Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization

Abstract

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.

Evaluation of SHARP signal system for enzymatic detection of amplified hepatitis B virus DNA

“…Alternative formats involving microplate hybridization of amplified products have been developed, and some have been applied to the detection of HBV DNA. The microplate sandwich hybridization assay described by Keller et al (7), for example, provides a high degree of sensitivity (50 HBV genomes per ml), but the hybridization reaction requires 4 h (whereas it was 30 min in the present study), one false-positive reaction was recorded, and the assay has not yet become available in kit form. In the DNA enzyme immunoassay, the hybrids that form between amplified DNA and a biotinylated probe bound to an avidin-coated plate are detected enzymatically with a monoclonal antibody to double-stranded DNA.…”

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

“…The duration of the post-PCR processing is 5.5 h, and many steps could be further automated. With a 5.5-h duration, the assay does not take more time than most rapid other tests (10,13), and post-PCR workup can be performed more quickly than several other procedures (8,19,21,31). If a thermocycler with quick temperature changes is used, the whole test can probably be accomplished in 1 day.…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…Whilst Southern blot detection of amplicon by hybridisation with a labelled oligonucleotide probe (oligoprobe) increases the specificity of amplicon detection, it is time consuming, frequently uses radioactive labels and requires multiple PCR product handling steps, increasing the risk of spreading amplicon throughout the laboratory (Holland et al, 1991). Alternatively, PCR -ELISA has been used to capture amplicon onto a solid phase via biotin or digoxigenin-labelled primers, oligoprobes or by direct capture after incorporation of the biotin or digoxigenin into the amplicon (van der Vliet et al, 1993;Keller et al, 1990;Kemp et al, 1990;Kox et al, 1996;Dekonenko et al, 1997;Watzinger et al, 2001). Once captured, amplicon is detected using an enzyme-labelled avidin or anti-digoxigenin reporter molecule in a manner similar to a standard ELISA format.…”