重组酶介导的蓝氏贾第鞭毛虫特异性等温核酸扩增方法的建立及评价

Establishment and evaluation of a novel DNA detection method based on recombinase-aided isothermal amplification assay for Giardia lamblia

目的建立基于重组酶介导的等温扩增技术(RAA)的蓝氏贾第鞭毛虫核酸检测方法,并评价其检测敏感性和特异性。方法选择蓝氏贾第鞭毛虫β-贾第素(β-giardin)基因作为检测靶基因,设计、合成特异性检测引物及荧光探针,建立荧光RAA检测体系。分别以不同拷贝数的重组质粒(含β-giardin基因靶序列)和不同浓度蓝氏贾第鞭毛虫基因组DNA为模板进行荧光RAA扩增,评价其检测敏感性;分别以蓝氏贾第鞭毛虫、日本血吸虫、华支睾吸虫、微小隐孢子虫、似蚓蛔线虫、沙门氏菌及志贺氏菌基因组DNA为模板进行扩增,评价其检测特异性。结果成功建立了蓝氏贾第鞭毛虫荧光RAA检测方法,其可在等温(39℃)条件下实现对靶基因片段的快速、特异性扩增(20 min内)。分别以重组质粒和蓝氏贾第鞭毛虫基因组DNA为模板,该方法的检测敏感性可达10~2拷贝/μL和1 pg/μL;以日本血吸虫、华支睾吸虫、微小隐孢子虫、似蚓蛔线虫、沙门氏菌及志贺氏菌基因组DNA为模板的RAA检测结果均为阴性,具备较好特异性。结论建立了一种操作简便、敏感、特异的可用于蓝氏贾第鞭毛虫核酸检测的荧光RAA方法。

Objective To establish a novel nucleic acid assay for detection of Giardia lamblia based on the recombinase-aided isothermal amplification(RAA) assay,and evaluate its sensitivity and specificity for detection of G.lamblia.Methods The specific primer sequences and florescent probes were designed and synthesized based on the G.lamblia β-giardin gene as the target gene,and a fluorescent RAA assay was established.The recombinant plasmids at various copies(containing the β-giardin gene target sequence) and the genomic DNA of G.lamblia at various concentrations were used as templates for the fluorescent RAA assay to assess the sensitivity,and the genomic DNA from G.lamblia,Schistosoma japonicum, Clonorchis sinensis,Cryptosporidium parvum,Ascaris lumbricoides, Salmonella and Shigella was used as templates to assess the specificity of the fluorescent RAA assay.Results A novel fluorescent RAA assay was successfully established for detection of G.lamblia,which allowed the rapid and specific amplification of the target gene fragments at 39 ℃ within 20 min.The sensitivities of the fluorescent RAA assay were10~2 copies/μL and 1 pg/μL for detection of the recombinant plasmid and G.lamblia genomic DNA,respectively,and the fluorescent RAA assay was negative for detection of the genomic DNA from S.japonicum, C.sinensis,C.parvum, A.lumbricoides, Salmonella and Shigella,which showed a high specificity.Conclusion A fluorescent RAA assay,which is simple,sensitive and specific,is successfully established for nucleic acid detection of G.lamblia.