DNA的扩增结果均为阴性。mRAA方法成功检出所有EgG1和Em混合模拟犬粪样、棘球绦虫感染动物肝脏组织、棘球绦虫阳性犬粪样,且检测结果与mPCR一致。结论建立的mRAA方法可用于EgG1和Em DNA的快速检测,敏感性、特异性和可靠性均较好。

Objective To establish a multiplex recombinase-aided isothermal amplification(mRAA)method for rapid detection of Echinococcus granulosus G1(EgG1)and E.multilocularis(Em)DNA.Methods The mRAA rapid detection method for Echinococcus was established by designing primers based on the mitochondrial genes of EgG1 and Em as target sequences.The sensitivity of the mRAA method was assessed by amplifications of EgG1 and Em genomic DNA at concentrations of 10.00,5.00,1.00,0.50,0.10,0.05,and 0.01 ng/μl and of pMD19-T(Simple)recombiant plasmids at 10^5,10^4,10^3,10^2 and 10 copies/μl.The specificity of the mRAA method was evaluated by using genomic DNA of Taenia saginata,T.asiatica,T.multiceps,Dipylidium caninum,Toxocara canis,Trichuris trichiura Linnaeus,Giardia lamblia,Fasciola hepatica,Paragonimus westermani,Fasciola gigantica and Clonorchis sinensis as templates for amplification.To verify the reliability and feasibility,optimized mRAA method was used to examine three fecal samples of dogs with simulated infection of mixed EgG1 and Em.19 liver tissue samples of infected animals(10 with EgG1 infection and 9 with Em infection)collected from field,7 fecal samples of infected dogs(5 EgG1 infected and 2 Em infected)collected from field.Results The established m RAA method could specifically amplify the EgG1 and Em mitochondrial gene fragments with the sequence lengths of 250 and 500 bp,respectively.The detactable limit of EgG1 and Em genomic DNA by m RAA was 2 pg/μl,and that of the EgG1 and Em recombinant plasmids was 200 copies/μl.The mRAA method showed negative results of amplification on genomic DNA of T.saginata,T.asiatica,T.multiceps,D.caninum,T.canis,T.trichiura Linnaeus,G.lamblia,F.hepatica,P.westermani,F.gigantica and C.sinensis.It was demonstrated that the mRAA method successfully detected EgG1 and Em DNA from the fecal samples of dogs with simulated infection of mixed EgG1 and Em,the liver tissue of infected field animals,and fecal samples of infected field dogs.Conclusion The mRAA method displays evident sensitivity,specificity and reliability,and could be used for rapid detection of EgG1 and Em DNA.