Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Mar;35(3):708–713. doi: 10.1128/jcm.35.3.708-713.1997

Prenatal diagnosis of rubella virus infection by direct detection and semiquantitation of viral RNA in clinical samples by reverse transcription-PCR.

M G Revello 1, F Baldanti 1, A Sarasini 1, M Zavattoni 1, M Torsellini 1, G Gerna 1
PMCID: PMC229655  PMID: 9041417

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.

Full Text

The Full Text of this article is available as a PDF (445.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagnarelli P., Menzo S., Valenza A., Manzin A., Giacca M., Ancarani F., Scalise G., Varaldo P. E., Clementi M. Molecular profile of human immunodeficiency virus type 1 infection in symptomless patients and in patients with AIDS. J Virol. 1992 Dec;66(12):7328–7335. doi: 10.1128/jvi.66.12.7328-7335.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banatvala J. E., Best J. M., O'Shea S., Dudgeon J. A. Persistence of rubella antibodies after vaccination: detection after experimental challenge. Rev Infect Dis. 1985 Mar-Apr;7 (Suppl 1):S86–S90. doi: 10.1093/clinids/7.supplement_1.s86. [DOI] [PubMed] [Google Scholar]
  3. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bosma T. J., Corbett K. M., Eckstein M. B., O'Shea S., Vijayalakshmi P., Banatvala J. E., Morton K., Best J. M. Use of PCR for prenatal and postnatal diagnosis of congenital rubella. J Clin Microbiol. 1995 Nov;33(11):2881–2887. doi: 10.1128/jcm.33.11.2881-2887.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bosma T. J., Corbett K. M., O'Shea S., Banatvala J. E., Best J. M. PCR for detection of rubella virus RNA in clinical samples. J Clin Microbiol. 1995 May;33(5):1075–1079. doi: 10.1128/jcm.33.5.1075-1079.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braun C., Kampa D., Fressle R., Willke E., Stahl M., Haller O. Congenital rubella syndrome despite repeated vaccination of the mother: a coincidence of vaccine failure with failure to vaccinate. Acta Paediatr. 1994 Jun;83(6):674–677. doi: 10.1111/j.1651-2227.1994.tb13111.x. [DOI] [PubMed] [Google Scholar]
  7. Carman W. F., Williamson C., Cunliffe B. A., Kidd A. H. Reverse transcription and subsequent DNA amplification of rubella virus RNA. J Virol Methods. 1989 Jul;25(1):21–29. doi: 10.1016/0166-0934(89)90097-9. [DOI] [PubMed] [Google Scholar]
  8. Cone R. W., Hobson A. C., Huang M. L. Coamplified positive control detects inhibition of polymerase chain reactions. J Clin Microbiol. 1992 Dec;30(12):3185–3189. doi: 10.1128/jcm.30.12.3185-3189.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cradock-Watson J. E., Miller E., Ridehalgh M. K., Terry G. M., Ho-Terry L. Detection of rubella virus in fetal and placental tissues and in the throats of neonates after serologically confirmed rubella in pregnancy. Prenat Diagn. 1989 Feb;9(2):91–96. doi: 10.1002/pd.1970090203. [DOI] [PubMed] [Google Scholar]
  10. Eggerding F. A., Peters J., Lee R. K., Inderlied C. B. Detection of rubella virus gene sequences by enzymatic amplification and direct sequencing of amplified DNA. J Clin Microbiol. 1991 May;29(5):945–952. doi: 10.1128/jcm.29.5.945-952.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerna G., Revello M. G., Dovis M., Petruzzelli E., Achilli G., Percivalle E., Torsellini M. Synergistic neutralization of rubella virus by monoclonal antibodies to viral haemagglutinin. J Gen Virol. 1987 Jul;68(Pt 7):2007–2012. doi: 10.1099/0022-1317-68-7-2007. [DOI] [PubMed] [Google Scholar]
  12. Gerna I., Zannino M., Revello M. G., Petruzzelli E., Dovis M. Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies. J Clin Microbiol. 1987 Jun;25(6):1033–1038. doi: 10.1128/jcm.25.6.1033-1038.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grangeot-Keros L., Pillot J., Daffos F., Forestier F. Prenatal and postnatal production of IgM and IgA antibodies to rubella virus studied by antibody capture immunoassay. J Infect Dis. 1988 Jul;158(1):138–143. doi: 10.1093/infdis/158.1.138. [DOI] [PubMed] [Google Scholar]
  14. Ho-Terry L., Terry G. M., Londesborough P. Diagnosis of foetal rubella virus infection by polymerase chain reaction. J Gen Virol. 1990 Jul;71(Pt 7):1607–1611. doi: 10.1099/0022-1317-71-7-1607. [DOI] [PubMed] [Google Scholar]
  15. Ho-Terry L., Terry G. M., Londesborough P., Rees K. R., Wielaard F., Denissen A. Diagnosis of fetal rubella infection by nucleic acid hybridization. J Med Virol. 1988 Feb;24(2):175–182. doi: 10.1002/jmv.1890240206. [DOI] [PubMed] [Google Scholar]
  16. Morgan-Capner P., Rodeck C. H., Nicolaides K. H., Cradock-Watson J. E. Prenatal detection of rubella-specific IgM in fetal sera. Prenat Diagn. 1985 Jan-Feb;5(1):21–26. doi: 10.1002/pd.1970050105. [DOI] [PubMed] [Google Scholar]
  17. Revello M. G., Baldanti F., Furione M., Sarasini A., Percivalle E., Zavattoni M., Gerna G. Polymerase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection. J Med Virol. 1995 Dec;47(4):462–466. doi: 10.1002/jmv.1890470428. [DOI] [PubMed] [Google Scholar]
  18. Terry G. M., Ho-Terry L., Warren R. C., Rodeck C. H., Cohen A., Rees K. R. First trimester prenatal diagnosis of congenital rubella: a laboratory investigation. Br Med J (Clin Res Ed) 1986 Apr 5;292(6525):930–933. doi: 10.1136/bmj.292.6525.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Thomas H. I., Morgan-Capner P., Enders G., O'Shea S., Caldicott D., Best J. M. Persistence of specific IgM and low avidity specific IgG1 following primary rubella. J Virol Methods. 1992 Sep;39(1-2):149–155. doi: 10.1016/0166-0934(92)90133-x. [DOI] [PubMed] [Google Scholar]
  20. Weber B., Enders G., Schlösser R., Wegerich B., Koenig R., Rabenau H., Doerr H. W. Congenital rubella syndrome after maternal reinfection. Infection. 1993 Mar-Apr;21(2):118–121. doi: 10.1007/BF01710747. [DOI] [PubMed] [Google Scholar]
  21. Zipeto D., Baldanti F., Zella D., Furione M., Cavicchini A., Milanesi G., Gerna G. Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction. J Virol Methods. 1993 Sep;44(1):45–55. doi: 10.1016/0166-0934(93)90006-d. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES