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 hybridisation assay was developed and applied for detection of enteroviruses in environmental samples. Following reverse transcription, viral cDNA was labelled with digoxigenin (DIG)-dUTP during the PCR amplification step. The labelled PCR products were then hybridised with enterovirus-specific biotinylated oligonucleotide probe and captured in streptavidin-coated microtitre wells. Hybridised enteroviral PCR products were detected by an anti-digoxigenin peroxidase conjugate using either a colourimetric or a chemiluminescent substrate and automated measurement. Standard curves were established for poliovirus and other enteroviruses. The chemiluminescent assay was more sensitive than the colourimetric assay for detection of poliovirus, and was specific for enteroviruses. The chemiluminescent ELISA assay was used to confirm the presence of enteroviruses in environmental water samples.
Greening
G E
GE
Communicable Disease Group, ESR Ltd, Kenepuru Science Centre, Porirua, New Zealand. gail.greening@esr.cri.nz
Woodfield
L
L
Lewis
G D
GD
D000818:Animals; D001331:Automation; D019921:Biotinylation; D002460:Cell Line; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005618:Fresh Water / Q000821:virology; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017186:Titrimetry; D010948:Viral Plaque Assay
D015342:DNA Probes; D012367:RNA, Viral