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 Reviews 11, 83-107). Important candidates are the enteroviruses, in particular coxsackieviruses B3 and B4. The latter can cause diabetes in animals (Clements et al., 1995, Lancet 346, 221-223).


OBJECTIVE
We have developed a quantitative PCR method for the detection of enterovirus genomes in biological samples. The quantitative PCR will be used to screen for enteroviruses in blood of diabetes patients and their relatives by testing a Blood Diabetes Register.


METHODS
A substantial amount 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 only the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal study, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method.


RESULTS
The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other picornavirus genera scored negative (even up to 3 x 10(6) genome copies). An equal detection limit of 10 genome copies was found for all enteroviruses.


CONCLUSIONS
The developed method will permit us to generate quantitative and longitudinal data of enterovirus genomes in blood of diabetes patients and their relatives, which might help in the elucidation of the relationship between enteroviruses and IDDM.
Lauwers
S
S
Department of Microbiology and Hygiene, Vrije Universiteit Brussel, Belgium.
Bissay
V
V
Rombaut
B
B
D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000821:virology*; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000097:blood / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay; D016679:Genome, Viral; D006801:Humans; D008137:Longitudinal Studies; D010850:Picornaviridae Infections / Q000097:blood / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D012680:Sensitivity and Specificity
D012367:RNA, Viral