

@article {811,
	title = {An integrated cell culture and reverse transcription quantitative {PCR} assay for detection of infectious rotaviruses in environmental waters.},
	journal = {Journal of microbiological methods},
	volume = {82},
	number = {1},
	year = {2010},
	pages = {59-63},
	abstract = {Rotaviruses exist widely in water environments and are the major cause to the gastroenteritis in children. To overcome the limitations associated with the current methods for detecting rotaviruses in environmental samples, such as long duration with the traditional cell culture-based plaque assay, inability to detect infectivity with {RT-PCR-based} molecular methods and lower sensitivity with {ELISA} tests, we developed an integrated cell culture and reverse transcription quantitative {PCR} {(ICC-RT-qPCR)} assay to detect infectious rotaviruses based on detection of viral {RNA} during replication in cells. The cell culturing step before {qPCR} allows the infectious rotaviruses to replicate and be detected because they are the only ones that can infect cells and produce {RNA.} The results showed that as low as 0.2 {PFU/ml} rotaviruses were detected by {ICC-RT-qPCR} after 2 days of incubation. With samples, the copy numbers of {VP7} gene of rotaviruses linearly correlated (with a coefficient {(R(2))} of 0.9575) with initial virus concentrations ranging from 0.2 to 200 {PFU/ml.} In parallel comparing tests, the {ICC-RT-qPCR} exhibited higher sensitivity than both the plaque assay and the {RT-qPCR} when applied to field samples. {ICC-RT-qPCR} detected infectious rotavirus in 42\% (10/24) of secondary effluents, while only 21\% (5/24) and 12\% (3/24) of samples were positive with either the plaque counting or the {RT-qPCR} method, respectively. Concentrations of rotaviruses in secondary effluent samples were determined to be 1-30 {PFU/l.} The results demonstrated that the developed {ICC-RT-qPCR} method reduced test duration and improved sensitivity towards infectious rotavirus and therefore can be an effective and quantitative tool for detecting infectious rotaviruses in water environments.},
	doi = {10.1016/j.mimet.2010.04.003},
	url = {http://dx.doi.org/10.1016/j.mimet.2010.04.003},
	author = {Li, Dan and Gu, April Z and Yang, Wan and He, Miao and Hu, Xiu-Hua H and HC C Shi}
}
