Establishment of three-way junction formation and primer generation-rolling circle amplification for detection of EV71
Abstract：Objective To establish a method of combining three-way junction (3WJ) formation and primer genera-tion-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 suc-cessfully with good detection effect.
Keywords：enterovirus 71isothermal amplificationrolling circle amplificationthree-way junction
Publication Date：2015-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 21-23,111 )