| Objective:To quantitatively evaluate the diagnostic efficacy of various nucleic acid detection techniques of Schistosoma japonicum,and to establish a sensitive,specific and rapid method of nucleic acid detection of Schistosoma japonicum by using the recombinase polymerase amplification(RPA)technique according to the result of comprehensive evaluation,and to preliminarily evaluate its potential value for detecting early infection.Methods:Articles related to nucleic acid tests of schistosomiasis published during January 1990 to June 2019 were searched in China National Knowledge Infrastructure(CNKI),Wan Fang,Science Direct and PubMed database by computer,supplemented by manual retrieval and literature tracing.In accordance with the strict inclusion and exclusion criteria,eligible articles were included and relevant data were extracted to be comprehensively evaluated by Meta-analysis.Primers targeting for the gene fragment of schistosoma japonicum Sj28S were designed by manual selection and assisted by the Primer Premier 5 software.The temperature and time were optimized to determine the optimal reaction conditions of established RPA method,The specificity of RPA were evaluated through testing the extracted genomic DNA of Schistosoma japonicum and other parasites.Then,the sensitivity were tested,and compared with that of PCR and LAMP.In addition,the detection performance of RPA was assessed through detecting the genomic DNA of different stages of Schistosoma japonicum was evaluated.Genomic DNA of schistosome infected or uninfected Oncomelania snails were extracted and mixed with different ratio and then detected by RPA to evaluate the minimum mixed ratio and repeatability of RPA method.5,10,20 and 40 cercariae infected mice models were established(referred to as 5-tail group","10-tail group","20-tail group" and "40-tail group"),fecal and sera samples were stored in frozen state after numbering on the 3rd day,1st,2nd,3rd,4th,5th and 6th week respectively.The snails were attacked by miracidia and 10 snails were collected on the 1st,3rd and 5th day,1st,2nd,3rd,4th,5th,6th,7th,8th,9th,10th and 11th week respectively,and the samples were collected and stored in refrigerator after received microscopic examination.Then the DNA of Mice and snails were extracted and tested in parallel by PRA and PCR.The efficacy of RPA method in detecting early infection of Schistosoma japonicum was comprehensively evaluated.Results:A total of 19 publications covering 24 groups of studies were enrolled into analysis,including 5 Chinese publications and 14 English publications.There were 17 groups of studies reporting the comparison between the variable-temperature nucleic acid amplification techniques and the golden standard method,and 7 groups of studies showing the comparison between the isothermal nucleic acid amplification techniques and the golden standard method.Assessment of the literature quality indicated a minor overall bias of the included literatures,while the Deek funnel plot showed a possible publication bias in the variable-temperature nucleic acid amplification techniques.There was a heterogeneity caused by non-threshold effect among the studies associated with the variable-temperature amplification technique,then the random effects model was used to combine the effects.The pooled sensitivity and specificity of the variable-temperature amplification technique were 0.81(95%CI:0.79,0.83)and 0.73(95%CI:0.71,0.74)for the diagnosis of schistosomiasis japonica,and area under the SROC curve was 0.944 3 There was no heterogeneity among the studies associated with the isothermal amplification techniques,and the fixed effects model was therefore used to combine the effects.The pooled sensitivity and specificity of the isothermal amplification technique were 0.96(95%CI:0.94,0.98)and 0.95(95%CI:0.94.0.97)respectively,and area under the SROC curve was 0.989 9The RPA method can successfully amplify the 216 bp target gene fragment of Schistosoma japonicum.The optimum reaction conditions of RPA were 39? and 20min This method had no cross reaction with the genomic DNA of Schistosoma mansoni,Schistosoma haematobium,single-tale cercariae within oncomelania snails,Clonorchis sinensis,Fasciola gigantica,Paragonimus westermani,Taenia saginata and uninfected oncomelania snails.The minimum detection limit of RPA was 100 fg/?l for adult genome of Schistosoma japonicum,and 100 copies/?l for recombinant plasmids,presenting higher sensitivity than that of PCR and LAMP.Furthermore,genomic DNA samples of different stages of Schistosoma japonicum could be accurately detected by RPA.The RPA method could detect the infection when the ratio of infected and overall snails DNA was set up as 1:1000,and presenting good repeatability and high accuracy.Infected animal model tests showed that the positive results could be detected in feces of mice with 20 cercariae group in the 4th week,10 and 40 cercariae groups in the 5 th week,5 cercariaegroup only in the 6 th week.The fecal DNA of 10,20 and 40 cercariae groups could be detected by PCR in the 5 th week,and only in the 6 th week in 5 cercaria group.Positives tested by RPA were presented in the sera samples of 5 cercaria group in the 5th and 6th week,10 cercaria group in the 2nd,3rd,4th and 6th week,20 cercaria group in the 3rd,5th and 6th week,40 cercaria group in the 3rd day,1st,2nd,4th,5th and 6 th week None positive results were detected in the sera of mice in 5 and 10 cercariae groups by PCR.The sera of mice in 20 cercariae group were detected in the 4th week,while those in 40 cercariae group were detected in the 6th week.The results of dissection and microscopic examination showed that no positive snails were observed before the 6th week,and the daughter sporocyst of schistosome was observed since the 7th week.The positive rates of Oncomelania hupensis detected by microscopy at each time point were:0%,0%,0%,0%,0%,0%,0%,0%,0%,0%,10%,30%,20%,40%,30%,respectively.The positive rates by RPA method were:80%,60%,50%,60%,40%,60%,60%,50%,20%,20%,80%,50%,50%,respectively.The positive rates by PCR at each time point were 60%,70%,60%,50%.20%,0%,30%,0%,20%,0%,10%,30%,50%and 50%,respectively.There were a total of 13 snails that were positive by microscopy,and all of which were also positive by RPA test.PCR method only detected positive 9(69%)of them,failed to detect all positive snails by microscopy.Conclusion:Both variable-temperature and isothermal nucleic acid amplification techniques have a high efficiency for the diagnosis of schistosomiasis japonica.The isothermal amplification techniques showed a relatively higher accuracy than the variable-temperature amplification techniques.In this study,an RPA method was constructed to detect the nucleic acid of Schistosoma japonicum,presenting advantages with high sensitivity,specificity,simplicity and rapidity.This method is expected to be used for rapid detection and risk monitoring of schistosomiasis. |
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