| At present,China is in the critical stage of eliminating schistosomiasis,and the overall situation of Schistosoma japonicum is at a very low level.However,imported cases have been reported from time to time,so there is an urgent need for more rapid and sensitive detection methods for case diagnosis or risk identification.Objective:By combining Lateral flow dipstick(LFD)and Recombinase polymerase amplification(RPA),to establish a visual detection technology that can simply and quickly identify nucleic acids of Schistosoma japonicum and Schistosoma mansoni,to preliminarily evaluate its value in early detection of S.japonicum intermediate host or final host infection,and to analyze the cost through reagents and time conversion.Methods:Specific primers were designed by Primer Premier 5 software assisted by manual methods,specially targeting for SjR2 and SjG55A genes.Primer sequences were determined by simple detection limit and specificity,and a preliminary method for RPA detection was established.Based on the RPA detection,the probe was designed to determine the best reaction conditions,and the LFD-RPA detection method was preliminarily established.PCR method was used as parallel control to evaluate the minimum detection limit,sensitivity,specificity and results of different termination methods of the established detection method.Positive snails and negative snails were prepared in different proportions to evaluate the lowest detection mixing degree of LFD-RPA method.The snail samples of different stages after the infection of metacercariae were taken to determine the infection status of Oncomelania hupensis by anatomic microscopy,and the DNA of the snail samples was extracted to evaluate the ability of LFD-RPA method to detect the nucleic acid of Schistosomiasis in different stages of infection of Oncomelania hupensis.The parallel detection of RPA and PCR was conducted.LFD-RPA method was used to detect the samples collected in the field.RPA and PCR were used as parallel controls to evaluate the detection effect of LFD-RPA method.With the target gene of S.mansoni COX1,Primer Premier 5 software was used to design and screen primers with manual assistance,and the final Primer sequence was determined to establish a preliminary RPA detection method.Based on the RPA detection,the probe was designed to determine the best reaction conditions,and the LFD-RPA detection method was preliminarily established.Based on the primers designed for the Sm1-7 gene of S.mansoni in the literature,a specific probe was designed to explore the optimal reaction temperature and time,and a LFD-RPA detection method was established.PCR was used as parallel control to evaluate the minimum detection limit and specificity of the established method.The mouse models of 40 and 80 S.mansoni cercariae were established(referred to as "40 group" and "80 group").The feces and serum of mice were collected at different times after infection,and DNA was extracted.PCR,RPA and LFD-RPA were used for parallel detection.To comprehensively evaluate the efficacy of LFD-RPA for detection of S.mansoni early infection.Subsequently,the basic data of the biological techniques for nucleic acid detection of schistosoma commonly used in our laboratory were collected,including the specific name of the required kit or main reagents,the price of each box,the number of reactions per box and the source.Calculate the input cost of each sample for different detection methods,including reagent cost and labor cost required for each sample test.Based on the reagent cost and labor cost of each sample,the reagent cost and labor cost of each test were calculated under the condition that a single sample of ginseng and ginseng was set.At the same time,96 reactions were used as the total quantity of different detection methods.Under the condition of batch testing,reagent cost and labor cost are input for each sample testing.According to the number of instruments required for the experiment,the technical requirements of each method were divided into four levels:simple,general,complex and extremely complex.The cost and technical requirements of LFD-RPA and other nucleic acid detection methods were compared and analyzed.Results:The LFD-RPA detection methods targeting SjR2 and SjG55A of Schistosoma japonicum and the LFD-RPA detection methods targeting SmCOX1 and Sm1-7 of Schistosoma mansoni were successfully established.The optimal reaction parameters of each method were 39℃ and 20min.The LFD-RPA reaction was terminated by standing on ice,adding DNA extract or phenolic chloroform.The lowest detection limits of LFD-RPA method based on SjR2 and SjG55A for plasmids and adult genomes were 1 copies/μL,102 copies/μL,1 fg/μL and 10fg/μL,respectively.SjR2-LFD-RPA was better,it is better than corresponding RPA and PCR methods.The SjG55A-LFD-RPA method had no cross-reaction with the genomic DNA of S.mansoni,infected Biomphalaia spp.,S.haematobium,Clonorchis sinensis,Fasciola gigantica and Paragonimus westermani.The SjR2-LFD-RPA method did not cross-react with the genomic DNA of S.haematobium,Clonorchis sinensis,Fasciola gigantica and Paragonimus westermani,but it could detect S.mansoni and infected Biomphalaia spp.,suggesting a cross-reaction with S.mansoni.SjR2-LFD-RPA and SjG55A-LFD-RPA could detect 1 positive snail out of 2000 and 1500 negative snails,respectively,and the mixed degree of minimum detection was higher than that of corresponding RPA and PCR.SjR2-LFD-RPA,SjR2-RPA and PCR could detect 20 groups of 1:50 mixed positive Oncomelania hupensis,and the sensitivity of all three methods was 100%.The detection of S.japonicum infected snails in laboratory showed that all the snails 6 weeks before S.japonicum infection were negative by microscopic examination,and positive snails appeared since 7 weeks.The positive rates of snails 7 to 11 weeks were 10%,30%,20%,40%,30%,respectively.The positive rates of SjR2-RPA were 90%、70%、50%、70%、60%、70%、60%、50%、20%、30%、70%、50%、50%and 50%at 1D,3D,5D and 1-11W after snail infection,respectively.The positive rates of SjR2-LFD-RPA were 90%、80%、70%、70%、60%、60%、70%,70%、30%、30%、70%、50%、50%、50%,respectively at 1D,3D,5D,and 1-11W after snail infection.Statistical analysis showed that the total detection rate of SjR2-LFD-RPA,PCR and RPA was higher than that of anatomical microscopy.A total of 1550 Oncomelania snails were collected from 31 tubes of mixed DNA,and the detection rate of SjR2-LFD-RPA was 12.90%,while other methods were negative.The established SmCOX1-LFD-RPA detection limits of plasmid and adult genomic DNA of Schistosoma mansoni were 10copies/μL and 1fg/μL,which were higher than those of the corresponding RPA and PCR.The established Sm1-7-LFD-RPA had a higher detection limit of 1copies/μl and 1fg/μl for plasmid and adult genomic DNA.SmCOXl-LFD-RPA and Sm1-7-LFD-RPA were specific to S.mansoni and infected Biomphalaia spp.,and had no cross-reaction with S.japonicum,positive Oncomelania hupensis,S.haematobium,Clonorchis sinensis,Fasciola gigantica and Paragonimus westermani.It was found that SmCOX1-RPA and PCR had no positive results on the mixed blood and faecal samples 1 to 8W after infection,no matter in the 40 group or 80 group.When SmCOX1-LFD-RPA and Sm1-7-LFD-RPA detected mixed blood samples and mixed feces samples of the 40 tail groups,shallow bands began to appear 3W after infection,and the bands became more and more obvious with the increase of infection time.The color of the bands reached the deepest at the 8 W and 6-8W after infection,respectively.SmCOX1-LFD-RPA and Sm1-7-LFD-RPA appeared lighter bands from 1W when mixed blood samples and mixed faecal samples were detected in the 80 tail group,and the bands gradually darkened during 1W to 8W,and the bands reached the deepest at 6W to 8W after infection.In general,the stripe color of Sm1-7-LFD-RPA is clearer than that of SmCOX1-LFD-RPA.In the cost comparison and analysis of different detection methods,it was found after calculating the total cost of each test,PCR(453 RMB/time)>LFD-RPA(400 RMB/time)>freeze-dried powder RPA(330 RMB/time)>LAMP(300 RMB/time)and liquid RPA(300 RMB/time).The sequence of each test time was as follows:PCR(2-2.5 h/test)>RPA(0.8～1 h/test)>LAMP(0.5～1 h/test)>LFD-RPA(0.4～0.6 h/test).With 96 reactions as a batch test,the detection time of each batch of different detection methods was calculated as follows:PCR(2-2.5 h/batch test)>RPA(3.2-4 h/batch test)>LAMP(2-4 h/batch test)>LFD-RPA(1.6-2.4 h/batch test).In the case of batch testing,the total cost of each sample testing is as follows:LFD-RPA(112.77 RMB/batch per sample)>freeze-dried powder RPA(61.92 RMB/batch per sample)>LAMP(60.64 RMB/batch per sample)>liquid RPA(51.06 RMB/batch per sample)>PCR(5.81 RMB/batch per sample).PCR and RPA were classified as "complex",while LAMP and LFD-RPA were classified as "simple" according to the technical requirements of experimental operators for the need of special instruments and equipment.Conclusion:In this study,the LFD-RPA technology for nucleic acid detection of Schistosoma japonicum and Schistosoma mansoni was established,which has a low detection limit,simple operation and direct method for obtaining results.SjG55A-LFD-RPA can be used for the detection of mixed Oncomelania japonicum infection,and SjR2-LFD-RPA has a cross reaction to S.mansoni.The detection method of LFD-RPA based on SmCOX1 and Sm1-7 target genes has a low detection limit and no cross reaction with other species,which is expected to be used for the identification of early infected host of S.mansoni.As a relatively new nucleic acid detection technology,LFD-RPA has a high detection cost at present.However,due to its simple operation,high sensitivity and strong specificity,LFD-RPA has a broad application prospect in the field detection and risk monitoring of schistosomiasis in the future. |
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