Abstract
BackgroundHand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71.MethodsSix primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60°C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis.ResultsA total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples.ConclusionOur results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.
Highlights
Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China
The optimum reaction system of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay The optimal temperature for RT-LAMP reaction was determined to be 60°C, which could provide the optimal activity of Bst DNA polymerase (Figure 2A)
Restriction enzyme analysis of RT-LAMP products The specificity of RT-LAMP was confirmed by the digestion using HinfI and TaqI restriction enzymes
Summary
Foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. We have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71. Traditional EV71 infection is primarily dependent on serodiagnosis, and virus culture and identification These methods are time-consuming and easy to produce cross-immune response with CVA16. Reverse transcription-PCR (RT-PCR) and real-time PCR assays have been used for EV71 detection [16,17,18]. These nucleic acid amplification methods with intrinsic disadvantages of requiring sophisticated instrumentations and expensive reagents may not be the best choice for basic clinical settings in developing countries or in field situations. LAMP assays have been widely used to detect a variety of infectious diseases, such as bacterial, fungus and viral infections [20,21,22]
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