Quantitative reverse transcription-PCR assay with an internal standard for the detection of prostate-specific antigen mRNA.

BACKGROUND: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. METHODS: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. RESULTS: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to approximately 1-100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mL of blood for the 2 males]. CONCLUSIONS: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry.

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

Population screening for the common G985 mutation causing medium-chain acyl-CoA dehydrogenase deficiency with Eu-labeled oligonucleotides and the DELFIA system.

We have screened 10171 neonatal blood spots from the Trent and West Midlands regions of the UK for the common G985 mutation to more accurately establish the incidence of medium-chain acyl coenzyme (Co)A dehydrogenase (MCAD) deficiency. We have used a technique involving PCR and Eu-labeled allele-specific oligonucleotides detected by using time-resolved fluorometry on the dissociation-enhanced fluorescence immunoassay (DELFIA) system for the detection of the G985 mutation. We have also evaluated the feasibility of neonatal screening with this technique. We identified 158 G985 heterozygotes and no G985 homozygotes. The calculated incidence of MCAD deficiency in the population studied (all mutations, assuming 90% of MCAD mutations are G985) is 1 in 13426 (95% confidence limits 1 in 10070-1 in 18791). At the optimum cutoff criteria, the technique has a sensitivity of 97.5%, specificity of 99.6%, and positive predictive value of 80.2%. We conclude that this study confirms that MCAD deficiency is a common inherited metabolic disease and is a candidate for neonatal screening. The methodology used is robust and suitable for large-scale population studies such as this. The technique is also potentially suitable for screening.

Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism.

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes.

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph1). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new sensitive hybridization method combined with polymerase chain reaction (PCR), based on the detection of the europium (Eu3+) label by time-resolved fluorescence, was applied for the detection of Ph1 chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA synthesis by reverse transcriptase, the PCR amplification (30 cycles) was carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was measured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukocytes could be detected. Six patients with CML confirmed by clinical and cytogenetic criteria were studied. Three of the patients underwent an allogeneic BTM 6-18 months before the investigation and all of them were Ph1-negative. The other three patients who were nontransplanted were positive as expected.

Time-resolved fluorometry in the genetic diagnosis of familial defective apolipoprotein B-100.

A novel technique for screening point mutations has been developed for diagnosis of familial defective apolipoprotein (apo) B-100 (FDB). In FDB, an amino acid exchange occurs at position 3500 in apoB-100 due to a point mutation. Polymerase chain reaction (PCR) was performed on the appropriate region of the apoB gene, and the PCR products were hybridized in solution with europium-labeled oligonucleotides, complementary to either the wildtype or the mutant genome. The presence or absence of the apoB-3500 mutation was monitored by time-resolved fluorescence of the europium chelate. The method allows a larger number of samples to be processed simultaneously, and the detection system displays a high level of sensitivity without the hazards connected to the use of radioactivity. When 127 Swedish patients, clinically diagnosed as suffering from heterozygous familial hypercholesterolemia, were screened for the presence of the apoB-3500 mutation, two patients, unrelated to each other, were found to be heterozygotes. These patients are the first reported cases of FDB from Sweden, and the frequency rate observed among hypercholesterolemic patients, 1.6%, is in accordance with the figures reported for several other patient population in Europe and the United States.

Simultaneous detection of two cystic fibrosis alleles using dual-label time-resolved fluorometry.

A simple dual-label hybridization test for normal and mutant cystic fibrosis (CF) alleles is described. The assay is based on time-resolved fluorometry (TRF), which allows the simultaneous detection of DNA probes labelled with different lanthanides from one hybridization reaction. DNA was liberated from dried blood disks, normally used in neonatal screening programmes, by boiling in alkaline solution. A 138 bp region including the site of deletion, delta F-508, which is present on about 70% of cystic fibrosis chromosomes, was amplified using the polymerase chain reaction (PCR). The presence or absence of normal and mutant alleles was then determined in a solution hybridization using allele specific oligonucleotide probes labelled either with europium (Eu) or with samarium (Sm) chelates. A common biotinylated probe was used for binding the hybrids onto microtitration wells coated with streptavidin. Some 5 x 10(7) molecules of the normal allele (Eu) and 5 x 10(8) molecules of the mutant allele (Sm) could be detected simultaneously in a single hybridization reaction. The assay was simple to perform and made it possible to reduce the number of hybridizations needed to interpret the sample as being normal, carrier or mutant with regard to the mutation, delta F-508.

Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.

Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry.

Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.