三向连接构造组合引物介导的滚环扩增技术检测肠道病毒71型方法的建立

Establishment of three-way junction formation and primer generation-rolling circle amplification for detection of EV71

目的建立三向连接构造(3WJ)组合引物介导的滚环扩增(PG-RCA)技术检测肠道病毒71型(EV71)的方法,并验证其检测效果。方法根据EV71的VP1基因保守区设计引物模板、引物以及环状探针前体,确立反应体系及反应条件,实时荧光PCR仪监测扩增结果。采用已建立的3WJ组合PG-RCA技术检测16例手足口病患儿咽拭子标本(EV71阳性12例、柯萨奇病毒A阳性2例、柯萨奇病毒B阳性2例)。结果已建立的3WJ组合PG-RCA技术能检测到amol级的EV71 RNA,说明方法建立成功。EV71阳性标本扩增曲线荧光强度均超过阈值;柯萨奇病毒A、B阳性标本扩增曲线荧光强度均低于阈值。结论成功建立3WJ组合PG-RCA技术检测EV71的方法,检测效果好。

Objective To establish a method of combining three-way junction( 3WJ) formation and primer generation-rolling circle amplification( PG-RCA) for the detection of enterovirus 71( EV71) and to verify its detection effect.Methods According to conservative EV71 VP1 gene,we designed template of primer,primer and circular probe precursor to detect EV71 with an isothermal reaction format. Real-time fluorescence PCR was used to detect the amplification. Using the established 3WJ combined with PG-RCA technology to detect the pharyngeal swab specimens in 16 cases of children with hand,foot and mouth disease( 12 cases of positive EV71,2 cases of positive coxsackie virus A and 2 cases of positive coxsackie virus B). Results The 3WJ combined PG-RCA technology could detect the amol level of synthetic EV71 RNA.The detection method had good specificity and sensitivity. The amplification curve fluorescence intensity of EV71 positive specimens was stronger than the threshold,and the amplification curve fluorescence intensity of Coxsackie virus A and B positive specimen was lower than the threshold. Conclusion The 3WJ combined PG-RCA technology is established successfully with good detection effect.