Quantitative PCR for hepatitis B virus with colorimetric detection.

A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.

Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal disease caused by inadequate aspartylglucosaminidase (AGA) activity. The disease is prevalent in the genetically isolated Finnish population. We have used a new method, solid-phase minisequencing, to determine the frequency of two missense mutations in the AGA gene in this population. In samples from 70% of the Finnish AGU families, we found that the two nucleotide changes were always associated, and they were identified in 98% of the AGU alleles analyzed. Thus, the high prevalence of AGU in the Finnish population is the consequence of a founder effect of one ancient mutation. The identification of asymptomatic carriers by the minisequencing test proved to be unequivocal. The method also allowed quantification of a mutated nucleotide sequence present in less than 1% of a sample. The frequency of AGU carriers in this population was 1/36 when estimated by quantifying the mutated AGU allele in a pooled leukocyte sample from 1350 normal Finnish individuals.

N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing.

Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.

A rapid and quantitative solution hybridization method for detection of HBV DNA in serum.

A sensitive and convenient solution hybridization technique was adapted for the semiquantitative detection of hepatitis B virus DNA in serum. The assay utilizes 35S-isotope as label and biotin-avidin interaction for collection of the hybrids onto microtitre plate wells. Results are obtained as numerical values, which allow quantification. 10(6) molecules of HBV DNA/ml serum could be detected by a 3-h hybridization followed by a 2-h collection reaction. By analyzing 500 microliters of serum, 30 (88%) of 34 patient sera positive for HBeAg were also positive for HBV DNA and 17 HBsAg-positive sera, 16 of which were anti-HBe-positive, were DNA-negative. The amount of HBV DNA varied from 5 x 10(6) to 3 x 10(9) molecules/ml. The solution hybridization method which was developed allows fast and accurate quantification of HBV DNA in serum providing an estimate of the virus titre.

Quantification of polymerase chain reaction products by affinity-based hybrid collection.

We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.