The Establishment Of Recombinase-mediated Nucleic Acid Amplification Technology To Detect WNV And The Experimental Evaluation Of CV-A6 Inactivation

Background:People generally did not catch disease who was infected by West Nile Virus(WNV).A small proportion people may develop diseases such as West Nile Fever(WNF)or West Nile Encephalitis or even death.The widespread prevalence of WNV has posed severe challenges to public health and safety issues worldwide.Although the prevalence and spread of WNV has not yet occurred in China,WNV has been detected in mosquito specimens in Xinjiang and other places.The rapid detection of the virus is of great significance for the early clinical diagnosis and prevention of the spread of the virusRecombinase aided amplification(RAA)technology is a new type of recombinase-based isothermal nucleic acid amplification technology.RAA can achieve a large number of target fragments within 30 minutes at a temperature of 37-42 0 C when combined with fluorescence technology,electrophoresis technology and lateral flow dipstick(LFD)technology.RAA technology is well suitable for use in the field and in areas with remote districts and large-scale rapid screeningCoxsackie virus group A type 6(CV-A6)is one of the important etiology of hand,foot and mouth disease in China in recent years.The CV-A6 mainly infects children and is one of the main pathogens that threaten children's health.Therefore,CV-A6 has also became a research hotspot problem in recent years.However,due to the continuous occurrence of laboratory safety incidents,people pay more and more attention to laboratory safety.Therefore,it is very important to conduct virus inactivation and routine disinfection in the research process of CV-A6,Objective:To establish two RT-RAA detection methods for West Nile virus.One is a real-time fluorescent RT-RAA detection method in combination with fluorescence technology,and another is a visual detection method in combination with lateral flow test strips.and evaluated the sensitivity and specificity of the two methods,and used mosquito specimens for evaluation and real-time fluorescence RT in parallel for comparison.To evaluate the thermal inactivation of Coxsackievirus group A type 6(CV-A6)and compare the thermal inactivation effects with two commonly used chlorine-containing high-level disinfectants in the laboratory,and to provides basic data on disinfection after enterovirus experiments in the bio-safety level 2 laboratory.Methods:1.Real-time fluorescent RT-RAA detection method for West Nile virus:(1)obtained the gene sequences of different types of West Nile virus(mainly type 1 and type 2)from NCBI,and aligned gene sequences,designed primers and probes in conservative and specific sequences;(2)According to the principle of RAA primer probe design and used corresponding software(Amplif X 1.5.4 and Oligo 7)to design three set of primers pair and one probe;(3)Used the recombinant plasmid standard to conduct primer probe screening,selected the best primer and probe combination,and then performed sensitivity and specificity evaluation.(4)Added a section of the designed rosette virus as a internal reference probe,and constructed its recombinant plasmid standard as an internal reference(IAC-plasmids),and then used the internal reference plasmids and the target plasmids to evaluate the sensitivity and specificity of the dual RAA methods.(5)The established RT-RAA method for detecting WNV and the real-time fluorescent RT-qPCR method simultaneously detected 32 mosquito specimens,and a preliminary evaluation of the method was made.RT-RAA-LFD detection method for West Nile virus:(1)According to the principle of RAA-LFD primer and probe design,modified it on the basis of fluorescent RT-RAA optimal primer and probe to establish RT-RAA-LFD method to detect WNV.(2)The standard recombinant plasmid of the target fragment of WNV was used for sensitivity evaluation,and other virus were used for specificity evaluation.(3)Compared the detection of 32 mosquito specimens with the RT-qPCR method and evaluated the method.2.Treated a certain titer of CV-A6 virus at different temperatures,different disinfectant concentrations and different inactivation time,inoculated the inactivated virus in 96-well cell culture plates for 7 days,observed and recorded the cells daily CPE,and finally calculated the virus titer after inactivationResults:1.In this study,The real-time fluorescence RT-RAA and RT-RAA-LFD isothermal nucleic acid amplification method for detecting West Nile virus were established.This method can detect West Nile virus within 30 minutes at a temperature of 39?,The sensitivity of the dual-channel real-time fluorescent RT-RAA was 10 copies per reaction.The sensitivity of the RT-RAA-LFD was 1000 copies per reaction,there is no cross reaction with other common flaviviruses and arboviruses that proved there two methods had a good specificity.These methods were used to detect 32 mosquito specimens,and tested 5 positive and 27 negative result,which was consistent with RT-qPCR test results2.Inactivation at 56? for 20 minutes could be effective(the difference between the virus titers before and after inactivation is?4 that is considered effective)inactivation of CV-A6.The virus could be completely inactivated at 65? for 30 minutes and 72 ? for 10 minutes;The commercially available Lierkang trichloroisocyanuric acid effervescent disinfection tablet and 84 disinfectant could completely inactivate the CV-A6 in 1 minute at the recommended effective chlorine concentration of the productConclusion:1.In this study,a real-time fluorescent RT-RAA and a visual RT-RAA-LFD method for detecting West Nile virus were established.This method has the advantages of high sensitivity,good specificity,rapidity,easy to operate and low cost.It overcomes the disadvantages of traditional PCR that requires expensive and large-scale instruments to achieve accurate temperature control,this method has the potential to be applied in the field and in remote regions,and provides technical reserve for the possible importion and prevalence of West Nile virus in China.,helps to detect WNV infection early and quickly.2.The inactivation of CV-A6 with chlorine-containing chemical disinfectants and heat inactivation were related to time,effective chlorine concentration and temperature.The two commonly used disinfectants in the laboratory were heat disinfected at 65? for 30 minutes at the recommended effective concentration and The disinfection effect at 72? for 10 minutes could completely inactivate the virus,and could be used as a routine inactivation condition for the virus in the intestinal laboratory.