An improved method for the detection of Norwalk-like caliciviruses in environmental samples.
An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.
Loisy
F
F
Laboratoire de Microbiologie, IFREMER, Nantes, France.
Le Cann
P
P
Pommepuy
M
M
Le Guyader
F
F
D002139:Caliciviridae / Q000302:isolation & purification*; D004783:Environmental Microbiology; D008280:Magnetics; D009663:Norwalk virus / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012722:Sewage / Q000382:microbiology*
D012722:Sewage