title: Prenatal diagnosis of rubella virus infection by direct detection and semiquantitation of viral RNA in clinical samples by reverse transcription-PCR.
abstract: A reverse transcription-nested PCR (RT-nPCR) method for prenatal diagnosis of rubella virus (RV) infection was developed. In the first step of RT-nPCR a synthetic RNA molecule (pRRV) differing from the RV target sequence by having a 21-nucleotide insertion was used as the internal control of amplification for the detection of PCR inhibitors. In addition, comparison of pRRV and RV-specific PCR signals allowed for the semiquantitation of RV input target sequences (range, 10 to > and = 1,000 RV genomes). In parallel, a complete RT-nPCR assay was performed with the same samples in the absence of the internal control to confirm the results of the first step and to detect RV RNA-positive samples containing < 10 RV genomes. Subsequently, the RT-nPCR method was used to examine retrospectively clinical samples (direct RT-nPCR) from eight congenitally infected and eight uninfected fetuses for RV RNA. RT-nPCR was also used to detect RV RNA in cell cultures (culture-RT-nPCR) 96 h after inoculation with the same specimens. With amniotic fluid (AF) samples, direct RT-nPCR identified eight of eight cases of RV transmission (sensitivity, 100%), whereas culture-RT-nPCR and virus isolation detected only six of eight cases (sensitivity, 75%). However, when the culture-RT-nPCR results were positive, culture-RT-nPCR confirmed the direct RT-nPCR results 3 days to 3 weeks earlier than virus isolation. The specificity of direct RT-nPCR was 100%, with eight of eight uninfected fetuses being negative. Semiquantitation showed only small amounts (< and = 100 copies) of viral RNA in clinical samples. In conclusion, direct RT-nPCR with AF samples (i) shows 100% sensitivity and specificity for prenatal diagnosis of RV infection and (ii) is a rapid technique, giving results in 24 to 48 h after sampling.
authors.affiliation: Institute of Infectious Diseases, University of Pavia, Italy.
authors.forename: M G
authors.initials: MG
authors.lastname: Revello
authors.forename: F
authors.initials: F
authors.lastname: Baldanti
authors.forename: A
authors.initials: A
authors.lastname: Sarasini
authors.forename: M
authors.initials: M
authors.lastname: Zavattoni
authors.forename: M
authors.initials: M
authors.lastname: Torsellini
authors.forename: G
authors.initials: G
authors.lastname: Gerna
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods* / Q000706:statistics & numerical data; D011786:Quality Control; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D012410:Rubella Syndrome, Congenital / Q000175:diagnosis* / Q000821:virology; D012412:Rubella virus / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data
chemical_list: D017931:DNA Primers; D012367:RNA, Viral