Development and Evaluation of a Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Enterovirus 71

Jiang, Tao; Liu, Juan; Deng, Yong-Qiang; Xu, Lijuan; Li, Xiaofeng; Han, Jianfeng; Cao, Ruiyuan; Qin, E-De; Qin, Cheng‐Feng

doi:10.1128/jcm.02045-10

Cited by 29 publications

(19 citation statements)

References 36 publications

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“…Rapidity and simplicity of the methods for the EV71 detection are critical for community hospital laboratory or field use, RT-LAMP assays with RNA extraction for the rapid diagnosis of EV71 infection have been reported previously [11], [12], [13], [14]. As the RNA extraction step requires approximately 30 min, omission of RNA extraction could save both time and labor for preparing the samples for RT-LAMP, a major advantage for rapid diagnosis in hospital laboratories or field use.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Nie

Zhang

et al. 2012

PLoS ONE

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Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID50) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

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Section: Discussionmentioning

confidence: 99%

“…This technique is being used increasingly for rapid detection and typing of emerging viruses [5], [6], [7], [8], [9], [10]. The detection of EV71 by RT-LAMP with RNA extraction was developed recently in our laboratory [11] and other groups [12], [13], [14].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Nie

Zhang

et al. 2012

PLoS ONE

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“…Apart from traditional gel electrophoresis, the RT-LAMP products can be detected through spectrophotometric equipment to measure turbidity (15) or through direct a visual inspection of turbidity (16) and color changes (17)(18)(19). To date, several RT-LAMP-based detection methods for EV71 and CVA16 have been established in our laboratory and with other groups (18,(20)(21)(22). Although the RT-LAMP assay can achieve a high detection limit with the artificial plasmid templates, its detection limit is not adequate for assessing clinical specimens with a low viral load in the early phase of HFMD, leading to a certain false-negative diagnosis rate (23).…”

mentioning

confidence: 99%

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

et al. 2014

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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...

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“…HFMD epidemics cause serious public health threats among infants throughout the world (Lee et al 2009 ;McMinn 2002 ). Jiang et al ( 2011 ) developed an RT-LAMP assay targeting the VP3 gene of EV71 and further evaluated with 40 clinical stool samples collected during the epidemics of HFMD in Guangxi province of China in 2009. This assay was found to be more sensitive and accurate, so it may become a useful alternative in resource-limited hospital laboratories and rural clinics for diagnosis of EV71.…”

Section: Human Virusesmentioning

confidence: 99%

Rapid Detection of Viruses Using Loop-Mediated Isothermal Amplification (LAMP): A Review

Saharan

Khatri

Dingolia

et al. 2013

Biotechnology: Prospects and Applications

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Most of the diseases caused by viral infection are found to be fatal, and the diagnosis is diffi cult due to confusion with other causative agents. So, a highly effi cient molecular-based advance detection technique, i.e., loopmediated isothermal amplifi cation (LAMP) method, is developed for diagnosis of viral infections by various workers. It is based on amplifi cation of DNA at very low level under isothermal conditions, using a set of four specifi cally designed primers and a DNA polymerase with strand displacement activity. This technique is found to be superior than most of the molecular techniques like PCR, RT-PCR, and real-time PCR due to its high specifi city, sensitivity, and rapidity. Major advantage of LAMP method is its cost-effectiveness as it can be done simply by using water bath or dry bath. Here, in this review information regarding almost all the effective LAMP techniques which is developed so far for diagnosis of numerous viral pathogens is presented.

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Development and Evaluation of a Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Enterovirus 71

Cited by 29 publications

References 36 publications

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

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

Rapid Detection of Viruses Using Loop-Mediated Isothermal Amplification (LAMP): A Review

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