

@article {812,
	title = {Detection of infectious adenovirus in cell culture by {mRNA} reverse {transcription-PCR.}},
	journal = {Applied and environmental microbiology},
	volume = {69},
	number = {12},
	year = {2003},
	pages = {7377-7384},
	abstract = {We have developed and evaluated the reverse transcription {(RT)-PCR} detection of {mRNA} in cell culture to assay infectious adenoviruses {(Ads)} by using Ad type 2 {(Ad2)} and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce {mRNA} during replication in cell culture. Three primer sets for {RT-PCR} amplification of {mRNA} were evaluated for their sensitivity and specificity: a conserved region of late {mRNA} transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early {mRNA} transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The {mRNAs} of infected A549 and Graham 293 cells were recovered from cell lysates with {oligo(dT)} at different time periods after infection and treated with {RNase-free} {DNase} to remove residual contaminating {DNA,} and then Ad {mRNA} was detected by {RT-PCR} assay. The {mRNA} of Ad2 was detected as early as 6 h after infection at 10(6) infectious units {(IU)} per cell culture and after longer incubation times at levels as low as 1 to 2 {IU} per cell culture. The {mRNA} of Ad41 was detected as soon as 24 h after infection at 10(6) {IU} per cell culture and at levels as low as 5 {IU} per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no {mRNA} was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) {UV} radiation. Detection of Ad2 {mRNA} exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but {mRNA} detection occurred sooner. These results suggest that {mRNA} detection by {RT-PCR} assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.},
	author = {G Ko and Cromeans, Theresa L and Sobsey, Mark D}
}
