Establishment And Evaluation Of Cell-free Schistosoma DNA Detection Kit Based Nested PCR Targeting SjCHGCS19 Retrotransposon For Diagnosis Of Schistosomiasis Japonica

Part I Establishing of rapid and simple cf DNA extractionmethod of schistosomiasis japonica detection kitObjective: Establishing a quick and easy method for isolating Schistosoma cell-free DNA from serum.Method: According to the survey of the present technical of DNA extraction,take Si O2-Spin column as basis to build a fast and simple DNA extraction method.The princple is that nucleic acid is adsorbed to Si O2 in high salt and acidic PH condition,and desorbed from within low salt and high PH buffer.The method contain plastic material,a series of buffer,and a protocol.And then assess the performance of extracting,the simplicity and costing of it,in comparision with two control methods.Result:Base on the Si O2-Spin column,Method I and II were built.The volume of serum which they can treat is 100 ul and 200 ul.Method I with the limit of recover 10-7 dilution adult DNA from serum,identical to Phenol method;In preparation of five infected mice serum,the templates isolated by Method I have two positive by nested PCR detection,but these of Phenol method have four positive.And in the extraction of DNA of infected rabbit serum,modified method II have 6 positive in seven sample,is superior to method I(3),Phenol method(5)and QIAamp Kit(3).In simplicity,method II protocol have six step,24 times of operation.The manipulation is simple and standardized.So its simplicity is identical to QIAamp Kit.There is seven step in the protocol of Phenol method,29 times of operation.And the manual-operation need experience,not standardized.The speed of method II,have fastvelocity,within 1.5 h or 2.5 h for preperating 12 or 24 samples.The speed is identical to QIAamp Kit,superior to Phenol method with more than seven hours.The cost of isolation,method II take 3.3 yuan per sample,Phenol method 3.1 yuan per sample,QIAamp Kit 28.3 yuan per sample.Conclusion: In this stuy,method II established based Si O2-spin column,have high performance of circulating DNA extraction.And it is simple and quick,with low cost.Part II Optimizing the primers of SjCHGCS19 of schistosomiasis japonica detection kitObjective: Optimizing the nested primers of Sj CHGCS19 for more suited the detecting of the short fragment DNA,Schistosoma circulating DNA.Method: On the basis of research about the high specific and sensitive Sj CHGCS19 PCR with 303 bp target discovered by our lab,this study will go forward designing a new primers from it.The primer designed in two principle,the amplicon of primers less than 166/143 bp,and it’s optimal to locat the primer in the high identical location of the multicopy sequence.The protocol of primer design,according the instruction of Primer-BLAST,reset the amplicon length and Tm dependent the practice.Evaluating the sensitivity of the primers using the dilution template of Schistosoma DNA,and specificity using template of DNA of Schistosoma mansoni,Schistosoma haematobium,Clonorchis sinensis,Paragonimus westermani,Trichinella spiralis.Result: According to the amplicon band brightness,clear backgroud principle,from six pairs of outer primers and seven pairs of inner primers,screened out a set of nested primers.FOP19/BOP19 and FIP19/BIP19,the amplicon of them is 139 bp and 117 bp.The new primers have same sensitivity with the former,at 8 fg/ul adult Schistosoma DNA.In specificity,they have cross-reaction with Schistosoma mansoni,have cross-reaction with Clonorchis sinensis and Trichinella spiralis,at concentration of 76.5 ng/ul and 108.1 ng/ul,not cross-reaction at concentration of 0.765 ng/ul and 10.81 ng/ul.But haven’t cross-reaction with Schistosoma haematobium and Paragonimus westermani,at concentration of 40.7 ng/ul and 54.6 ng/ul.The new primers improved the speed of PCR,the amplification program can accomplish in two hour.Conclusion: The primers optimized amplify the products with 139 bp and 117 bp.The sensitivity of them is 8.29 fg/ul,at the same level with the former.The cross-reaction with Schistosoma mansoni make the detection by them cannot discriminate between schistosomiasis monsoni and japonica.The cross-reaction with Clonorchis sinensis and Trichinella spiralis,at or above nanogram level,dosen’t interference the detection of Schistosoma DNA in the blood.Although the optimized primers haven’t improved the sensitivity and specificity,they designed with short-length amplicon more suited for detection of DNA from circulating body fluid.And the new primers can accelerate the speed of detection.Part III Evaluating the performance of SjDNA detection Kit at schistosomiasis japonica model of rabbitObjective: Evaluating the detecting sensitivity and the effects of chemotherapy of the Kit at model of schistosomiasis japonica rabbit.Method:Establishing schistosomiasis japonica model,9 rabbits were randomly devided into 3 group,every group have 3 rabbits.Group I were infected with 250 pair of S.japonica cercariae,rabbits were respectively numbered 1-3;Group II were infected with 100 pair of S.japonica cercariae,rabbits were respectively numbered 4-6;Group III were infected with 100 pair of S.japonica cercariae,rabbits were respectively numbered 7-9.At 7 w and 8 w after infected,Group I and II had two treatments with praziquantel.At the end of experiment,all rabbit were sacrificed for investigation the worm.Blood were collected weekly and serum were contrifuged from blood at 2000 rpm for 10 min with twice.The Sj DNA of each sera collected were detected by the Kit,the result data were statistical and analysis for detecting sensitivity and the effect of chemotheray.At the step of DNA extraction,the performance of Kit was evaluated with Phenol method and QIAamp method as control.The serum template isolated by three DNA isolating methods were detected in three time by nested PCR.Result:It is 23.7% of the sensitivity of Kit for early phase 1-5 w,84.9% for mid-phase6-11 w,79.6% for late phase.The data of three group presented that the positive ratio goes raise weekly before 9 w,goes down or turn negative of group I and II after 9 w,but didn’t change in group III after 9 w.The trends of the detecting method is in the same.For low-intensity infection,before 6 w post infection,at the same sensitivity of Kit is late one week the medium,after 6 w,the sensitivity is at the same.The effect of chemotherapy,by detection of Kit,the positive ratio of the first week post treatment is at the highest level,second week with 17%,and fifth week with 0.And the time point of turn negative is fifth week of group I,second week of group II.Conclusion: The result of detection of schistosomiasis japonica rabbit serum by the Kit,it sensitivity is superior to the other two.For low-intensity infection,its sensitivity have decreased a little before 6 w,but equal after 6 w.The effect of chemotherapy,at second week post treatment almost rabbit(5/6)turn negative.On the basis of the research about Schistosoma DNA detection by our lab,this stuy established a Sj DNA detection Kit,which content a Si O2 spin column based DNA isolating method and a nested PCR based Sj CHGCS19 target.The Kit have high sensitivity for low-intensity infection,at acute phase and chronic phase.Its quick turn negative after successful treatment,make it have good performance to discriminate persistent infection and re-infection.The Kit have high value in application of detection of schistosomiasis japonica and evaluating the effect of treatment.