Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay

Abstract

The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.

Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy

“…First, the DNA fragment encompassing the mutation site is amplified with biotinylated primers according to a standard PCR protocol (step 1). The double-stranded biotinylated PCR product is then collected onto streptavidincoated microtitration wells prepared as described by Dahlen et al (1991) (step 2)) and denatured with alkali (step 3). In screening for a specific point mutation, two separate solution hybridization reactions are carried out using allele-specific Eu-labelled oligonucleotide probes (step 4).…”

“…To introduce reactive sites for the labelling with Eu, a 20nucleotide long tail of modified cytidines was inserted at the 5'-ends of the probes (Sund et al, 1988). The labelling with Eu was performed as described by Dahlen et al (1991). The mutationspecific MUT probe had 15 Eu chelates per oligonucleotide, and the wild-type specific WT probe had 19 Eu chelates, respectively.…”

“…In order to introduce a specific and simple diagnostic test for LHON, we have applied a timeresolved fluorometry (TRF) method (Soini and Lovgren, 1987) for the identification of the ND4/ 11778 mutation and for the quantification of heteroplasmy. TRF has earlier been used for the detection of viral DNA in clinical specimes (Dahlen et al, 1988(Dahlen et al, , 1991 and of the mutations in nuclear genes (Iitia et al, 1992a, b). Here we demonstrate that the TRF method can also be used in the detection of mitochondrial point mutations and quantification of heteroplasmy.…”

Advances in the diagnosis of respiratory virus infections

“…We used microtitration strips coated with streptavidin and then with biotinylated probes in our liquid-phase hybridization for the assay of PCR products. However, if streptavidin is coated directly on polystyrene, it is in only partially active form, We coated the strips first with biotinylated bovine albumin followed by streptavidin (Dahlen et al, 1991) and then biotinylated probes. These triple-coated strips can be stored refrigerated for months.…”

Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens

“…Despite the high efficiency of the PCR, direct visualization of amplified HIV-1 DNA in ethidium bromide-stained agarose gels has been hampered by the fact that HIV-1 is present at very low titer in most infected individuals [Sonnerborg et al, 1990;Simmonds et al, 19901. Thus several methods have been described for detection of amplified HIV-1 DNA, mainly relying on solid-phase or solution hybridization of the PCR product to a labeled probe [Abbott et al, 1988;Carman and Williamson, 1989;Kumar et al, 1989;Coutlee et al, 1990;Ou et al, 1990;Dahlen et al, 1991;Keller et al, 19911. Although usually sensitive, most detection systems may be lengthy or require additional equipment and training or yield some indetermi- nate results.…”