多酶恒温快速扩增技术结合荧光探针快速检测日本血吸虫基因方法的建立

Establishment of multienzyme isothermal rapid amplification assay combined with fluorescent probe for rapid detection of Schistosoma japonicum

目的结合多酶恒温快速扩增技术(MIRA)和荧光探针法,建立一种快速检测日本血吸虫特异性基因片段的方法。方法以日本血吸虫非长末端重复序列反转录转座子(SjR2)片段为靶序列,设计并合成3对引物和荧光探针,建立荧光MIRA法反应体系,PCR检测,绘制扩增曲线。比较并筛选扩增效果较好的引物对和探针浓度;通过检测1 fg/µl、5 fg/µl、10 fg/µl、100 fg/µl、1 pg/µl和10 pg/µl等不同浓度的日本血吸虫成虫基因组DNA,评价该法的灵敏度;提取卫氏并殖吸虫、华支睾吸虫、东方次睾吸虫、颚口线虫及刚地弓形虫基因组DNA,并用荧光MIRA法检测,评价其特异度。耳缘静脉采集兔血,分离血清,制备含1 fg、5 fg、10 fg、100 fg、1 pg和10 pg日本血吸虫成虫DNA的兔模拟阳性血清并进行DNA提取,以荧光MIRA法检测,评估该法检测血清中日本血吸虫靶基因的最低限度。结果引物对1(SjR2‑1)的扩增效率较高,在反应第22个循环时(11 min)扩增出荧光产物,荧光值最高达170000;探针量为0.6µl/反应时具有较好的荧光强度和低荧光背景。建立的荧光MIRA法在39℃时第26个循环(13 min)时扩增出荧光产物,检测血吸虫成虫DNA的最低检出限为1 fg/反应。扩增产物电泳显示,血吸虫基因组DNA模板含量为10 pg、1 pg、100 fg、10 fg时,均有电泳条带出现,电泳显示的检测限为10 fg/反应,产物大小为186 bp。仅含有日本血吸虫成虫DNA的反应管出现荧光扩增曲线,检测卫氏并殖吸虫、华支睾吸虫、东方次睾吸虫、颚口线虫及刚地弓形虫等基因组DNA未出现明显荧光扩增曲线。不同浓度的血吸虫DNA模拟阳性血清中,DNA含量为1 pg和10 pg时,有阳性扩增曲线出现,最快于第16个循环(8 min内)即出现阳性荧光扩增信号,该法检测模拟阳性血清中血吸虫成虫DNA的最低检测限为1 pg/反应。结论成功建立了一种检测日本血吸虫特异性基因片段的荧光MIRA法,该法反应快速、敏感性高、特异性强,具有潜在的日本血吸虫病诊断应用价值。

Objective To develop a method for rapid detection of specific gene fragments of Schistosoma ja⁃ponicum using a multienzyme isothermal rapid amplification(MIRA)combined with fluorescent probing.Methods The S.japonicum non‑long terminal repeat retrotransposons(SjR2)fragment was selected as the target sequence,and three pairs of primers and fluorescent probes were designed and synthesized to establish a fluorescent MIRA reaction system,with which,fluorescence quantitative PCR was performed,and amplification curves were plotted to compare and screen out primer pairs and probe concentrations with better amplification effects.To evaluate the sensitivity of this method,adult S.japonicum genomic DNA at different concentrations of 1 fg/µl,5 fg/µl,10 fg/µl,100 fg/µl,1 pg/µl,and 10 pg/µl,were detected,respectively.To evaluate the methd specificity,genomic DNA extracted from Paragonimus wes⁃termani,Clonorchis sinensis,C.orientalis,Gnathostoma,and Toxoplasma gondii were examined using the fluorescence MIRA method.To assess the detactable limit of serum gene DNA of the method,rabbit blood was collected from the ear vein for separating serum to prepare simulated positive serum samples containing 1 fg,5 fg,10 fg,100 fg,1 pg,and 10 pg DNA of adult S.japonicum,which were detected using the fluorescence MIRA method.Results The am‑plification efficiency of primer pair 1(SjR2‑1)was high,and the fluorescent product was seen at the 22nd cycle(11 min)with a maximum fluorescence value of 170000;the probe amount at 0.6µl/reaction displayed better fluorescence intensity with low fluorescence background.The fluorescent products were amplified at the 26th cycle(13 min)at 39℃.The mini‑mum detection limit of the established fluorescence MIRA method for detecting Schistosoma adult worm DNA was 1 fg/reaction.The electrophoresis of amplification products showed that electrophoresis bands appeared when the template of Schisto⁃soma genomic DNA was 10 pg,1 pg,100 fg,and 10 fg,respectively,and the detection limit was 10 fg/reaction and the product size was 186 bp.The reaction tube containing only the DNA of S.japonicum adult worm showed fluorescence amplification curves,while no significant fluorescence amplification curves were observed in the detection of genomic DNA of P.westermani,C.sinensis,C.orientalis,Gnathostomum and T.gondii.When the DNA content in the simu‑lated positive serum of different concentrations of Schistosoma was 1 pg and 10 pg,positive amplification curves ap‑peared,and positive fluorescence amplification signals appeared as early as the 16th cycle(within 8 min).The minimum detection limit for detecting Schistosoma adult worm DNA in simulated positive serum by this method was 1 pg/reaction.Conclusion A fluorescent MIRA method detecting specific gene fragments of S.japonicum was successfully developed.The method is rapid,sensitive and specific in use,showing potential diagnostic application value for schistosomiasis japonica.