Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA

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.

Comparison of the Membrane-Filtration Fluorescent Antibody Test, the Enzyme-Linked Immunosorbent Assay, and the Polymerase Chain Reaction to Detect Renibacterium Salmoninarum in Salmonid Ovarian Fluid

“…The internal primers used in the second round amplification also act as an internal control by confirming the presumptive product of the initial amplification, thereby minimizing the opportunity for false-positive reactions. 36 The correlation between ELISA absorbances and MF-FAT counts sug-gests that the ELISA was not compromised by falsepositive reactions, and the detection of R. salmoninarum by the nested PCR in ovarian fluids categorized as negative on the basis of the ELISA confirms an earlier finding that an as yet uncharacterized factor associated with ovarian fluid affects detection of the soluble antigenic fraction by the ELISA.…”

In a Transgenic Model of Spontaneous Autoimmune Diabetes, Expression of a Protective Class II MHC Molecule Results in Thymic Deletion of Diabetogenic CD8+ T Cells

“…To determine whether or not transcription of the InsHA transgene was occurring in the thymus, the highly sensitive technique of nested RT-PCR was performed (35). Total nucleic acid was isolated from the thymus of InsHA mice.…”

Determination of clonality in patients who present with diagnostic dilemmas: a laboratory experience and review of the literature

“…39 Therefore, negative results by the polymerase chain Thus, in summary, antigen receptor configuration analysis appears to be useful in the context of differentiating clonal reaction should be interpreted with some caution. A few reports have documented false positive results by proliferative disorders from reactive cases.…”