Study Of Pathogen Of Hand, Foot, And Mouth Disease(HFMD) By Isothermal Amplification

A rapid expansion of Hand, Foot, and Mouth Disease(HFMD) outbreak has occurred in China since2004and large scale outbreaks of HFMD have occurred nationally every year since2008, affecting more than one million children and causing several hundred children deaths since2009. HFMD has become important public health issue for China. Human entero virus71(HEV71) and Coxsackievirus A16(CVA16) are the major causative agents of HFMD and the other human enterovirus infections are also responsible for partial HFMD cases. So it is important to development rapid and simple assays for HEV71and CVA16, and ensure detection of all HEVs causing HFMD. This study contain three parts including the development and evaluation of RT-LAMP assay for the detection of HEV71and CVA16, evaluation of a direct RT-LAMP method without RNA extraction for the detection of HEV71and the development and evaluation of RT-GEAR Assay for the detection of pan-enterovirus (pan-EV).Part1. Development and evaluation of RT-LAMP Assay for the detection of HEV71and CVA16 A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HEV71Subgenotype C4(HEV71-C4) and CVA16infection respectively. The reaction was performed in one step in a single tube at65℃for60minutes with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. A positive reaction is indicated by the turbidity of the reaction solution or the color change of the added HNB dye from violet to skt blue. The detection limit of the RT-LAMP assay was0.3350%tissue culture infective dose (TCID50) and1.58TCID50per reaction based on a serried of ten-fold dilutions of a titrated HEV71or CVA16stain respectively, and no cross-reaction was observed with Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,24), Cosackievirus B(CVB) viruses (CVB1,2,3,4,5) or ECHO viruses (ECHO3,6,11,19). The assay was further evaluated with47clinical stool specimens with HEV71or CVA16or other human enterovirus infection differentiated previously using virus isolation followed by neutralization test and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of HEV71subgenotype C4and CVA16.The improved RT-LAMP assay for HEV71and CVA16using a commercial loopamp RNA amplification kit and a fluorescent detection reagent was further evaluated. The one step reaction was performed in a single tube at65℃for45min for HEV71and35min for CVA16. The detection limits of RT-LAMP assays for both HEV71and CVA16were0.1of a50%tissue culture infective dose (TCID50) per reaction, based on10-fold dilutions of a titrated HEV71or CVA16strain. In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71and CVA16, the RT-LAMP assay was evaluated with515clinical specimens from Hunan Province during outbreak in2011, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for513/515(99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of HEV71and CVA16in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.Part2. Evaluation of a direct RT-LAMP method without RNA extraction for the detection of HEV71A 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 HEV71subgenotype C4in 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 was1.6of a50%tissue culture infective dose (TCID50) per reaction based on a series of ten-fold dilutions of a titrated HEV71strain and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16,24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4,5) or ECHO viruses (ECHO3,6,11,19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting HEV71infection with145nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be90.3%and100%respectively, compared to RT-LAMP, and86.83%and100%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 HEV71infection in China.Part3. Development and evaluation of RT-GEAR Assay for the detection of pan-enterovirus (pan-EV)Enteroviruses are ubiquitous pathogen worldwide and most individuals encounter numerous serotypes during their lifetime. There were18serotypes of enterovirus which cause epidemics of hand, foot, and mouth disease in young children base on current molecular virologic investigation of the disease. Conventional diagnostic techniques are often too slow and too insensitive to benefit the patient optimally. A total522whole genomic sequences of enterovirus were obtained from the NCBI database and were further analysed by Multiple Sequence Alignment software. Several oligomeric regions of great homology among the enterovirus5’ Untranslated Regions(UTR) were identified and designated as primer pairs for a modified reverse transcription isothermal amplification technique, the name of which is abbreviated to RT-GEAR and was applied to the detection of pan-Enterovirus in this study. The reaction was performed in one step in a single tube at63℃for60minutes with the addition of HNB dye prior to amplification in which enteroviral RNA can be amplified to be detected by naked eye. A total53serotypes of the enterovirus and6viruses associated with viral gastroenteritis were evaluated for this molecular diagnosis assay. All of the enteroviral RNA were successfully amplified and no cross-reation was observed with the6viruses which adenovirus, astrovirus, bocavirus, norovirus, rotavirus, sapovirus were included. In comparison with the conventional realtime RT-PCR assay using pan-Enterovirus PCR primers based on a series of ten-fold dilutions of viral RNA from2serotypes of the human enterovirus A,2serotypes of the human enterovirus B, and 2serotypes of the human enterovirus C, the result showed that the sensitivity of both assays were approximately equal. The RT-GEAR assay for pan-Enterovirus was further evaluated with a total of58clinical specimens obtained from pediatric patients suffering from HFMD during outbreak in2012, of which33specimens were HEV71infected and25specimens were CVA16infeted which confirmed by realtime RT-PCR, the result showed that all the samples tested positive. The molecular assay developed in this study provides a sensitive, specific, rapid, and high throughput diagnostic method for enterovirus infections, especially for the detection of hand, foot, and mouth disease, which will be useful in future outbreaks.