Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA
- 1 February 1990
- journal article
- research article
- Published by Wiley in Journal of Medical Virology
- Vol. 30 (2), 85-91
- https://doi.org/10.1002/jmv.1890300202
Abstract
The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.Keywords
This publication has 26 references indexed in Scilit:
- Use of the Polymerase Chain Reaction for Early Detection of the Proviral Sequences of Human Immunodeficiency Virus in Infants Born to Seropositive MothersNew England Journal of Medicine, 1989
- Avoiding false positives with PCRNature, 1989
- Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.JCI Insight, 1989
- Early diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA-DNA hybridisation assayJournal of Medical Virology, 1989
- Enzymatic Gene Amplification: Qualitative and Quantitative Methods for Detecting Proviral DNA Amplified in VitroThe Journal of Infectious Diseases, 1988
- Detection of Cytomegalovirus DNA in Peripheral Blood of Patients Infected with Human Immunodeficiency VirusThe Journal of Infectious Diseases, 1988
- Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA PolymeraseScience, 1988
- Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaScience, 1985
- Human Cytomegalovirus DNA Sequences with Homologies to the Cellular GenomeJournal of General Virology, 1984
- Homology between mammalian cell DNA sequences and human herpesvirus genomes detected by a hybridization procedure with high-complexity probeCell, 1982