RT-PCR and chemiluminescent ELISA for detection of enteroviruses

Reverse transcription followed by polymerase chain reaction amplification ( RT-PCR) is now used commonly to detect the presence of enteric RNA viruses in environmental. samples. A sensitive, non-isotopic microtitre plate hybri disation assay was developed and applied for detection of enteroviruses in environmental samples. Following reverse transcription, viral cDNA was labe lled with digoxigenin (DIG)-dUTP during the PCR amplification step. The lab elled PCR products were then hybridised with enterovirus-specific biotinyla ted oligonucleotide probe and captured in streptavidin-coated microtitre we lls. Hybridised enteroviral PCR products were detected by an anti-digoxigen in peroxidase conjugate using either a colourimetric or a chemiluminescent substrate and automated measurement. Standard curves were established for p oliovirus and other enteroviruses. The chemiluminescent assay was more sens itive than the colourimetric assay for detection of poliovirus, and was spe cific for enteroviruses. The chemiluminescent ELISA assay was used to confi rm the presence of enteroviruses in environmental water samples. (C) 1999 E lsevier Science B.V. All rights reserved.