PRENATAL-DIAGNOSIS OF RUBELLA-VIRUS INFECTION BY DIRECT-DETECTION ANDSEMIQUANTITATION OF VIRAL-RNA IN CLINICAL-SAMPLES BY REVERSE TRANSCRIPTION-PCR

A reverse transcription-nested PCR (RT-nPCR) method for prenatal diagn osis of rubella virus (RV) infection was developed, In the first step of RT-nPCR a synthetic RNA molecule (pRRV) differing from the RV targe t sequence by having a 21-nucleotide insertion was used as the interna l control of amplification for the detection of PCR inhibitors, In add ition, comparison of pRRV and RV-specific PCR signals allowed for the semiquantitation of RV input target sequences (range, 10 to greater th an or equal to 1,000 RV genomes). In parallel, a complete RT-nPCR assa y was performed with the same samples in the absence of the internal c ontrol to confirm the results of the first step and to detect RV RNA-p ositive samples containing <10 RV genomes, Subsequently, the RT-nPCR m ethod was used to examine retrospectively clinical samples (direct RT- nPCR) from eight congenitally infected and eight uninfected fetuses fo r RV RNA, RT-nPCR was also used to detect RV RNA in cell cultures (cul ture-RT-nPCR) 96 h after inoculation with the same specimens, With amn iotic fluid (AF) samples, direct RT-nPCR identified eight of eight cas es 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-nP CR confirmed the direct RT-nPCR results 3 days to 3 weeks earlier than virus isolation, The specificity of direct RT-nPCR was 100%, with eig ht of eight uninfected fetuses being negative. Semiquantitation showed only small amounts (less than or equal to 100 copies) of viral RNA in clinical samples. In conclusion, direct RT-nPCR with AF samples (i) s hows 100% sensitivity and specificity for prenatal diagnosis of RV inf ection and (ii) is a rapid technique, giving results in 24 to 48 h aft er sampling.