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Concern about the possible secondary spread of variant Creutzfeldt-Jakob disease (vCJD) through blood transfusion and blood products has increased the need for a sensitive and rapid test for the identification of PrP(Sc) in specimens collected non-invasively from living persons. Furthermore, an accurate estimate of the prevalence of pre-clinical vCJD in the British population would be possible if there were such a test that could be applied to specimens available readily (e.g. blood and urine). As a first step towards that goal, we have developed a simple and sensitive test for the detection of PrP(Sc) in peripheral tissues and brain of vCJD patients, based on the differential extraction of PrP(Sc) with guanidine hydrochloride. The prion protein (PrP) isoforms are extracted sequentially from homogenized tissue by applying two different concentrations of this chaotropic agent. Each extraction yields a fraction of the PrP isoforms with different solubilities in guanidine hydrochloride. Quantitation of the two fractions (relatively insoluble or relatively soluble) using time resolved fluorescence (DELFIA) as a reporter system allows differentiation between PrP(Sc) infected and non-infected tissues. The assay has a detection limit of 10 pg PrP, is robust and could be automated.

A heteroduplex mobility assay (HMA) using 753 and 446 base pair (bp) amplicons of the p17/p24 region of the gag gene of HIV-1 has been developed and validated with reference clones and clinical samples representative of subtypes A, B, C, D, E, G, and H. There was complete concordance between the gag HMA assigned subtype and the subtype known from gag or env sequence data or env HMA. The heteroduplexes from both amplicons can be clearly resolved on either MetaPhor XR agarose or MDE polyacrylamide gels. The MetaPhor XR gel system was the more convenient and is the preferred choice for routine HMA subtyping. This gag HMA provides a rapid, simple and inexpensive method for subtyping HIV-1 based on a genomic region other than the commonly used env gene target. The incorporation of gag HMA into subtype determination algorithms should allow the detection of gag/env recombinant strains of HIV-1.

A nested PCR was designed using primers from the pol and tax genes of human T-cell leukaemia virus type I (HTLV-I). The assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1 x 10(5) Peripheral blood mononuclear cells (PBMCs). Using serial dilutions of sample DNA, the assay was applied prospectively to study proviral load in patients with HTLV-associated disease and carriers. The median proviral load expressed as number of copies/100 PBMCs was found to be 14.0 copies in patients with HAM and 1.55 copies in initially asymptomatic carriers. The assay was used to test for low proviral load in subjects who may have HTLV-I infection, and to monitor response to therapy.

The feasibility of using DNA heteroduplex mobility analysis (HMA) as a rapid and reproducible method for routine subtyping HIV-1 in clinical specimens was examined by comparison with subtype determination by sequencing in both the gag and env genes. The heteroduplexes formed were examined by conventional polyacrylamide gel electrophoresis (PAGE) and also by electrophoresis in the Pharmacia PhastSystem. The significance of the HMA results was determined by the Kruskal-Wallis test, a non-parametric one way analysis of variance. It was possible to obtain an HMA profile rapidly (1-2 days) using fast PCR conditions and the PhastSystem. The HMA bands were generally sharper and more satisfactory on the Phast gels than on conventional polyacrylamide gels and the use of Phast gels was an improvement over conventional PAGE. Non-B subtype viruses could be distinguished from B subtypes, but it was more difficult to distinguish between the non-B subtypes and to assign a subtype to them. Thus, HMA can be adapted to offer a rapid screening method for HIV-1 subtyping, but sequencing is still necessary to assign a definitive subtype. This reflects the empirical nature of the subtype definitions and the quasispecies nature of the HIV genome population.

The Roche Amplicor PCR kit was used to detect HIV-1 DNA in UK patients of known serostatus. Four false-negative and/or equivocal results were obtained from patients who were known to be anti HIV seropositive (Tosswill et al., 1994). Cells from the blood of these patients were shown to contain HIV DNA after extraction, concentration and amplification by nested PCR using primers flanking those in the kit. To determine whether DNA sequence divergence was the cause of these discrepancies, the gag region targeted by the primers in the kit was sequenced for specimens giving positive, equivocal and false-negative results. No greater degree of sequence divergence was found within the primer and probe target regions among the equivocals and false-negatives than among the positive control specimens. The few misleading results were probably attributable to low copy numbers of proviral DNA in these specimens. Sequences obtained from the target and flanking regions of the kit were sufficient to allow the genotype of the virus to be determined.

We have used a simple method for detecting HIV-1 in the serum of infected individuals using the polymerase chain reaction (PCR). This is useful if only serum, or other specimens which would not be expected to harbour proviral DNA, is available for diagnostic testing. Viral RNA present in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any proteins present, specifically ribonucleases. After washing the RNA/silica pellet, the RNA is eluted in water and reverse transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is amplified using nested PCR and the products of amplification are detected by gel electrophoresis and ethidium bromide staining. The identity of bands on the gel is confirmed using a digoxigenin-labelled oligomer probe. The method is a general one applicable to amplification of any RNA species.

A simple, sensitive and specific method using the polymerase chain reaction (PCR) for amplification of human immunodeficiency virus type 1 (HIV-1) is described. The method involves minimal manipulations. Peripheral blood mononuclear cells (PBMC) were prepared by a rapid Ficoll-Paque gradient method. Lymphocytes were lysed in PCR buffer containing Proteinase K and detergents, and subjected to amplification under stringent conditions, using two primer pairs. Amplified DNA sequences were hybridized with a 3'-end labelled probe, electrophoresed on agarose gels and visualised by ethidium bromide staining. Identification of amplified HIV-1 proviral DNA sequences was confirmed by autoradiography. HIV-1 sequences were amplified in all samples from 103 HIV-1 seropositive individuals, but not in 40 HIV-1 seronegative controls. The absence of contamination may be attributable in part to minimisation of manipulations before amplification.

The polymerase chain reaction (PCR) is a powerful method for the in vitro amplification of specific nucleic acid sequences. As very small amounts of a virus genome can be detected it has obvious diagnostic applications. The background to the reaction and its use for human immunodeficiency virus (HIV) detection are described. The problems likely to be encountered in using PCR as a diagnostic assay (false positives and negatives) and the practical measures which can be taken to overcome them are discussed.