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 reaction (RT-nPCR) method for rubella virus (RV) RNA detection was assessed in a series of African green monkey kidney (AGMK) cell cultures inoculated with clinical samples from patients with suspected RV infection. Results were compared with those of conventional virus isolation/identification. The assay included an internal control of amplification consisting of a synthetic RNA molecule mimicking the RV E1 target sequence. A semiquantitation of RV RNA was achieved by comparing the relative 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 samples. 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. When 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 RV was isolated in only 7/16 (44%) cell cultures. Semiquantitation showed that: i) viral RNA amount progressively increased with time; ii) cell cultures containing very low levels of viral RNA one week p.i. either required a few blind passages for virus recovery or remained negative for RV isolation. Finally, PCR inhibitors were found in 10/164 (6%) cell cultures examined. In conclusion, RT-nPCR proved to be very sensitive and very specific and greatly reduced RV detection time in inoculated cell cultures.
Revello
M G
MG
Istituto di Malattie Infettive, Università di Pavia, Italy.
Sarasini
A
A
Baldanti
F
F
Percivalle
E
E
Zella
D
D
Gerna
G
G
D000328:Adult; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000175:diagnosis; D011296:Prenatal Diagnosis / Q000379:methods; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012409:Rubella / Q000175:diagnosis*; D012411:Rubella Vaccine; D012412:Rubella virus / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014709:Vero Cells
D012367:RNA, Viral; D012411:Rubella Vaccine; D012194:RNA-Directed DNA Polymerase