| Schistosoma japonicum is a parasitic disease that seriously harms human health.Our country used to be a seriously endemic area of S.japonicum.With the years of prevention and control,the prevalence of S.japonicum have successfully been reduced at a low intensity.Diagnosis is an important element in the work of schistosomiasis control.The current field control work is in urgent need of detection methods with high sensitivity,good specificity,high compliance of the public and easy operation for detection.With the development of molecular biology technology,a variety of nucleic acid detection methods have appeared in the diagnosis of S.japonicum infection,showing good sensitivity and specificity.In current studies,feces and serum have been used as the detection samples,and we intend to establish a molecular detection method for S.japonicum with DNA fragments in urine,which is a non-invasive biological sample.Cell-free DNA(cf DNA)is a kind of fragmented extracellular DNA which has been used as a biomarker in tumor,pregnancy,organ transplantation,autoimmune diseases and many other fields.Cell-free DNA mainly exists in body fluids,such as blood,urine,synovial fluid,cerebrospinal fluid,etc.,which is easily degraded by nuclease,and most of the degraded products can be cleared by liver and kidney.Previous studies have shown that DNA fragments in urine are mostly fragmented with very low concentration,and it is necessary to develop and optimize detection and extraction method based on DNA fragments in urine samples to improve the accuracy and reliability of their clinical applications.Therefore,we carried out the following four parts in order to improve the sensitivity of urine cf DNA detection:Establishment of qPCR method for detection of specific gene fragments of Schistosoma japonicumObjective:Four genes(Sj G28,Sj R2,COI and nad1)of Schistosoma japonicum were screened to determine the most suitable target gene,so as to establish a fluorescence quantitative PCR method for the detection of small molecular DNA fragments of Schistosoma japonicum.Methods:Four gene fragments Sj G28,Sj R2,COI and nad1 were selected as target sequences to be amplified.The above four genes and their corresponding primers were screened through PCR amplification and agarose gel electrophoresis to determine the optimal target genes.Based on the principle of Taq Man probe,81 bp primers and probes were designed to establish the qPCR detection method of exogenous short DNA fragments of S.japonicum to evaluate the sensitivity of qPCR.The genomic DNA of S.japonicum,S.mansoni,S.haematobium,Babesia microti,A.duodenale,C.sinensis,and P.westermani were used as templates to evaluate the specificity of the method.Results:The results showed that the sensitivity of Sj G28 was 0.05ng/μL,which was higher than that of Sj G28(0.1 ng/μL)and COI(0.5 ng/μL)genes.At the low concentration of S.japonicum DNA(0.5 ng/μL),the Sj G28 gene was clearly amplified,and the Sj G28 gene was finally selected as the target gene for qPCR,which could amplify the minimum 1 fg/μL of S.japonicum genome DNA.The detection method of exogenous short DNA fragments by qPCR can amplify about the minimum 100 copies/μL of S.japonicum 81bp DNA fragments.The genomic DNA of S.mansoni,S.haematobium,Babesia microti,A.duodenale,C.sinensis,and P.westermani were used as the template for detection,and only S.japonicum DNA showed an increasing fluorescence signal value.Conclusions:In this study,we screened and identified S.japonicum Sj G28 gene as the target gene,designed probe and primers and established qPCR method for the detection of 81bp short DNA fragments of Sj G28.The method has high specificity and sensitivity,and can be used for the detection of exogenous short DNA fragments of Sj G28 in urine.Establishment and evaluation of a method for extracting short DNA fragments of exogenous Schistosoma japonicum from urineObjective:In order to establish an effective method for the extraction of 81 bp small molecular DNA fragments from Schistosoma japonicum in urine,different methods were used to extract the small molecular DNA fragments from Schistosoma japonicum.Methods:The 81bp exogenous short DNA fragments of S.japonicum was prepared by PCR amplification using Sj G28 gene as target sequence.The 81 bp exogenous S.japonicum short DNA fragments were diluted to 1.0×10~8,1.0×10~7,1.0×10~6,1.0×10~5,1.0×10~4,1.0×10~3,1.0×10~2 copies/μL.2μL of the above short DNA fragments were used as templates for qPCR amplification,and the detection was repeated for three times,with three multiple wells set for each time.Linear regression analysis was performed on the Ct value and concentration of the samples,and standard curves were established.Exogenous 81bp DNA fragments were added to artificial urine,healthy human urine sample and rabbit urine sample.After p H value adjustment,centrifugation,concentration and other urine treatment,81bp DNA fragments were extracted from urine using QIAmp Viral RNA mini kit(Qiagen)and BIOG cf DNA easy kit(BIOG),and the effects of different treatments and extraction methods were compared.According to the detection results of Ct value,the initial copy number of the samples were calculated from the standard curve equation.Then the effects of different processing methods and extraction methods of short DNA fragments in urine samples were calculated.Results:The Ct value results of the different concentration range from1from 1.0×10~8 to1.0×10~2 copies/μL were 10.37,13.95,16.34,19.75,22.58,24.57,27.85,respectively.There was a linear relationship between Ct value and logarithm of copy number(y=-0.382 x+11.689,R~2=0.994).When p H values of artificial urine were 5,6,7 and 8,the nucleic acid recoveries of artificial urine were(49.12±2.09)%,(84.52±4.96)%,(89.38±3.32)%and(87.82±3.90)%by Qiagen kit,respectively.The nucleic acid recoveries of artificial urine were(2.30±0.07)%,(8.11±0.26)%,(13.35±0.61)%,and(20.82±0.68)%by BIOG kit,respectively.The recoveries of the two methods were statistically significant(t=38.702,26.955,39.042,29.571,P<0.01).The recovery of nucleic acid was the lowest at p H 5(P<0.05).There was no significant difference in nucleic acid recovery at p H 6,7 and 8(P>0.05).Before and after centrifugation,the recoveries of exogenous short DNA fragments in artificial urine were(64.30±1.00)%and(58.87±0.26)%,respectively.After centrifugation,the recovery effect of exogenous short DNA fragments was decreased(t=12.033,P<0.05).The Ct values of exogenous short DNA fragments were 15.19±0.60,15.33±0.40 and 10.82±0.27,respectively,extracted from unconcentrated urine,urine concentrated by Amicon Ultra-100k tube and urine concentrated by Amicon Ultra-10k tube.Using Amicon Ultra-10k tube can effectively improve the recovery effect of artificial urine(t=25.355,P<0.01).The concentration of exogenous short DNA fragments in healthy people urine before and after centrifugation were about(31 165±1 017)copies/μL,(28 471±818)copies/μL,respectively,and the recovery effect of exogenous short DNA fragments was decreased after centrifugation(t=23.164,P<0.05).The concentration of exogenous short DNA fragments in healthy people urine were(7 126±497)copies/μL,(27 067±1 461)copies/μL,(688±118)copies/μL,respectively,extracted from unconcentrated urine,urine concentrated by Amicon Ultra-100k tube and urine concentrated by Amicon Ultra-10k tube.Using Amicon Ultra-100k tube can effectively improve the recovery effect of exogenous short DNA fragments urine from healthy people(t=-33.508,P<0.01).The concentration of exogenous short DNA fragments in healthy rabbits urine before and after centrifugation were about(191.47±3.04)copies/μL and(110.62±5.51)copies/μL,respectively,and the recovery effect of exogenous short DNA fragments was decreased after centrifugation(t=22.257,P<0.01).The concentration of exogenous short DNA fragments in rabbits urine were(16.21±1.02)copies/μL and(193.04±3.94)copies/μL,extracted from unconcentrated urine,urine concentrated by Amicon Ultra-100k tube.Using Amicon Ultra-100k tube can effectively improve the recovery effect of of exogenous short DNA fragments urine from healthy rabbits(t=-75.344,P<0.01).Conclusions:In this study,we successfully established a method for the extraction of short DNA fragments of exogenous S.japonicum in urine.The extraction effect of urine samples from healthy people and healthy rabbits could be significantly improved by using the Qiagen kit,p H of urine samples adjusted to 6～8,and Amicon Ultra-100k concentration tube.Detection of specific short molecular DNA fragments in urine of rabbits infected with Schistosoma japonicumObjective:The urine samples of rabbits at different stages of Schistosoma japonicum infection were detected to evaluate the feasibility of the specific small molecule DNA fragment detection method developed earlier in the detection of Schistosoma japonicum infection host urine samples.Methods:Firstly,a rabbit model of S.japonicum infection was established,and urine samples were collected before infection,after infection of 3d,1 w,2 w,3 w,4 w,5 w and 6 w.The urine of rabbits infected with S.japonicum was detected by the method established in Part Two.The urine p H value was adjusted to about 7,then centrifuged at 1500 g for 10 min.The above rabbit urine nucleic acids were detected by qPCR using Amicon ultra 100 k concentration tube and Qiagen kit.The experiment was repeated three times with three multiple wells each time.Results:After S.japonicum infection of 3d,1 w,2 w,3 w,4 w,5 w and 6 w,the Ct values of qPCR were 36.48,34.14,33.36,34.25,31.51,38.31,37.03,respectively.Ct value<35 is considered positive.There was no amplification without S.japonicum infection in rabbit urine samples.From 1w to 4w after S.japonicum infection,Ct values showed a decreasing trend,suggesting that the cf DNA content in early infection urine gradually increased.S.japonicum DNA wasn’t detected from 5 to 6 weeks after infection.Conclusions:In this study,the established qPCR method and the extraction method of exogenous S.japonicum short DNA fragments in urine were initially applied to the urine of infected rabbits.The short DNA fragments of S.japonicum at infection of 1w,2w,3w and 4w were successfully detected,showing a linear relationship(R~2=0.7528).This method is expected to provide suitable support for the field control of schistosomiasis.In conclusion,the Sj G28 gene 81bp short DNA fragments was used as the target sequence to establish a real-time fluorescence quantitative PCR detection method for S.japonicum specific short DNA fragments.Then the extraction method for S.japonicum short DNA fragments in urine was optimized and established.Adjusting the p H value of urine samples to6～8,using the Amicon Ultra-100k concentration tube and the Qiagen kit could improve the extraction effect of short DNA fragments in urine.Urine samples of rabbits infected with S.japonicum at different times were detected by the method of extraction of S.japonicum short DNA fragments and the qPCR method established in this study.The urine samples of rabbits infected with S.japonicum at different times were detected and results showed that the small molecular DNA fragments of S.japonicum in urine was increased by qPCR from 1w to 4 w after infection.The detection method of specific gene fragment of S.japonicum based on urine sample is expected to provide a new method for rapid and accurate diagnosis of schistosomiasis. |
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