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

c Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-footand-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-selfmatching-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. Hand-foot-and-mouth disease (HFMD) is a childhood syndrome and is characterized by tiny blisters on the skin and oral mucosa together with fever and poor appetite. The two major causative agents of HFMD are human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). EV71-related HFMD commonly accounts for the fatal cases, due to severe complications, such as brainstem encephalitis and rapid fatal pulmonary edema, whereas CVA16-related HFMD usually presents with mild symptoms (1-3). Since no vaccine or antiviral drugs are currently available to treat HFMD, the early and rapid detection of EV71 and CVA16 are critical for the prevention and control of HFMD infection.Presently, laboratory detection of EV71 and CVA16 includes traditional virus isolation, neutralization, and nucleic acid amplification techniques. It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4-6). Nucleic acid a...

Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I fromGeobacillus stearothermophilusby Site-Directed Mutagenesis at the Active Site

“…The primers for the IMSA assay was designed with the aid of Primer Explorer V4 [8] shown in Table 2 and synthesized by Huada Gene Co., Ltd. (Shenzhen, China). The optimal condition of IMSA assay was carried out in a 25  μ L volume containing the components: 12.5  μ L of 2x isothermal reaction mixture consisted of reaction buffer and dNTPs (Deaou Biotechnology, Guangzhou, China), 1.0  μ L of each primer (DsF and DsR, 5.0 mM; FIT and RIT, 20.0 mM; and SteF and SteR, 40.0 mM), 0.3  μ L of EV71 DNA template, and 1.0  μ L of commercial Bst 2.0 DNA polymerase (8 U/ μ L; New England Biolabs, MA, USA) as positive control while the other tubes contained equal amounts of WT or mutant proteins, finally adding ddH 2 O up to 25  μ L in reaction tubes.…”

“…LAMP technique has been widely used in the virus detection for infectious diseases, such as avian influenza [3], HFMD [4], Dengue Virus [5], the human immunodeficiency virus [6], and Ebola virus [7]. Presently, a novel and simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) was developed to achieve rapid detection of EV71 in the early phase of HFMD through visual color change by addition of hydroxyl naphthol blue dye [8]. Similar to the LAMP assay, the IMSA assay is an in vivo nucleic acid amplification technique depending on Bst DNA polymerase, dNTPs, magnesium ion betaine, and three pairs of primers consisting of one pair of stem primers (SteF and SteR), one pair of outer primers (DsF and DsR), and one pair of inner primers (FIT and RIT).…”

Digital Nucleic Acid Detection Based on Microfluidic Lab-on-a-Chip Devices

“…Herein, an easy-to-use and cost-efficient smartphone-based dLAMP POCT device platform is also established by our laboratory. SPC cdIMSA is an updated version of SPC cdLAMP, in which the LAMP is replaced by IMSA and a mixed dye is used to establish a label-free and sensitive dual-fluorescence detection for on-chip IMSA [70,71]. The used SPC chip for dIMSA is the same as that for dPCR without any modifications.…”

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

“…Between all capsid proteins, VP1 is the most external capsid proteins and is the main part in structural of the canyon on the surfaces of Picornavirus, and potentially implicated for pathogenicity of these viruses (De Jesus, 2007;Wu et al, 2013). Moreover, genetic diversity at VP1 is closely associated with the difference in viral serotypes (Wu et al, 2013) and often used as a target for molecular detection of Picornavirus viral family (Yaqing et al, 2012;Nie et al, 2012;Wang et al, 2014;Chen et al, 2014;Ding et al, 2014).…”

“…Also, reverse transcription LAMP (RT-LAMP) assay which is carried out in a single tube is a simple, high specific, rapid, and cost-effectiveness method . Less time-consuming of RT-LAMP than conventional PCR-based methods has been used successfully for rapid detection of pathogenic RNA viruses such as Enteroviruses (Arita et al, 2009;Jaianand et al, 2010Jaianand et al, , 2011Shi et al, 2011;Nie et al, 2012;Yaqing et al, 2012;Zhang et al, 2012;Zhao et al, 2013;Chen et al, 2014;Ding et al, 2014;Jiang et al, 2014;Zhao et al, 2014;Wang et al, 2014) and less prone to inhibit from impurity of template sample (Caipang et al, 2004;Kaneko et al, 2007;Tomita et al, 2008;Mori and Notomi, 2009;Ghosh et al, 2015). The RT-LAMP assay is very specific in compared to other molecular detection methods, because in this method four primers are used to recognize six specific regions of the target gene for amplification .…”