DETECTION OF COXSACKIEVIRUS B3 IN INTESTINAL TISSUE OF ORALLY-INFECTED MICE BY A STANDARDIZED RT-PCR ASSAY

Background: Previous studies have reported the role of enteroviruses i n chronic diseases, using in-house RT-PCR protocols. A well-standardiz ed PCR assay (Amplicor enterovirus, Produits Roche) designed for the d iagnosis of enterovirus meningitis in cerebrospinal fluids (CSF) was r ecently described. Objectives: To evaluate this commercially-available PCR assay for the detection of enterovirus in intestinal biopsies. St udy design: In order to obtain large quantities of infected material, eight mice were inoculated orally with 2 x 10(5) 50% tissue culture in fective doses (TCID50) of coxsackievirus B3 (CBV3); two mice were sacr ificed every day from day 1 to day 4 post-infection. Stool specimens a nd small bowel fragments were taken from infected animals and controls . Four protocols of RN4 extraction from intestinal tissue were compare d. Extracted RNA was then tested by the Amplicor assay and by a semine sted in-house PCR. Results: The best results were obtained with a comm ercial reagent using a combination of guanidium thiocyanate and phenol (TRI Reagent, Sigma). This procedure allowed the detection of enterov iral RNA in intestinal samples of 7/8 and 8/8 infected mice by Amplico r assay and seminested PCR, respectively, whereas only five samples we re tested positive by conventional cell culture. When tested on serial dilutions of CBV3 mixed with intestinal tissue, a sensitivity of 0.2 TCID50/mg was achieved with both PCR assays. Conclusions: The data dem onstrate that the Amplicor enterovirus assay, which is designed to avo id false-positive amplifications, can be used, with a slight modificat ion of the RNA extraction step, for the detection of enterovirus in sp ecimens different from CSF such as intestinal tissue. (C) 1997 Elsevie r Science B.V.