The Study Of Recombinase Aided Isothermal Amplification (RAA) Nucleic Acid Dipstick Assay For Detection Of Schistosoma Japonicum Gene Fragment

Schistosomiasis is a kind of zoonotic parasitic disease which seriously harms human health and affects social and economic development.Snails are the only intermediate host of Schistosoma japonicum.Snails investigation and control are of great significance in the prevention and control of schistosomiasis.At present,schistosomiasis is in a low epidemic state in China.The traditional methods of snail detection,such as crushing microscopy and release cercariae,are easy to cause missing detection.In recent years,the development of molecular biology provides more sensitive and simple technical support for snails detection.Meanwhile,the detection of gene fragments is expected to solve the problem that it is difficult to find the snails infected with Schistosoma japonicum in the development stage before the body contains sporocysts by traditional methods.Our team has carried out the research of Recombinase Aided isothermal Amplification(RAA)in the early stage,and initially established a fluorescent RAA method for rapid detection of snails infected with Schistosoma japonicum.However,this method requires specific fluorescence detection equipment,which limits its application in the field.Therefore,it is necessary to establish a rapid,convenient and visual detection method that does not rely on specific equipments to read amplification results.So it can provide technical support for monitoring risk factors of schistosomiasis transmission and accurate control of schistosomiasis.In this study,a rapid nucleic acid dipstick assay(RAA nucleic acid dipstick assay)for detection of Schistosoma japonicum gene fragments was established based on the principles of RAA method and nucleic acid dipstick assay,which mainly carries out the following three parts:Part 1 Establishment of Recombinase Aided isothermal Amplification and nucleic acid dipstick assayCombined with the previous research and literature search of the research group,SjG28,SjR2 and SjG40 gene fragments were selected as the target sequences to be amplified.Firstly,primers were designed according to the principles of RAA method and nucleic acid dipstick assay,in which the upstream primer 5’end was labeled with fluorescein,and the downstream primer 5’ end was labeled with biotin;nucleic acid test strip was designed according to the principle of lateral flow test strip,and the project was commissioned Jiangsu Qitian Gene Biotechnology Company prepared and used a commercially product as a control.Through two rounds of screening experiments,the false-positive primer pairs were excluded,and the best primer pairs were gradually selected and the amplification system 1 for RAA nucleic acid dipstick assay was established.SjG28 gene fragments were selected as the target sequences to be amplified.Primers and probes were designed,in which the downstream primer5’end was labeled with biotin,and the probe 5’ end was labeled with fluorescein.The amplification system 2 for RAA nucleic acid dipstick assay was established.According to the stability of the experimental results,the amplification system for RAA nucleic acid dipstick assay was determined.The genomic DNA of Schistosoma japonicum adult worms and eggs was used as the template and amplified by RAA at39 ℃ for 15 minutes,nucleic acid dipstick assay was used to detect the amplification products and observe whether the detection line of nucleic acid dipstick was colored.Nine pairs of primers were designed according to SjG28,SjR2,and SjG40 gene fragments.Four pairs of primers were selected through the first round of experiment,and one pair of primers was further selected through the second round of experiments.According to the stability of repeated experiments,the amplification system 2 with fluorescein labeled probe was selected to establish the RAA nucleic acid dipstick assay.RAA nucleic acid dipstick assay were used to detect the genomic DNA of adults worms and eggs of Schistosoma japonicum.The quality control line and test line showed clear bands,while only the quality control line appeared in the negative control.RAA nucleic acid dipstick assay that can be used to detect the specific gene fragment SjG28 of Schistosoma japonicum in this part.It is simple to operate and takes a short time.It does not require special equipment and visualizes the detection results.It is expected to become an new technology for on-site detection.Part 2 Evaluation of the detection efficiency of Recombinase Aided isothermal Amplification and nucleic acid dipstick assayThe nucleic acid dipstick assay method was applied to detect the specific gene fragment in the gradient dilution of SjG28 recombiant plasmid,and then nucleic acid dipstick assay method and fluorescent RAA method were applied to detect Schistosoma japonicum adult worm genomic DNA at different concentrations to evaluate the sensitivity of the method.The nucleic acid dipstick assay method and fluorescent RAA method were applied to detect genomic DNA of Clonorchis sinensis,S.mansoni,Ancylostoma duodenale,S.haematobium,Babesia,Paragonimus westermani to evaluate the specificity of the method.The temperature of RAA amplification was set at 15 ℃,20 ℃ and 25 ℃.The genomic DNA of Schistosoma japonicum adult worms was detected by nucleic acid dipstick assay.After RAA amplification within 30 minutes,observe whether the test line was clearly colored.And then observe the nucleic acid dipstick assay to detect the lowest temperature of the target sequence after reduce the RAA amplification temperature difference.In the reaction system of 50 μL,the nucleic acid dipstick assay method could detect 10 copies of recombinant plasmids.The nucleic acid dipstick assay method could detect at least 1 pg/μL of genomic DNA using Schistosoma japonicum adult worm genomic DNA as template.The genomic DNA of Schistosoma japonicum,Clonorchis sinensis,S.mansoni,Ancylostoma duodenale,S.haematobium,Babesia,Paragonimus westermani were used as templates.The results of nucleic acid dipstick assay method showed that the quality control line and detection line appeared in the Schistosoma japonicum genomic DNA.and only the quality control line appeared in the genomic DNA of other species.Only the genomic DNA of Schistosoma japonicum was amplified by fluorescence RAA method.After the genomic DNA of Schistosoma japonicum adult worms was amplified by RAA at 25 ℃ for 25 minutes.,the results of nucleic acid test strip showed that both the control line and the test line were colored.In this part,we evaluated the nucleic acid dipstick assay for specific gene fragment of Schistosoma japonicum.This method has good sensitivity and specificity.In the field detection of schistosomiasis,we only need to provide a constant temperature equipment for amplification.For amplification,the corresponding results can be detected by nucleic acid test strips,with fast detection speed,simple operation,and suitable for on-site use.Part 3 Recombinase Aided isothermal Amplification and nucleic acid dipstick assay for rapid detection of infected snailsThe group samples of infected snails are divided into 4 groups with 50 snails in each group,of which contain different numbers infected snails respectively.Nucleic acid was extracted from infected snails by crushing and removing the shell method.Nucleic acid dipstick assay and fluorescent RAA method were used to detect the nucleic acid,and compared detection results.Snails samples at different infection times and group samples of infected snails were collected respectively.20 snails were randomly collected at different times after miracidium infection.And preserved with anhydrous ethanol.Nucleic acid dipstick assay and fluorescent RAA method were used to detect the nucleic acid,and compared detection results.The results of nucleic acid dipstick assay method showed that only the quality control line appeared in the samples without infected snails,and the quality control line and detection line appeared in the samples with 1,2 and 3 infected snails respectively.The results of fluorescence RAA showed that the amplification results of the samples without infected snails were negative,and the samples including 1,2 and3 infected snails respectively were positive.The results of RAA nucleic acid dipstick assay showed that only the quality control line appeared in the snails with 1 and 5days after infection,and the quality control line and detection line appeared in the snails with other infection days.The snails with 1 and 5 days after infection were negative by fluorescent RAA method,and others were positive.The results of the two methods were consistent.In this part,nucleic acid dipstick assay was applied to detect snails infected with Schistosoma japonicum.The detection speed is fast and does not rely on equipments to reads the results.The detection of snails population samples can detect at least one in fifty infected snails.In the early detection of infected snails,the results were positive3 days after the infection.In summary,this study is the first to combine RAA amplification method and nucleic acid dipstick assay to establish a detection method for Schistosoma japonicum specific gene fragments,and evaluate the sensitivity and specificity of the method,The RAA nucleic acid dipstick assay was applied to detect infected snails.The preliminary study showed that the RAA nucleic acid dipstick assay can be used to detect the group samples of infected snails and the early detection of infected snails,which is expected to provide effective technical support for the field control of schistosomiasis.