USE OF REVERSE-TRANSCRIPTION POLYMERASE CHAIN-REACTION FOR DETECTION OF RUBELLA-VIRUS RNA IN CELL-CULTURES INOCULATED WITH CLINICAL-SAMPLES

A recently developed reverse transcription-nested polymerase chain rea ction (RT-nPCR) method for rubella virus (RV) RNA detection was assess ed in a series of African green monkey kidney (AGMK) cell cultures ino culated with clinical samples from patients with suspected RV infectio n. Results were compared with those of conventional virus isolation/id entification. The assay included an internal control of amplification consisting of a synthetic RNA molecule mimicking the RV El target sequ ence. A semiquantitation of RV RNA was achieved by comparing the relat ive band intensity of internal control and RV E1 fragment. RT-nPCR was positive in 15/16 (94%) RV isolation-positive cultures and in 12/60 ( 20%) RV isolation-negative cultures. All 27 cell cultures positive by RT-nPCR had been inoculated with clinical samples taken from patients with ascertained RV infection or given RV vaccination and consisted of cells harvested 1-10 days after primary inoculation of clinical sampl es. No RV RNA was found in any of the cell cultures inoculated with 14 clinical samples from 6 patients in whom RV infection was excluded. W hen considering the time-course of RV infection, it was found that RV RNA could be detected as early as 4 days p.i. in 10/21 (48%), and by 7 -10 days p.i. in 27/28 (96%) cell cultures, whereas by the same time R V was isolated in only 7/16 (44%) cell cultures. Semiquantitation show ed that: i) viral RNA amount progressively increased with time ii) cel l cultures containing very low levels of viral RNA one week p.i. eithe r required a few blind passages for virus recovery or remained negativ e for RV isolation. Finally, PCR inhibitors were found in 10/164 (6%) cell cultures examined. In conclusion, RT-nPCR proved to be very sensi tive and very specific and greatly reduced RW detection time in inocul ated cell cultures.