| Nowadays,schistosomiasis is still a serious parasitic disease of humans and animals and it is also a major public health problem in the middle and lower reaches of the Yangtze River in China.Diagnosis is the central link in the prevention and treatment of schistosomiasis,which can provide necessary information and basis for many aspects of schistosomiasis prevention and treatment,such as the determination of chemotherapy targets,the evaluation of chemotherapy effects,the planning and implementation,etc.The traditional diagnosis method of schistosomiasis was a pathogen known as the “gold standard” for schistosomiasis detection.However,the existing methods of pathogenic detection have less sensitivity and specificity,and most patients with fecal test have lower compliance.Although the immunological diagnosis has high sensitivity and specificity,it is more suitable for large-scale screening and sero-epidemiological investigation of chemotherapy subjects in infected areas.The detection of antibody was commonly used in diagnosis of schistosomiasis,however,the test results can not distinguish between current infections and past infections duing to the differences in the level and outcome of antibodies individually.The immunological diagnosis is not satisfactory in evaluation of treatment efficacyWith the development of molecular biology technology,a variety of nucleic acid detection methods have been applied to the detection of schistosomiasis infection.For example,Polymerase chain reaction(PCR)technology has high sensitivity and specificity,but relies on instruments and equipment,and has high temperature denaturation reaction time.It has high technical requirements for operators and is difficult to promote and apply in on-site prevention and control work.Recent years,nucleic acid isothermal amplification technology is gradually maturing.For example,the Loop-mediated isothermal amplification(LAMP)method has become one of the relatively extensive isothermal amplification techniques currently studied and applied due to its high specificity and sensitivity.However,the primer of the LAMP method is more complicated and difficult.It is necessary to design 4 pairs of primers according to 6 specific regions,and the amplification products are fragments of different sizes,which are easy to form aerosols and cause environmental pollution in the laboratory.Therefore,the LAMP reaction is highly susceptible to contamination and produces false positive results,which have high requirements for operators and testing environments.Recombinase Aided Amplification(RAA)is a novel in vitro amplification technique with simple primer design,and the reaction can be obtained at37-42 ℃ for 5-20 min.The amplified fragment can be detected by agarose gel electrophoresis,which is equivalent to the PCR reaction.The RAA reaction is simple and fast,does not require high temperature cycling,and the demand for the instrument is greatly reduced.In order to determine the sensitivity,specificity and operability of schistosomiasis diagnosis,this study established a rapid detection method for Schistosoma japonicum specific gene fragment RAA technique based on the principle of recombinase-mediated isothermal amplification technology,and carried out the following three parts:Part Ⅰ Establishment of the basic reaction system of Recombinase Aided isothermal Amplification(RAA)of Schistosoma japonicum specific gene fragment nucleic acidThe SjG28 gene fragment of S.japonicum was selected as the target sequence,and the synthetic primers were designed according to the RAA principle.The RAA reaction system was optimized from three aspects: reaction temperature,primer concentration and magnesium acetate concentration.This method was used to simultaneously amplify different copies of SjG28 gene fragment TA cloning plasmid and different concentrations of S.japonicum adult genomic DNA,and evaluated thesensitivity of the method.The method was applied to detect Schistosoma mansoni,Ascaris lumbricoides,Ancylostoma duodenale,Echinococcus granulosus,Clonorchis sinensis,and healthy human genomic DNA for their specificity.The established basic RAA method can specifically amplify the genomic DNA of adult worms and eggs of S.japonicum,and the reaction conditions were 37 ℃,30 min,and gel electrophoresis was completed for 1 h.The optimization results of RAA reaction system showed that the optimal amplification temperature of basic RAA reaction was 37℃,optimal primer concentration was 0.4μmol/L,and the optimum magnesium acetate concentration was 14mmol/L.Using the recombinant plasmid as a template,the lowest detectable plasmid copy number in the 50 μL reaction system was 1000 copies,and the sensitivity was comparable to the PCR method.The lowest detectable concentration of RAA was1 ng/μL using the genomic DNA of S.japonicum adults as a template.The RAA method can specifically amplify the adult genome of Schistosoma japonicum,and didn’t cross-react with the whole human genomic DNA and other parasite species.PartⅡ Establishment of the fluorescent reaction system of Recombinase Aided isothermal Amplification(fluorescent RAA)of Schistosoma japonicum specific gene fragment nucleic acidA fluorescent RAA method was established by combining fluorescent probe and Recombinase Aided isothermal amplification of nucleic acids.Fluorescent RAA method was used to amplify the gradient-diluted SjG28 gene fragment TA cloning plasmid with different copy to evaluate its sensitivity.The fluorescence RAA method was also specifically evaluated with different parasite species.Finally,fluorescent RAA method was used to detect of genomic DNA extracted from experimental rabbit feces,serum and infected snails.The reaction condition of the fluorescent RAA method was 39℃ for 30 min,and the nucleic acid amplification process was performed simultaneously with the fluorescence detection,shortening the time.Fluorescent RAA amplification with different copies of SjG28 recombinant plasmid as template can observe obviousfluorescence signal within 5min,and 10 copies of recombinant plasmid can be detected in 50μL reaction system.Sensitivity is 100 times higher than basic RAA and PCR.When the genomic DNA of S.japonicum adults as template,and the lowest detectable concentration of RAA was 1 fg/μL.The fluorescent RAA method can specifically amplify the adult genome of S.japonicum,and didn’t cross-react with other parasite species.Fluorescent RAA detection of S.japonicum adult,eggs,infected rabbit feces,serum,and infected snail extracted genomic DNA as templates can be specifically amplified.Part Ⅲ Rapid detection of infected snails of Schistosoma japonicum with fluorescent RecombinaseAided isothermal Amplification assayThe snail samples were divided into 3 groups,each group consisting of 7subgroups,6 of which were mixed with 50 negative snails,1,2,3,4,5,10 infected snails,1 subgroup was 1infected snail mixed into the 100 negative snail.The three groups of snails were extracted by crushing and husking kit nucleic acid extraction,crushing and husking nucleic acid rough extraction,directly crushed and directly crushing without husking,nucleic acid rough extraction,then detected by fluorescent RAA and PCR respectively.The fluorescent RAA method was applied to the detection of a group of infectious snails,which can be completed within 30 minutes at 39 ℃.The snail samples with crushing and husking kit nucleic acid extraction,can detect a minimum of 100 negative snails and mix 1infected snail,the PCR method can detect a minimum of 50 negative snails mixed with 1 infected snail;the snail samples with crushing and husking nucleic acid rough extraction can detect a minimum of 100 negative snails mixed with 1 infected snail,PCR method can detect 50 negative snails mixed 3infected snails;the snail samples with directly crushing without husking,nucleic acid rough extraction detected by fluorescent RAA,can detect a minimum of 50 negative snails mixed with 3 infected snail,only 50 negative snails mixed 10 infected snails by PCR was positive.At the same time,the detection time of the established fluorescentRAA method was shorter than that of the normal PCR method by 4-5 hours to 30 minutes.It is the first to apply RAA isothermal amplification technology to the detection of S.japonicum-specific nucleic acid fragments.The basic reaction system of S.japonicum-specific gene fragment isothermal amplification(RAA)was established and the system was optimized.The basic RAA method was comparable to the classical PCR method with good specificity and no cross-reaction with other trematode.Secondly,combined with fluorescent probe and Recombinase Aided isothermal amplification of nucleic acid to establish a fluorescent RAA detection method for S.japonicum-specific gene fragments.The sensitivity of detection was improved 100 times compared with the basic method while maintaining good specificity.The detection method of amplification results was improved,and the observation method of agarose gel electrophoresis under ultraviolet light was replaced by fluorescent signal,which made the detection method more rapid and simple.The fluorescent RAA method was successfully applied to the detection of different samples of schistosomiasis.Furthermore,the intermediate host-nail snail of S.japonicum was detected by fluorescence RAA method,and the treatment of snail samples was optimized.Preliminary studies have shown that the established fluorescent RAA method can be used for the detection of different samples of schistosomiasis,and it is expected to provide efficient and rapid technical support for the on-site prevention and control of schistosomiasis. |
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