DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction.

Abstract:

:We developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids and applied it to the detection of hepatitis B virus (HBV) DNA amplified from serum samples by means of the polymerase chain reaction (PCR) technique. The method is based on the ability of an anti-DNA monoclonal antibody to discriminate between single-stranded and double-stranded DNA. A solid phase was coated with a specific oligonucleotide probe, internal to the amplified region of HBV DNA, via an avidin-biotin bridge. The denatured PCR product was hybridized with the solid-phase probe, and the amplified DNA probe hybrid was then incubated with a monoclonal antibody specific for double- but not single-stranded DNA. Colorimetric detection of the DNA-antibody complex was achieved by adding an anti-mouse Ig antibody labeled with horseradish peroxidase. The combined use of DEIA and PCR can reveal a few HBV genome copies present in a serum sample. This method has several advantages: (a) the sensitivity is adequate for the detection of amplified DNA; (b) the signal is associated with the hybridization event, independently of modifications of the probe or of the amplification primers; and (c) the test is simple and rapid and, most importantly, requires only the standard facilities of a routine clinical laboratory.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Mantero G,Zonaro A,Albertini A,Bertolo P,Primi D

subject

Has Abstract

pub_date

1991-03-01 00:00:00

pages

422-9

issue

3

eissn

0009-9147

issn

1530-8561

journal_volume

37

pub_type

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