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Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability.

ABSTRACT: Human enterovirus 71 (EV71) is the primary pathogen of hand, foot, and mouth disease (HFMD). EV71 infection may lead to neurologic damage, with higher incidence of fatality compared with other HFMD pathogens. An effective drug or vaccine against EV71 infection is currently unavailable. It is desirable to determine the pathogen of HFMD accurately and quickly for early treatment. In the current study, reverse-transcription and loop-mediated isothermal amplification (RT-LAMP) technology were developed to detect EV71. The efficacy of detecting EV71 was compared with regular nested reverse-transcription polymerase chain reaction (RT-PCR). After detecting 108 clinical specimens, results showed that RT-LAMP can specifically detect EV71, but not Coxsackie virus A16, and exhibited a specificity of 100% and a sensitivity of 97.1%, which was higher than regular RT-PCR. The findings indicate that RT-LAMP is a practical method for EV71 diagnostic applications, particularly in small county institutes of medical service. The detection ability of RT-LAMP was significantly affected by cryopreservation as the clinical specimens were repeatedly subject to freezing and thawing treatments.

SUBMITTER: Xia JF

PROVIDER: S-EPMC7127202 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability.

Xia Ju-Feng JF Yan Xiu-Feng XF Yu Hui H Qu Di D Long Jian-Er JE

Diagnostic microbiology and infectious disease 20110906 3

Human enterovirus 71 (EV71) is the primary pathogen of hand, foot, and mouth disease (HFMD). EV71 infection may lead to neurologic damage, with higher incidence of fatality compared with other HFMD pathogens. An effective drug or vaccine against EV71 infection is currently unavailable. It is desirable to determine the pathogen of HFMD accurately and quickly for early treatment. In the current study, reverse-transcription and loop-mediated isothermal amplification (RT-LAMP) technology were develo ...[more]

PMID: 21899976

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Project description: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.

Dataset Information

Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability.

Publications

Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability.

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