

@article {3596,
	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{\textendash}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 \{\{\.I}U\{\}\} per cell culture. The \{\{\}mRNA\{\}\} of Ad41 was detected as soon as 24 h after infection at 10(6) \{\{\.I}U\{\}\} per cell culture and at levels as low as 5 \{\{\.I}U\{\}\} 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}
}
