Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay

Abstract

Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.

FIG 1

Principle of the RT-IMSA assay versus principle of the RT-LAMP assay with regard to primer design and initial amplification step. (A) Primer design of RT-IMSA reaction. Six primers are used in the RT-IMSA assay, including two stem primers (SteF and SteR) and two pairs of nested hybrid-primers (two outer primers of DsF and DsR and two inner primers of FIT and RIT). The primers specifically recognize seven distinct regions of the target cDNA labeled F3, F2, F1, T, R1c, R2c, and R3c from the 5′ end. The DsF and DsR primers consist of the F3 and R3 and F1c and R1c sequences, the FIT and RIT primers consist of F2 and R2 and Tc and T sequences, and the SteF and SteR primers are the R1c and F1c sequences, respectively. (B) Primer design of RT-LAMP reaction. If taking into account the loop primers, six primers are also required in the RT-LAMP assay, including the two outer primers F3 and B3, the two inner hybrid-primers FIP and BIP, and the two loop primers LoopF and LoopB. The primers recognize eight distinct regions labeled F3, F2, LF, F1, B1c, LBc, B2c, and B3c from the 5′ end. (C) The initial step of RT-IMSA. For ease of explanation, DNA synthesis initiated from DsF, and FIT is set as the starting process (DNA synthesis proceeds with DsR and BIT in a similar manner). Horizontal straight lines with arrows represent the direction of primer elongation. Angled lines with arrows represent primers annealing to sites on the target. Arcs with arrows represent the self-matching function of two regions. In the step, four basic self-matching structures (SMS-1 to -4) with different lengths are generated. (D) The initial step of RT-LAMP assay. Only one basic self-matching structure, namely, the dumbbell structure, is generated. F, forward direction (green); R, reverse direction (red); c, complementary sequence (blue, forward complementary; yellow, reverse complementary).

FIG 2

Visual RT-IMSA assays and the electrophoresis of amplified and digested products. (A) Visual detection by RT-IMSA assay with the addition of HNB dye prior to amplification. Sky blue indicates positive reactions and violet indicates negative reactions. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (B) Visual RT-IMSA by adding the modified HNB with a GeneFinder dye after amplification. The color of positive reaction was dark green, whereas the color of negative reactions was orange. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (C) Electrophoresis of the products of visual RT-IMSA (HNB), including those of real-time RT-LAMP assay when testing identical templates (the VP1 RNA copies of CVA16). M, DL2000 marker; lanes 1 to 3, the products by RT-IMSA to amplify RNA copies of 10³, 10⁵, and 10⁷, respectively; lanes 4 to 6, the products by RT-LAMP to amplify RNA copies of 10³, 10⁵, and 10⁷, respectively. (D) Electrophoresis of digested products of visual CVA16 RT-IMSA (HNB) in which the site of HindIII restriction enzyme was introduced into the DsF and FIT primers. M, DL2000 marker; Lanes 1 to 4, the digested product of a positive reaction (the site in DsF), the digested product of positive (the site in FIT), the digested product of negative, and the amplified product of positive, respectively.

FIG 3

Linearity of quantification of the real-time RT-IMSA and RT-LAMP assays to test EV71 and CVA16 established through a linear regression plot by plotting the time-to-positivity values against the values of log₁₀ RNA copies tested per reaction. Linearity of quantification of RT-IMSA for the detection of EV71 (A), linearity of quantification of RT-LAMP for the detection of EV71 (B), linearity of quantification of RT-IMSA for the detection of CVA16 (C), and linearity of quantification of RT-LAMP for the detection of CVA16. NTC, no-target control. OD, optical density.