Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)–enzyme-linked immunosorbent assay in sewage treatment effluent

Abstract

A colorimetric nucleic acid sequence-based amplification–enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA–RNA hybrids were colorimetrically detected by the addition of streptavidin–peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4×10 1 PFU ml −1) and 15 PFU (3×10 3 PFU ml −1) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of non-target bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.