DEVELOPMENT OF AN ENTEROVIRUS SPECIFIC PCR METHOD FOR THE QUANTIFICATION OF ENTEROVIRUS GENOMES IN BLOOD OF DIABETES PATIENTS

Background: Insulin-dependent diabetes mellitus or type 1 diabetes is a disease with a diverse aetiology. Epidemiological studies examining newly diagnosed, recent onset IDDM patients have suggested a role for viruses in the aetiology of IDDM (Yoon, 1995, Diabetes/Metabolism Revi ews 11, 83-107). Important candidates are the enteroviruses, in partic ular coxsackieviruses B3 and B4. The latter can cause diabetes in anim als (Clements et al., 1995, Lancet 346, 221-223). Objectives: We have developed a quantitative PCR method for the detection of enterovirus g enomes in biological samples. The quantitative PCR will be used to scr een for enteroviruses in blood of diabetes patients and their relative s by testing a Blood Diabetes Register. Study design: A substantial am ount of data has been collected on enterovirus induced IDDM, our study is original in so far as it will be: (1) a quantitative study, not on ly the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal st udy, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method. Re sults: The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other pico rnavirus genera scored negative (even up to 3 x 10(6) genome copies). An equal detection limit of 10 genome copies was found for all enterov iruses. Conclusions: The developed method will permit us to generate q uantitative and longitudinal data of enterovirus genomes in blood of d iabetes patients and their relatives, which might help in the elucidat ion of the relationship between enteroviruses and IDDM. (C) 1998 Elsev ier Science B.V. All rights reserved.