Premium
Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA
Author(s) -
PorterJordan Kathleen,
Rosenberg Eric I.,
Keiser John F.,
Gross Jeffrey D.,
Ross Allan M.,
Nasim Suhail,
Garrett Carleton T.
Publication year - 1990
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.1890300202
Subject(s) - polymerase chain reaction , loop mediated isothermal amplification , biology , nested polymerase chain reaction , microbiology and biotechnology , dna , primer dimer , virology , false positive paradox , contamination , multiple displacement amplification , multiplex polymerase chain reaction , dna extraction , gene , genetics , ecology , machine learning , computer science
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.