Development And Application Of Loop-mediated Isothermal Amplification Methods For Enterovirus 71 And Coxsackievirus A16 Detection

Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) are two major etiological agents of hand, foot and mouth disease (HFMD) in children. The spread of HFMD infections is mainly by the faecal-oral and oral-oral route. Infection is transmitted by contact with water, food and ground contaminated with infected feaces. Water is the essence of life. The lack of accessible clean and safe water has caused large numbers of the human morbidity and mortality in the world each year. There has been a significant increase in EV71 epidemic activity in the Asia-Pacific region since 1997, and has a propensity to cause severe neurological disease or death.Traditional method for routine detection of EV71 and CoxA16 is complex and time-consuming. It takes from 5 to 7 days with low sensitivity. This method can not fulfill the need for detecting EV71 and CoxA16 rapidly and sensitivity. The detection of viruses in the environmental water is a valuable work for preventing diseases and evaluating anitary quality of water and health condition of environment.Objective:Developt EV71 and CoxA16 loop-mediated isothermal amplification(LAMP) assays, apply the two assays to detect EV71 and CoxA16 in the stool samples. Developt real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect EV71 and CoxA16 in the source water samples of Tianjin.Method:1 In this study, the outer primers and the inner primers for LAMP were designed to target the VP1 regions of EV71 and CoxA16 by using software. The reaction conditions were optimized including temperature, Mg2+ concentration, betaine concentration and dNTPs concentration etc. LAMP was carried out in a total 25μL. The mixture of EV71 or CoxA16 LAMP system was heated at 62℃or 63℃for 60min and heated at 80℃for 10min to termination the reaction. Amplification products were detected by visual inspection, agarose gel electrophoresis. In addition, the field applicability of the LAMP assay was also demonstrated by standardizing SYBR Green I-based LAMP which was followed by monitoring gene amplification with the naked eye through color changes. Furthermore, LAMP products were fragmented by restriction enzyme. There were 6 enteroviruses to be detected by LAMP in order to evaluate the specificity of primers.2 A total of 60 stool samples from HFMD patients in Tianjin of China were evaluated by LAMP and compared to conventional PCR.3 In this research, we collected 18 drinking water source samples from multiple locations of Tianjin between 2010 and 2011 for analysis of EV71 and CoxA16 contamination respectively. Total viral nucleic acid was extracted from 500mL of drinking water source sample concentrated by Ultracel-100k centrifugal filter devices.Results:1 The LAMP systems developed in this study were proved to be high specificity methods. Cross-reactivity with Hepatitis A virus (HAV), Poliovirus, coxsackievirus B3 (CoxB3), coxsackievirus B5 (CoxB5), Echo30 and CoxA16/EV71 was not observed. The EV71 and CoxA16 LAMP assays were found to be 10-fold and 100-fold more sensitive than conventional PCR respectively with detection limits of both 101 copy numbers.2 The positive rate of the EV71 LAMP assay was higher (31/60=51.7%) than the PCR assay (27/60=45%), the positive rate of the CoxA16 LAMP assay was equivalent with PCR assay (7/60=11.7%). This result demonstrated that EV71 was the primary cause of HFMD during this epidemic, and CoxAl6 was low-level epidemic activity in Tianjin during 2010. These two LAMP systems have potential usefulness for rapid and sensitive diagnosis in outbreak of HFMD.3 The viral recovery rate using Ultracel-100k centrifugal filter devices was 34.83%. The results indicated that EV71 were detected at concentrations ranging from 0 to 1.9×106 copies/L and CoxA16 were detected at concentrations ranging from 0 to 5.4×105 copies/L by RT-LAMP in drinking water source, Tianjin.Conclusion:The LAMP assay of detection of EV71 and CoxA16 developed in this study is high specifical, sensitive and timesaving, which was applied in the general detection of EV71 and CoxA16 in stool samples and drinking water source samples successfully.