Real-time PCR Double-target Assay For Evaluation Of Short-term Treatment Efficacy With Detection Of Serum DNA In Hosts Infected With S.japonicum And Research Of Sentinel Mice Warning

Part 1 Real-time PCR double-target assay for evaluation of short-term treatment efficacy with detection of serum DNA in hosts infected with S.japonicum in different cyclesObjective: This study have applied Real-time PCR double-target method to explore the dynamic changes of serum DNA of different course of praziquantel treatment, exploring and evaluating the potential value of the short-time therapy evaluation of S.japonicum,which the time point that worms DNA of serum had become negative have been regarded as time of therapy evaluation.Method:Rabbits infected with 200 mixed sexual cercaria of S.japonicum were administered with PZQ(200mg/kg) at 4w 、 7w 、 12 w 、 16 w following infection respectively,in addition to the control group,collecting serum of 1-7d after treatment(every day),2th week to put to death(every week),with control group collection the matching rabbit serum. Real-time PCR double-target detect serum DNA.Results:Positive products were amplified in the serum of 3rd post-infection by Real-time PCR double-target. Serum DNA in rabbits at 3d to 42 w after infection could be detected positively by Real- time PCR double-target method.The testing results of serum DNA of different treatment duration shown that serum DNA detected by double-target Real- time PCR have peak at 3d to 4d after treatment.Serum DNA of the treatment groups at 4w、7w、12w、16w respectively after infection detected by Real-time PCR double-target have become negative at 6w to 7w、15w to 16w、18w to 20w、26w to 28 w respectively after treatment. In the study of the moment that worms DNA of serum of hosts infected with mixed sexual cercariae had become negative,this time point had shown a trend of negative for the treatment groups at 2w approximately after treatment by ROC analysis.Conclusion: Real-time PCR for S.japonicum had a value of early diagnosis.The testing results of Real- time PCR double-target have shown that they have higher sensitivity and complementary certainly.The early research has shown that serum DNA in infected hosts have derived primary from worm and eggs.The quantitative testing results have shown that serum DNA of different treatment duration detected by Real-time PCR double-target have been negative from 6w to 7w to 26 w to 28 w.However,depending on the dymanic change rules of worms DNA of serum,speculating the moment that worms DNA have become negative by ROC analysis could shorten largely the time of therapy evaluation.The research results have provided the experimental basis for exploring the potential application value of that the time point that worms DNA of serum after treatment have become negative could be regarded as time of therapy evaluation.Part 2 The application research:the nested PCR double-target method in the warning of the model of S.japonicum sentinel miceObjective: the method of nested PCR double-target have been applied to sentinel mice warning of S.japonicum,exploring the sensitivity of the method,and evaluating the value of early warning.Method: Establishment of sentinel mice model infected with mixed sexual cercarias:infected with 5,10 and 20 dual sexual cercarias of S.japonicum respectively, dividing randomly to two groups.One group used to evaluate the actual infection,the other group collected mice serum of three groups mices of different degree of infection at 0d,3d,5d,1w and 2w after infection,using nested PCR double-target and two immunology methods to detect mice serum.Results: Sj19 nested PCR could detect positive repeatedly for serum DNA of three groups of different degrees of infection at four time points after infection;Sj R2 nested PCR only could detect positive for serum DNA of the group infectted with 5 dual sexual cercaria at 5th day after infection,and serum DNA of the group infectted with 10 dual sexual cercaria at 3rd day,5th day and 7th day after infection,and serum DNA of the group infectted with 20 dual sexual cercaria at four time points after infection.Two methods of immunology(IHA and ELISA) could all not detect positive in mice serum of three groups of different degrees of infection at four time points after infection.Conclusion: The Sj19 and Sj R2 nested PCR could detect positive for serum DNA of the infection group with 5 dual sexual cercaria at 3rd or 5thday after infection,showing higher sensitivity and shorting the time of early warning.In particular,the Sj19 nested PCR could detect positive for serum DNA of the infection group with 5 dual sexual cercaria at 3rd day after infection, showing higher sensitivity than Sj R2 nested PCR. The research results have shown that serum DNA of sentinel mice model detected by Sj19 and Sj R2 nested PCR could shorten largely the warning time, having the potential application value of early warning of S.japonicum.Part 3 Gender identification of Schistosoma japonicum cercaria and the research of the folded rate of male and female wormObjective: To evaluate the PCR method for the identification effect of gender of S.japonicum cercaria,and explore the difference of male and female cercaria susceptibility,and the rat of survival and folded worms,with different infection degree of male and female cercaria with matching infection.Methods: Collecting the cercarias escaped by different single positive snails,taking two cercaria samples of each snail,getting the cercaria DNA base cracking,SCAR-PCR detect cercaria DNA and idientify gender of cercaria.All snails after identification raises separately on the basis of gender of cercaria, escaping cercaria after 20 days. The mice infected with came 5♂+5♀、10♂+10♀和 15♂+15♀ male and female cercarias,dissectting infected mices after 40 days and recording worms getted from each mice.Results: There were 23 positive snails.After identification,10 of it could escaped single female cercarias,9 of it could escaped single male cercarias,and 4 of it could escaped bisexual cercarias.In matching infection,the survival rates of famale and male worms of three groups of mice infected with came 5 ♂ +5 ♀、 10 ♂ +10 ♀和 15 ♂ +15 ♀ were respectively 72.0% and 82.0% 、 71.3% and 85.0% 、 62.0% and 72.7%,and the susceptibility of male and female cercaria has been no significant difference by statistical analysis. The overall survival rates of worms of three groups of mice were respectively 77.0%、78.0%、67.7%,and the group infected with came 15♂+15♀ male and female cercaria was lower slightly than the other two groups. The folded rates of famela and male worms of three groups were respectively 68.6%、71.3%、62.0%,no significant difference among the groups,remindering the folded rates of mela and female worm could be higher in infection of equal proportion of female and male cercaria.Conclusion: SCAR-PCR was a rapid,simple and reliable method identifying gender of S.japonicum cercaria.The accuracy of identification for snail escaped single male cercaria would be higher especially,which was of great significance to establish a animal models infected single male S.japonicum cercaria for the study of drugs. Observation of the folded rate have provide an important reference for the evaluation of actual infected degree with mixed infection.