PCR for detection of rubella virus RNA in clinical samples

Abstract

A reverse transcription nested PCR (RT-PCR) assay for the detection of rubella virus RNA using primers from the E1 open reading frame was established. This assay was found to be sensitive (detecting approximately two synthetic RNA copies and RNA extracted from 0.1 50% tissue culture infective dose of rubella virus) and specific; five wild-type rubella strains and four vaccine strains were detected, and no nonspecific amplification of 16 other RNA viruses or RNAs from seven cell types occurred. Rubella virus RNA was detected in 12 pharyngeal swabs from patients with serologically confirmed rubella; these RT-PCR results were in complete agreement with virus isolation. Analysis of products of conception obtained after confirmed primary maternal rubella infection by RT-PCR gave 92% agreement (12 of 13 samples) with virus isolation. No false-positive results were obtained. The potential use of this assay for prenatal diagnosis of congenital rubella infection and for investigating aspects of the pathogenesis of chronic disease is discussed.

Use of PCR for prenatal and postnatal diagnosis of congenital rubella

“…Although several investigators have reported the development of RV RT-PCR with a view to its use for prenatal diagnosis of CAR (7,9,16), there has been only one published preliminary report of its use for this purpose (24). Our RT-PCR has been shown to compare well with virus isolation for detection of RV in pharyngeal swabs and POCs (7). In the larger series of POCs presented here, we obtained evidence of intrauterine infection by RT-PCR in 67% of cases in which primary infection had occurred in the first 12 weeks of pregnancy.…”

“…Detection of RV RNA by RT-PCR. RV RNA was detected by a previously described RT-PCR (7), which has been shown to be specific for RV RNA and sufficiently sensitive to detect 0.1 50% tissue culture infectious dose or approximately two synthetic RNA copies. The oligonucleotides used were located within the E1 open reading frame of the RV genome.…”

“…We have previously described a reverse transcription (RT)nested PCR assay (RT-PCR) for detection of rubella virus (RV) RNA (7), and in the present study, we evaluated this assay for the prenatal diagnosis of CAR and the diagnosis of CAR in infancy.…”

Approach to Congenital Intrauterine in F Ections

“…Nested RT-PCR based diagnosis is awardable on amniotic fluid, urine, fetal blood or mother's blood. 13 Using these primers (first round R2 -5'CAA CAC GCC GCA CGG ACA AC 3' -8807-8826, R7 4'CCA CAA GCC GCG AGC AGT CA 3' -8991-8972. Nested R11 -5' CTC GAG GTC CAG GTC CYG CC 3' 8826-8845.…”

“…Nested R11 -5' CTC GAG GTC CAG GTC CYG CC 3' 8826-8845. R8C -5' GAA TGG CGT TGG CAA ACC GG 3' 8968-8949), authors 13 have demonstrated that the method was highly specific amplifying all the nine strains of rubella virus (including 4 vaccine strains). No false positive PCR products were generated from the RNAs of 16 other RNA viruses or RNAs from 7 cell types or human DNA.…”