%0 Journal Article
%T Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71
%A Weifeng Shi
%A Kun Li
%A Yun Ji
%A Qingbo Jiang
%A Mei Shi
%A Zuhuang Mi
%J BMC Infectious Diseases
%D 2011
%I BioMed Central
%R 10.1186/1471-2334-11-197
%X Six 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 (60ˇăC) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis.A 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.Our 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.HFMD, a common illness in children, can be caused by many human enteroviruses such as coxsackie viruses A4, A5, A6, A10, A16, B1, B3, and EV71 [1-4]. Among these viruses, human EV71 and CVA16 are major causative agents of HFMD. EV71 and CVA16 infections in HFMD are indistinguishable. However, EV71 infection is frequently associated with serious neurological complications and fatalities [5]. EV71 was initially isolated from the stool of a 9-month-old infant with fatal encephalitis in California in 1969 [6]. Subsequently, the prevalence of EV71 infection has been reported in many countries and regions, such as Taiwan, Hongkong, Malaysia and Singapore, as well a
%U http://www.biomedcentral.com/1471-2334/11/197