Detection of Types 40 and 41 Adenoviruses in Stool Samples of Diarrheal Children by Solid Phase PCR

Abstract

Adenoviruses (AdVs) types 40 and 41 are the causative agents of diarrhea in children. Hence, rapid sensitive and specific detection of these viruses are of clinical importance. The customary methods such as propagation of virus in cell culture suffer from limitations. Detection of immobilized amplified products in a one phase system (DIAPOPS) method has the potential to overcome these problems. A DIAPOPS method for detection of AdV types 40 and 41 was designed. Forward primers were covalently linked to the Nucleolink surface. After amplification of a 745 bp sequence of DNA binding protein gene, the amplified product was hybridized with the biotinylated probe. The hybrids were detected by the antibody-peroxidase conjugate. After optimization of the DIAPOPS conditions, 80 stool samples from children with clinical manifestation of viral diarrhea were tested. Their DIAPOPS results were compared with those of the conventional polymerase chain reaction (PCR) assay. Positive results were obtained in 11 samples. The comparison between conventional PCR and DIAPOPS showed a significant increase in sensitivity of the DIAPOPS test, 6 samples shown to be negative by conventional PCR, were demonstrated positive by DIAPOPS (p=0.00). The DIAPOPS assay presented in this study can provide a rapid, sensitive, specific and economic method for detection of viral infections. The assay can be performed for numerous samples simultaneously in a day. This DIAPOPS method can provide a practical and reliable tool for diagnosis of enteric adenoviruses. In addition, the risk of contamination in this assay is low.