Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.

Mismatch cleavage detects base deletion in cystic fibrosis gene.

The delta F508 is the most common defect in the cystic fibrosis (CF) gene; it involves in a 3-base deletion in codon 508 and results in the loss of a phenylalanine residue at amino acid position 508. Our previous results have shown the mismatch enzyme cleavage at the mismatch of a DNA duplex in identifying a specific DNA sequence or a point mutation. The assay is simple and reliable. By manipulating the melting temperature (Tm) for the hybrids of the DNA targets and the deoxynucleotide probes, the mismatch cleavage assays are able to detect the most common defective CF gene, delta F508. The assays with a delta F508 and a normal wild-type probe can differentiate the three genotypes, i.e., delta F508/delta F508, delta F508/normal and normal/normal. Furthermore, the addition of ammonium acetate amplifier to the assay for recycling the target DNA can increase the sensitivity to a level that is sufficient to detect the mutated target in a few micrograms of genomic DNA without the aid of PCR amplification. The detection of the base deletion, the amplification of sensitivity and the differentiation among the genotypes of normal, carrier delta F508 and mutant delta F508 suggest the useful application of mismatch cleavage in genetic diagnosis at the DNA level.

Nontuberculous mycobacteria in adult patients with cystic fibrosis.

Because patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual microorganisms, we screened the sputum of adult CF patients for mycobacterial organisms. Acid-fast bacilli (AFB) smears and mycobacterial culture were performed on 297 sputum specimens from 87 patients. Cultures for mycobacteria were frequently overgrown with other bacteria; 22.6 percent of cultures were contaminated. Despite this limitation of mycobacterial culture, 17 patients had at least one positive culture for a Mycobacterium other than tuberculosis (MOTT). Eleven patients were positive for Mycobacterium avium-intracellulare (MAI), two for MAI and M chelonei, three for M chelonei, and one for M fortuitum. None was positive for M tuberculosis. Patients with CF with MOTT were similar to patients with CF without MOTT; only a slightly different (older) age distribution was recognized. The clinical significance of MOTT was difficult to determine in any individual patient, but patients with positive AFB smears appeared more likely to suffer pathogenic effects. We conclude that MOTT is frequently recovered from adult CF patients in the southeastern United States. A specific risk factor for colonization and/or pathogenic infection in this patient group was not evident. The general prevalence and clinical pathogenesis in CF patients in the United States remains to be determined.

Improved enzymatic hydrolysis of conjugated bile acids.

The hydrolysis of conjugated bile acids by cholylglycine hydrolase (EC 3.5.1.24) using the standard procedure for a commercial enzyme preparation was found to be incomplete, as judged by the use of 24-14C-taurocholic acid as a tracer. A method is proposed that incorporates the nonionic detergent Triton X-100 into the reaction mixture to achieve almost complete hydrolysis. It is proposed that the observed enhancement of enzyme activity is due to the formation of micelles by the detergent.

Neonatal screening for biotinidase deficiency.

Children with juvenile-onset multiple carboxylase deficiency lack biotinidase activity (biotinamide amidohydrolase, EC 3.5.1.12) in the liver and other tissues. Hence, little free biotin is metabolically available, resulting in seizures, acidosis, and serious neurological damage. As the absence of hepatic biotinidase activity is reflected in serum, assessment of biotinidase status can easily be made from a blood sample. A convenient qualitative procedure for screening infants has been employed in order to estimate serum levels of biotinidase in as little as 10 microliters of sample. This colorimetric procedure detects the formation of free p-aminobenzoate cleaved from the substrate, N-biotinyl-p-aminobenzoate at pH 6.0. The assay is easily performed and has a low incidence of false positive results. A kinetic assay for serum biotinidase has also been developed using biotinyl-p-nitroanilide (BpNA) as substrate. When 50 microliters of biotinidase positive serum was incubated with 0.2 mM BpNA in phosphate buffer at pH 6.0, an increase in absorbance was observed at 405 nm. The rate of change in absorbance was followed kinetically on the Roche Cobas BIO analyzer at 37 degrees C. Monitoring the increase in absorbance of para-nitroanilide every 60 seconds over 30 minutes demonstrated linearity from 10 to 30 minutes. In comparing results from this kinetic assay on 48 randomly selected sera with those obtained using a colorimetric procedure, a correlation coefficient of 0.85 was obtained. Several false positive results were observed in clearly lipemic sera.

Detecting multiple cystic fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis.

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis.

We have developed a method for processing and detecting fungi in clinical specimens using the polymerase chain reaction (PCR) methodology. This PCR amplification of a segment of a ribosomal DNA gene results in a 310 bp product. The gene sequences used as amplimers have been highly conserved throughout the fungal kingdom and positive PCR results have been obtained for all genera and species of fungi tested (n = 42). Neither human nor a variety of pathogenic bacteria (n = 24) gave an amplified product. This PCR method can detect as few as 15 cells of Candida albicans in clinical specimens following a simple processing procedure. The sensitivity of the PCR for detecting other fungi remains to be determined. Analysis of the 310 bp amplified product, following digestion with the restriction enzyme HaeIII, can be used to further characterize the identity of the fungus involved into the following five groups: (i) Candida species and closely related yeasts; (ii) Cryptococci and Trichosporon species; (iii) Aspergilli and clinically related septate molds; (iv) the Zygomycetes; and (v) the dimorphic fungi.

Differences in gene expression between B-cell chronic lymphocytic leukemia and normal B cells: a meta-analysis of three microarray studies.

MOTIVATION: A major focus of current cancer research is to identify genes that can be used as markers for prognosis and diagnosis, and as targets for therapy. Microarray technology has been applied extensively for this purpose, even though it has been reported that the agreement between microarray platforms is poor. A critical question is: how can we best combine the measurements of matched genes across microarray platforms to develop diagnostic and prognostic tools related to the underlying biology? RESULTS: We introduce a statistical approach within a Bayesian framework to combine the microarray data on matched genes from three investigations of gene expression profiling of B-cell chronic lymphocytic leukemia (CLL) and normal B cells (NBC) using three different microarray platforms, oligonucleotide arrays, cDNA arrays printed on glass slides and cDNA arrays printed on nylon membranes. Using this approach, we identified a number of genes that were consistently differentially expressed between CLL and NBC samples.

Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection.

Single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are popular electrophoretic methods for the identification of sequences. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Here we review the theory and practice of SSCP and HA, including the factors contributing to the sensitivity of mutation detection. For SSCP analysis, these factors include: choice of gel matrix, electrophoretic conditions, presence of neutral additives, fragment size, and G+C content For HA, the principle factors influencing sensitivity are the gel matrix and the identity of the base mismatch.

Quantitative measurement of telomerase activity in lymphadenopathy: correlation with histologic features and human immunodeficiency virus-1 infection.

Telomerase is a ribonucleoprotein that uses its internal RNA component as a template for synthesis of telomeric DNA on the ends of chromosomes after each round of cell division. It is expressed in approximately 90% of all human cancers tested to date, as well as in most immortal cell lines. Recently, telomerase activity was detected in normal proliferating lymphoid tissue and in non-Hodgkin's lymphomas (NHLs) by use of the telomeric repeat amplification protocol assay, a qualitative measure of telomerase activity. In this study, we modified the assay to measure quantitatively the telomerase activity in lymph node biopsy specimens obtained from patients with lymphadenopathy. The lymph nodes either contained benign reactive changes, were involved by NHL of B-cell lineage, or were involved by Hodgkin's disease. Telomerase activity was detected in all of our samples, benign as well as malignant. The levels of activity were unaffected by the patient's human immunodeficiency virus-1 status. Although the specimens involved by NHLs showed a range in telomerase activity from low to high, the levels did not correlate strictly with the histologic grade according to the Working Formulation. All of the cases of Hodgkin's disease also expressed telomerase activity, and the levels were similar regardless of histologic subtype. Our results showed that telomerase activity was expressed in both benign and malignant lymphoproliferative processes.

Heteroduplex generator in analysis of Rh blood group alleles.

We describe the use of heteroduplex analysis to enhance the resolution of different rhesus-derived (Rh) isotypes. Heteroduplex analysis of different domains of the Rh D and Rh CE loci can be performed to diagnose a variety of blood group incompatibilities. One application of this technique is the ability to test for fetal-maternal blood group incompatibilities during pregnancy. Several new serotype-specific sequence variations were discovered in the mapping of the Rh locus, and used in the construction of artificial heteroduplex generators (HGs). HGs facilitate the resolution of the Rh isotypes by electrophoretic methods.

Optimization, assessment, and proposed use of a direct nested reverse transcription-polymerase chain reaction protocol for the detection of hepatitis C virus.

OBJECTIVES: To compare the performance of reverse transcription followed by the polymerase chain reaction (RT-PCR), without RNA purification, with the performance of classic protocols. STUDY DESIGN/METHODS: Direct and classic techniques were used to test three groups of samples: six hepatitis C virus (HCV) seroconversion panels (n = 90), a HCV RNA reference panel (n = 26), and serial dilutions of four HCV-positive sera (n = 24). These methods were then applied sequentially through a clinical diagnostic algorithm to test 268 samples from high-risk patients. RESULTS: For the three groups of samples, we found a 94% concordance between direct and purified RT-PCR methods. For the detection of HCV RNA in clinical samples, sensitivity was maximized and cost minimized using both protocols according to the proposed algorithm. CONCLUSIONS: The direct PCR method is reliable, sensitive, and can result in time and cost savings. The suggested testing algorithm can enhance sensitivity and time savings for populations with a high prevalence of infection.

Bile acid synthesis by cultured hepatocytes. Inhibition by mevinolin, but not by bile acids.

The ability of cultured hepatocytes to maintain a constant rate of bile acid synthesis and secretion in a chemically defined serum-free culture medium allowed us to examine the direct effects of bile acids on their synthesis. Mass quantitation of bile acids by gas liquid chromatography showed that adding taurochenodeoxycholate at concentrations which were from 2 to 20 times the concentration of bile acids found in rat portal blood increased rather than decreased the secretion of cholic acid. Using a more direct approach to measure relative rates of bile acid synthesis and secretion, we determined the rate of de novo 14C-bile acid synthesis. Adding taurocholate at concentrations ranging from 2 to 60 times the concentration found in rat portal blood did not inhibit 14C-bile acid synthesis. Furthermore, free and conjugated di- and tri-hydroxy bile acids did not inhibit bile acid synthesis. In contrast, mevinolin, a potent inhibitor of cholesterol biosynthesis, inhibited 14C-bile acid synthesis by 63%. Since previous demonstration of bile acid negative feedback regulation was achieved in vivo by infusing taurocholate into the intestines of bile-diverted rats, we examined the possibility that bile acid synthesis must be induced in order for bile acids to inhibit their synthesis. Hepatocytes obtained from bile-diverted rats exhibited a 5-fold increase in the synthesis and secretion of 14C-bile acids. However, taurocholate did not inhibit bile acid synthesis by cells from bile-diverted rats. These data show that bile acids do not interact directly with the hepatocyte to inhibit their synthesis.

A model for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic fibrosis.

Gene mutations responsible for the majority of Duchenne/Becker muscular dystrophy (DMD/BMD) and cystic fibrosis (CF) chromosomes have been identified. We describe a DNA-based strategy, rather than the traditional biochemical assays, for screening newborns. DNA sequences spanning the CF mutation and several DMD/BMD deletion-prone exons are amplified simultaneously via a multiplex polymerase chain reaction. The gel is visually inspected for DMD/BMD deletions and then blotted and hybridized with allele-specific oligonucleotides to determine the presence or absence of the CF mutation. We determined that blood spots provide sufficient DNA for the molecular analysis, so the procedure can be used in screening programs of newborns.

Molecular probe protocol for determining carrier status in Duchenne and Becker muscular dystrophies.

By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.

Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single-base changes, with the GC content being the predominant determinant of detection sensitivity.

Determination of carrier status in Duchenne and Becker muscular dystrophies by quantitative polymerase chain reaction and allele-specific oligonucleotides.

Detection of carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), in the deletion cases, involves calculating gene dosage from Southern blots. We show that the analysis of dosage can also be made from the polymerase chain reaction (PCR) with use of allele-specific oligonucleotides (ASOs). The deletion-prone exons are amplified, transferred to a membrane, and hybridized with ASOs complementary to the exons; the autoradiographic bands are then quantified with a densitometer. After determining the quantitative conditions of the amplification reaction, we were able to identify deletions in a DMD/BMD carrier female. The determination of carrier status via PCR removes several of the technical limitations of Southern analysis and is also cost- and labor-effective.

Sources of nonlinearity in cDNA microarray expression measurements.

BACKGROUND: A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides. RESULTS: We uncovered two sources of nonlinearity. The first, which led to discrepancies in analysis affecting the fluorescent signals, was signal quenching associated with excessive dye concentrations. The second, affecting the radioactive signals, was a nonlinear transformation of the raw data introduced by the scanner. Correction for this transformation was made by some, but not all, image-quantification software packages. CONCLUSIONS: The second type of nonlinearity is more troublesome, because it could not have been predicted a priori. Both types of nonlinearities were detected by simple dilution series, which we recommend as a quality-control step.