Establishment Of Real-time Quaking Induced Conversion (RT-QuIC) Assay And Its Primary Applications

Prion diseases are a kind of fatal neurodegenerative disease affecting central nervous system of humans and animals with long incubation,100%mortality.Prion diseases can be transmitted within same species.Prion diseases in human consist of Creutzfeldt-Jakob disease(CJD),fatal familial insomnia(FFI),Kuru,and Gerstmann-Straussler-Scheinker syndrome(GSS),etc.And CJD include sporadic,genetic,iatrogenic and variant CJD.The pathogen of prion diseases is prion(PrPSc),an infective protein which can self-propagate and have no nucleic acid.PrPSc comes from PrPC,which is a celluar prion protein.PrPC is the normal form of prion and can be found in human generally.PrPC and PrPSc have the same sequence of amino acid.However under some circumstances,the 2nd structure of PrPC protein changes and transforms into PrPSc.For now the diagnosis of prion diseases depend on detecting PrPSc in brain tissue.However brain tissue is not easy to accuire,especially in China.Most patients in China are diagnosis as clinical prion diseases according to their clinical manifestation,clinical examination and laboratory test.Therefore we need other tissue or body fluid rather than brain tissue to detect PrPSc.Real-time quaking induced conversion(RT-QuIC)assay bases on the characteristics of forming amyloid fibrils of prion proteins.Then the amount of PrPSc can be reflect through thioflavine T(ThT).RT-QuIC assay is of high-throughout,high speed,high sensitivity and high specificity.It was reported that RT-QuIC could ben used in detecting PrPSc in cerebrospinal fluid(CSF).Comparing with brain tissue,CSF is much easier to accuire.For now in China there is no research about RT-QuIC assay.In our study,we established RT-QuIC assay the first time based on recombinant hamster PrP 23-231 protein as the substrate.The sensitivity of the established RT-QuIC for PrPSc was evaluated as 100 ag according to the evaluation of PrPSc in the brain homogenates of scrapie strain 263K infected hamsters.The possible influences of some factors,such as the concentrations of ThT,SDS and the amounts of human CSF were tested.It was found that the reaction containing1xPBS,170mM NaCl,100mM EDTA,10 ?M ThT,1 ng SDS,10?g rPrP and 10 ?l CSF were optimized for the assay.The reactivities of different PrPSc strain in RT-QuIC assay were also examined.Besides the prion strains our lab has processed including hamster 263K strain,mouse ME7 and 139A strains,and S15 cell lines,we also prepared SMB-S15 cell inoculated mouse strains.Cell lysates of S15 were intracerebrally inoculated into three different strains of mice,namely C57,CD1 and Balb/C.All infected mice developed typical experimental prion diseases approximately 170 days post-infection.The typical neuropathological characteristics,such as spongiform vacuolation,PrPSc deposits,astrogliosis and activated microglia were all seen.Brain homogenate of three strains of mice were intracerebrally inoculated into next generation.And the incubation time of second generation decreased to 150 days.The results indicated we successfully obtained three S15 inoculated mouse strains.Strains had different reactivities in RT-QuIC assay.In our study,recombinant protein was based on hamster prion protein.Therefore hamster 263K strain had the best reactivity,followed by mouse and S15 cell strains.For S15 cell and S15 cell inoculated mouse strains,reactivities of animal brain homogenates were better than cell lysates.Under the established experimental condition of RT-QuIC assay,the CSF samples of 70 probable sCJD patients and 48 non-CJD cases were also preliminarily screened.Markedly high ratio of sCJD samples(57.14%)showed positive reactions in RT-QuIC,with short lag phase(<50 h post-reaction)and high peak ThT values(>25000 rfu).On the contrary,a few samples of non-CJD displayed weak positive reactions at the late stages(>50 h post-reaction)with much low ThT values.In China the most three frequent genetic prion diseases are FFI,T188K mutation and E200K mutation.The clinical manifestations,examinations,laboratory tests and neuropathology of those diseases vary largely.In our study,we explored the reactivities of the CSF of those three diseases in RT-QuIC assay.Totally,CSF samples from 24 patients with FFI,25 with T188K gCJD,and 16 with E200K gCJD were enrolled in our RT-QuIC assay.16.7%of FFI,52%of T188K and 62.5%of E200K gCJD were RT-QuIC positive.Calculation of the positive converting time post-reaction and maximal ThT fluorescent value of the individual positive samples identified that E200K gCJD showed shortest lag phase and highest peak ThT value,followed by T188K gCJD,whereas FFI showed very low maximal ThT value.Statistical analyses reveal positive correlations of the CSF RT-QuIC reactivities of those genetic prion diseases with the abnormalities on EEG,MRI and 14-3-3 protein.Our study established RT-QuIC assay the first time in China.RT-QuIC assay can be used in detecting PrPSc in CSF.Meantime RT-QuIC assay can be used to examine the infectivity of different prion strains.