ANALYSIS OF VIRAL-RNA PERSISTENCE IN SEAWATER BY REVERSE-TRANSCRIPTASE PCR

It is important to determine the stability of naked viral RNA in seawa ter, since false-positive results can occur when reverse transcriptase PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free R NA instead of RNA from intact viruses. An acid guanidinium thiocyanate phenol-chloroform method was used to extract total RNA from a filtere d poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted to tal RNA was seeded into filtered and unfiltered seawater, and the resu lting preparations were incubated at 4 degrees C and at room temperatu re (23 +/- 1 degrees C). Our results showed that the seeded RNA was mo re stable in filtered seawater than in unfiltered seawater at both tem peratures. The viral RNA could not be detected by the RT-PCR after 2 d ays of incubation in unfiltered seawater and after 28 days of incubati on in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in e nvironmental samples by the RT-PCR was mainly due to the presence of w ell-protected viral particles and not due to the presence of naked vir al RNA.