Abstract
A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blacklow N. R., Greenberg H. B. Viral gastroenteritis. N Engl J Med. 1991 Jul 25;325(4):252–264. doi: 10.1056/NEJM199107253250406. [DOI] [PubMed] [Google Scholar]
- Cukor G., Blacklow N. R. Human viral gastroenteritis. Microbiol Rev. 1984 Jun;48(2):157–179. doi: 10.1128/mr.48.2.157-179.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eiden J. J., Wilde J., Firoozmand F., Yolken R. Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction. J Clin Microbiol. 1991 Mar;29(3):539–543. doi: 10.1128/jcm.29.3.539-543.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gouvea V., Glass R. I., Woods P., Taniguchi K., Clark H. F., Forrester B., Fang Z. Y. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990 Feb;28(2):276–282. doi: 10.1128/jcm.28.2.276-282.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenberg H. B., Valdesuso J. R., Kalica A. R., Wyatt R. G., McAuliffe V. J., Kapikian A. Z., Chanock R. M. Proteins of Norwalk virus. J Virol. 1981 Mar;37(3):994–999. doi: 10.1128/jvi.37.3.994-999.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenberg H. B., Wyatt R. G., Valdesuso J., Kalica A. R., London W. T., Chanock R. M., Kapikian A. Z. Solid-phase microtiter radioimmunoassay for detection of the Norwalk strain of acute nonbacterial, epidemic gastroenteritis virus and its antibodies. J Med Virol. 1978;2(2):97–108. doi: 10.1002/jmv.1890020204. [DOI] [PubMed] [Google Scholar]
- Jansen R. W., Siegl G., Lemon S. M. Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2867–2871. doi: 10.1073/pnas.87.8.2867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaplan J. E., Gary G. W., Baron R. C., Singh N., Schonberger L. B., Feldman R., Greenberg H. B. Epidemiology of Norwalk gastroenteritis and the role of Norwalk virus in outbreaks of acute nonbacterial gastroenteritis. Ann Intern Med. 1982 Jun;96(6 Pt 1):756–761. doi: 10.7326/0003-4819-96-6-756. [DOI] [PubMed] [Google Scholar]
- Matsui S. M., Kim J. P., Greenberg H. B., Su W., Sun Q., Johnson P. C., DuPont H. L., Oshiro L. S., Reyes G. R. The isolation and characterization of a Norwalk virus-specific cDNA. J Clin Invest. 1991 Apr;87(4):1456–1461. doi: 10.1172/JCI115152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Persing D. H. Polymerase chain reaction: trenches to benches. J Clin Microbiol. 1991 Jul;29(7):1281–1285. doi: 10.1128/jcm.29.7.1281-1285.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robertson B. H., Khanna B., Nainan O. V., Margolis H. S. Epidemiologic patterns of wild-type hepatitis A virus determined by genetic variation. J Infect Dis. 1991 Feb;163(2):286–292. doi: 10.1093/infdis/163.2.286. [DOI] [PubMed] [Google Scholar]
- Rotbart H. A. Enzymatic RNA amplification of the enteroviruses. J Clin Microbiol. 1990 Mar;28(3):438–442. doi: 10.1128/jcm.28.3.438-442.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rychlik W., Rhoads R. E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 1989 Nov 11;17(21):8543–8551. doi: 10.1093/nar/17.21.8543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xi J. N., Graham D. Y., Wang K. N., Estes M. K. Norwalk virus genome cloning and characterization. Science. 1990 Dec 14;250(4987):1580–1583. doi: 10.1126/science.2177224. [DOI] [PubMed] [Google Scholar]