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Hemoglobin C (B6 Glu-Lys) can be identified by different techniques. Here we describe a restriction enzyme digestion protocol (BseRI GAGGAG N10) for direct detection of this variant.
Prenatal diagnosis was requested by a family carrying a 3 base-pair insertion in the dihydropteridine reductase (DHPR) coding region. A chorionic villus sample was obtained and fetal DNA was isolated directly from this. Diagnosis was performed by a polymerase chain reaction (PCR)-based technique, with a simple electrophoretic assay for the insertion. The fetus was found to be heterozygous for the insertion. This is the first time that prenatal diagnosis of DHPR deficiency has been performed by direct detection of the mutation.
A total of 234 urogenital specimens were investigated with a radioactive and a nonradioactive rRNA:cDNA-hybridization test for the direct detection of Chlamydia trachomatis (Gen-Probe, San Diego, Calif.). The results were compared with those yielded by the conventional cell culture technique (McCoy cells). Using cell culture a urogenital C. trachomatis infection could be detected in 37 of 234 patients (15.8%) at two outpatient sexually transmitted disease clinics. Referred to the detection rate, cell culture sensitivity was 83.8% for the radioactive test and 86.5% for the nonradioactive DNA-hybridization test. The specificity of the radioactive test was 94.8% and that of the nonradioactive test, 93.4%.
The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were designed to target the nuclease (nuc) and the attachment invasion locus (ail) genes of S. aureus and Y. enterocolitica, respectively and used in PCR. A combination of organic solvents, detergents and alkali in the DNA extraction method permitted a detection limit of 10 cfu ml(-1) milk for both the pathogens. When equal numbers of S. aureus and Y. enterocolitica were spiked in milk samples, the individual detection limit was determined to be 10(3) cfu ml(-1) milk. Simultaneous amplification of 482 and 359 bp fragments of the nuc and ail genes was obtained using the primer pairs in a single reaction. Multiplex PCR enabled the detection of 10(4) cfu ml(-1) milk of S. aureus and Y. enterocolitica without any pre-enrichment step. A combination of conventional isolation technique and PCR using DNA extracted by the proposed method was used to test raw milk samples for possible contamination with S. aureus and Y. enterocolitica. The presence of S. aureus in the tested samples was indicated by both the methods while Y. enterocolitica could not be detected in any of the samples. The template DNA extraction method developed in this study is rapid, sensitive and avoids interference from potential PCR inhibitors and demonstrates the potential of detecting multiple pathogens in milk samples without any enrichment.
Homocysteine (Hcy) is a biomarker for significant disorders including Alzheimer's and cardiovascular disease. The monitoring of Hcy levels in plasma is of current concern. We describe highly selective colorimetric methods for the direct detection of Hcy. Inexpensive, commercially available materials are employed. The results show potential application for the detection of Hcy in human blood plasma.
A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degrees C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.
An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.
A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.
A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.
BACKGROUND: Pseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum. METHODS: Adult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus. RESULTS: By sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4-17 months later, with a mean lag time of 4.5 months. CONCLUSIONS: This study indicates that molecular detection of PA in sputum employing the oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.
BACKGROUND: Phenylketonuria is as regards the genotype a very heterogenous disease. Successful prenatal and postnatal DNA diagnosis calls for knowledge of different mutations in a given population. The objective of the investigation was to introduce direct detection of 21 mutations in the gene for phenylalanine hydroxylase and to find the distribution and frequency of these mutations in the population of northern and southern Moravia. METHODS AND RESULTS: The authors analyzed a group of 95 patients where according to phenotypic classification classical phenylketonuria was involved which comprised 190 mutant alleles. The presence of mutations was assessed by means of a polymerase chain reaction of a Perkin Elmer DNA Thermal Cycler 480. From the total number of 21 mutations which were sought, 11 were identified in our population, which accounts for 80% of all mutations. It was revealed that mutation R408W is found in 55.3% of our patients. Twenty per cent of the mutations are still unknown. CONCLUSIONS: This investigation laid the foundations for direct DNA diagnosis of phenylketonuria in the Czech Republic. The results assembled in the Moravian region suggest that our population is as regards genotypes relatively homogenous. This gives great hope of successful prenatal diagnosis and postnatal genotype classification.
A new approach using combined liquid chromatography-mass spectrometry (LC-MS) with ionspray ionization is proposed for the direct detection of known and new toxins in mussels and phytoplankton. A first stage reversed-phase, negative ion mode, selected ion monitoring (SIM) LC-MS analysis was performed in order to detect DSP toxins in the same chromatographic run with a total run time of 20 min. The toxins analysed included yessotoxin (YTX), okadaic acid (OA) and four of its analogues, dinophysistoxins (i.e. DTX-1, DTX-2, DTX-2B, DTX-2C), and pectenotoxins (PTXs), involving PTX-2, two PTX-2 secoacids (PTX-2SAs), PTX-2SA, 7-epi-PTX-2SA, and AC1, the three isomeric toxins structurally related to PTX-2 recently identified in Irish phytoplankton. Positive samples can, therefore, be analyzed through reversed-phase, positive ion mode SIM LC-MS, in order to perform complete chromatographic separations of the structurally related toxins within the OA and PTX groups. Detailed toxin profiles of a number of toxic phytoplankton and shellfish, from different marine areas, were easily obtained through the new approach. PTX-2SAs and AC1 were found in phytoplankton and shellfish from Ireland as well as in Italian shellfish. Moreover, for the first time there was evidence of the presence of PTX-2 in Irish phytoplankton. YTX was present in Italian shellfish. Four isomeric OA toxins were detected in samples from Ireland with OA, DTX-2 and DTX-2B present in shellfish, and OA, DTX-2 and DTX-2C in phytoplankton. In contrast, OA was the only toxin from this group to be detected in Italian mussels.
The potential for development of a cost-effective protocol for selective use of the Gen-Probe probe competition assay (PCA) in conjunction with PACE 2 for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens that would not compromise patient care was investigated. To accomplish this, PCA data from testing performed over 12 months were retrospectively reviewed. Of 237 samples that were presumptively positive for C. trachomatis by initial probe assay and could be tested by PCA, positive PCA results were obtained for 100, 79, and 59%, respectively, of specimens that gave a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 relative light units (RLU). For the 141 specimens that were presumptively positive for N. gonorrhoeae and could be tested by PCA, positive PCA results were obtained for 99, 80, and 42%, respectively, of samples with a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 RLU. These data indicate that PCA should be a routine supplement to Gen-Probe PACE 2 for specimens with an initial signal by probe assay of less than 1,500 RLU and may not be necessary for samples yielding a signal of more than 1,500 RLU.
Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.
Histoplasmosis is the most common pulmonary mycosis in the United States. The responsible fungal pathogen, Histoplasma capsulatum, grows in soils contaminated with bird or bat droppings. Inhalation of dust from contaminated areas containing H. capsulatum spores is a primary route of infection. The ability to detect H. capsulatum in soil samples has been limited by the lack of fast, reliable and inexpensive methods. A polymerase chain reaction (PCR) method was developed that allows the direct detection of H. capsulatum in soil. A two-stage PCR protocol was followed employing both fungal-specific primers and nested primers specific for the internal transcribed spacer (ITS) region of the 5.8S rRNA gene of H. capsulatum. The estimated limit of detection of this method is 10 spores. In contrast to the more expensive and indirect mouse inoculum assay, which requires 6-8 weeks for sample analysis, PCR analysis of soil contaminated with H. capsulatum can be completed in less than 2 days.
A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.
OBJECTIVE: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients. METHODS: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS. RESULTS: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC. CONCLUSIONS: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.
1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.