Analysis of viral RNA persistence in seawater by reverse transcriptase-PCR.

Abstract

It is important to determine the stability of naked viral RNA in seawater, since false-positive results can occur when reverse transcriptase-PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free RNA instead of RNA from intact viruses. An acid guanidinium thiocyanate-phenol-chloroform method was used to extract total RNA from a filtered poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted total RNA was seeded into filtered and unfiltered seawater, and the resulting preparations were incubated at 4 degrees C and at room temperature (23 +/- 1 degrees C). Our results showed that the seeded RNA was more stable in filtered seawater than in unfiltered seawater at both temperatures. The viral RNA could not be detected by the RT-PCR after 2 days of incubation in unfiltered seawater and after 28 days of incubation in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in environmental samples by the RT-PCR was mainly due to the presence of well-protected viral particles and not due to the presence of naked viral RNA.