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PubMed1995

Rapid and sensitive streptavidin-biotin amplified fluorogenic enzyme-linked immunosorbent-assay for direct detection and identification of dengue viral antigens in serum.

Each of the four serotypes of dengue viruses is responsible for a spectrum of illnesses that range from nonspecific febrile syndrome with good prognosis to dengue haemorrhagic fever or dengue shock syndrome. Definite diagnosis of dengue is provided by the detection of virus in acute-phase sera of patients. Virus isolation can be accomplished with mosquito cell lines or mosquito inoculations. However, these methods are time consuming and labour intensive. The reverse-transcriptase polymerase chain reaction (RT-PCR) provides a potential means of rapid diagnosis but requires specialised facilities and equipment and is expensive. Therefore a rapid, simple, sensitive, and economical method for direct detection of viral antigens in viraemic sera is needed for clinical and epidemiological investigations. An amplified fluorogenic enzyme-linked immunosorbent assay (F-ELISA) is described for the detection and identification of dengue-3 viruses in serum specimens. This assay utilizes biotinylated mouse IgG antibody directed against dengue antigens captured by anti-dengue monoclonal antibody coated onto polystyrene microplate wells. It takes advantage of the high affinity of biotin for the multivalent binding sites of streptavidin-labelled beta-galactosidase, and combines the amplification effect of biotin-streptavidin interaction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing non-specific binding of proteins and enhancing the specific binding of antigens, F-ELISA was tested on 259 sera submitted routinely to our laboratory for confirmation of dengue diagnosis. The sensitivity of the F-ELISA was 90%, the specificity was 99% and the agreement rate was 98% between F-ELISA and virus isolation results.

Animals↗
PubMed1997

Direct detection of hepatitis B virus gene integrated in the Alexander cell using fluorescence in situ polymerase chain reaction.

Prolonged infection of hepatitis B virus (HBV) is reported to cause hepatocellular carcinoma (HCC) via liver cirrhosis. However, it is still unknown whether the HCC is induced by the HBV DNA integration or by inflammatory stimulation during the phase of liver cirrhosis. The aim of this study is to determine the intracellular or intranuclear distribution of HBV DNA with a highly sensitive assay. Here we directly detected the integration of HBV DNA by fluorescence in situ polymerase chain reaction (FISPCR). Since FISPCR products directly incorporate rhodamine-4-dUTP, the nucleus of Alexander cells integrated with HBV gene reacted with the HBV primers emits obvious fluorescence. The fluorescence values which were measured with an imaging analyzer show a significant difference between Alexander cells as compared to the controls. In conclusion, the target sequences of HBV were specifically amplified as fluorescent DNA after the present FISPCR procedure. This method could provide a novel and simple strategy for determining the quantitative role of viral DNA integration in oncogenesis.

Carcinoma, Hepatocellular↗
PubMed1992

Rapid and direct detection of the most frequent Mediterranean beta-thalassemic mutations by multiplex allele-specific enzymatic amplification.

A rapid nonradioactive method for the diagnosis of the most frequent Mediterranean beta-thalassemic mutations is described based on a multiplex allele-specific polymerase chain reaction (PCR). This method allows direct detection of normal or mutated alleles on genomic DNA. We have used this approach to detect the most frequent Mediterranean mutations: IVS-1 nt 110 (G----A) and 39 nonsense (C----T). For each mutation three allele-specific oligonucleotides were used: one common upstream primer and two downstream primers differing in their terminal 3' nucleotide (one specific for the normal allele and one for the mutant allele). For each sample two PCR reactions were performed in parallel using in one case IVS-1 nt 110 and codon 39 normal primers and in the second case using the corresponding mutated primers. In both cases the different PCR fragments were visualized. After optimization these primers directed only amplification of their complementary allele. A single blind study was performed on the DNA of 18 individuals who were homozygous or heterozygous for these mutations. In comparison with a parallel investigation, using oligonucleotide probes, all the results were unambiguous. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows the screening of a population group, including heterozygotes, which is required from an epidemiological and anthropological point of view. It could be extended to the large series screening of haplotypes before targeted diagnosis of various genetic diseases.

Algeria↗
PubMed1999

Inferring the fitness effects of DNA mutations from polymorphism and divergence data: statistical power to detect directional selection under stationarity and free recombination.

The fitness effects of classes of DNA mutations can be inferred from patterns of nucleotide variation. A number of studies have attributed differences in levels of polymorphism and divergence between silent and replacement mutations to the action of natural selection. Here, I investigate the statistical power to detect directional selection through contrasts of DNA variation among functional categories of mutations. A variety of statistical approaches are applied to DNA data simulated under Sawyer and Hartl's Poisson random field model. Under assumptions of free recombination and stationarity, comparisons that include both the frequency distributions of mutations segregating within populations and the numbers of mutations fixed between populations have substantial power to detect even very weak selection. Frequency distribution and divergence tests are applied to silent and replacement mutations among five alleles of each of eight Drosophila simulans genes. Putatively "preferred" silent mutations segregate at higher frequencies and are more often fixed between species than "unpreferred" silent changes, suggesting fitness differences among synonymous codons. Amino acid changes tend to be either rare polymorphisms or fixed differences, consistent with a combination of deleterious and adaptive protein evolution. In these data, a substantial fraction of both silent and replacement DNA mutations appear to affect fitness.

Adaptation, Biological↗
PubMed1997

Evaluation of the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.

Five hundred twenty processed respiratory specimens from 326 patients received for the diagnosis of tuberculosis or other mycobacterial infections were tested by means of the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, which uses ligase chain reaction technology for the direct detection of M. tuberculosis complex in respiratory specimens. The results of the LCx M. tuberculosis Assay were compared with the results of culture and staining techniques. After a combination of culture results and the patient's clinical data, a total of 195 specimens were collected from 110 patients who were positively diagnosed as having pulmonary tuberculosis. Twenty-three of these 195 specimens which corresponded to 10 patients with a history of pulmonary tuberculosis (TB) and anti-TB treatment ranging from 1 to 6 months were culture negative. The other 172 specimens were culture positive for M. tuberculosis. With an overall positivity rate of 37.5% (195 of 520 specimens), the sensitivity, specificity, and positive and negative predictive values were 90.8, 100, 100, and 94.7%, respectively, for the LCx M. tuberculosis Assay; 88.2, 100, 100, and 93.4%, respectively, for culture; and 82.6, 92, 72.9, and 97.6%, respectively, for acid-fast staining. For 161 specimens (82.6%) from patients smear positive for the disease and 34 specimens (17.4%) from patients smear negative for the disease, the sensitivity values for the LCx M. tuberculosis Assay were 98.8 and 53%, respectively. There were no statistically significant differences in the sensitivities and specificities between the LCx M. tuberculosis Assay and culture (P > 0.05). Conclusively, the LCx M. tuberculosis Assay has proved to have an acceptable sensitivity and a high specificity in detecting M. tuberculosis and has the potential of reducing the diagnosis time to an 8-h working day.

Bacterial Typing Techniques↗
PubMed2004

Direct detection of rifampin-resistant mycobacterium tuberculosis in respiratory specimens by PCR-DNA sequencing.

This study evaluated the feasibility of a molecular strategy based on identification of Mycobacterium tuberculosis by IS6110 PCR or Cobas Amplicor PCR, and rpoB PCR-DNA sequencing of the 81-bp rifampin resistance determining region (RRDR) for direct detection of rifampin resistance in respiratory specimens. A collection of 2,138 respiratory specimens and 352 nonduplicate M. tuberculosis isolates (including 233 isolates from the evaluated respiratory specimens and an additional collection of 119 stored isolates) from Southern China was investigated. Using culture as the reference test, the overall diagnostic sensitivities of an acid-fast bacillus (AFB) smear, Cobas Amplicor PCR, IS6110 PCR were 54.5% (156 of 286), 86.7% (248 of 286), and 89.2% (255 of 286), respectively. The sensitivities of the rpoB PCR for the specimens with positive AFB smears and with positive PCR results in the IS6110 PCR and/or Cobas Amplicor PCR were 100% (156 of 156) and 92.3% (239 of 259), respectively. Of the 352 nonduplicate M. tuberculosis isolates, the agar proportion method for rifampin reported 39 resistant strains. Full agreement (352 of 352) was found with the agar proportion method and the genotype inferred from the rpoB DNA sequencing data for rifampin. Thirty-nine mutations of nine distinct kinds, eight point mutations, and one deletion within the RRDR were found in the 39 resistant strains. For the direct DNA sequencing performed on rpoB PCR-positive respiratory specimens, the concordance with the agar proportion method and the subsequent PCR-sequencing for the culture isolate was 100%. This strategy has potential application for direct and rapid diagnosis of rifampin-resistant M. tuberculosis in IS6110 PCR or Cobas Amplicor PCR-positive respiratory specimens.

Antibiotics, Antitubercular↗
PubMed2004

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach.

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.

Blotting, Southern↗
PubMed1997

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Bacterial Outer Membrane Proteins↗
PubMed1998

Direct detection of Mycobacterium tuberculosis in respiratory specimens using an automated DNA amplification assay and a single tube nested polymerase chain reaction (PCR).

The performance of an automated DNA amplification assay (Roche Cobas Amplicor Mycobacterium Tuberculosis Test (aPCR) was compared with an in-house single tube nested polymerase chain reaction (nPCR) for the direct detection of Mycobacterium tuberculosis in respiratory specimens. Among 385 specimens, 56 were culture positive for mycobacteria (44 positive for Mycobacterium tuberculosis and 12 positive for non-tuberculosis mycobacteria). The diagnostic sensitivities of aPCR and nPCR were 86% and 91% whereas a 100% diagnostic specificity of both assays was attained. By aPCR, inhibitors were detected in 6% of the clinical samples. For nPCR, the usage of a new thermostable DNA polymerase facilitated pre-PCR decontamination using uracil-N-glycosylase and "hot start" in single step procedure. The results of the study indicated that DNA amplification assays, either manual or automated, were rapid and specific tools for diagnosing pulmonary tuberculosis.

Base Sequence↗
PubMed1999

Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction.

Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore 2000 biosensor based on surface plasmon resonance (SPR). PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides. We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity. It was shown that the target DNA was available for detection over the range of 40-160 nM. Therefore, the detection limit was 7.5 pmol of the target DNA (143 bases, applied volume 30 microliters). Our DNA detection system, the combination of BIAcore and the probe PNA, could detect the target DNA with good reproducibility. In this report, we show that our system is a powerful tool for the diagnosis of pathologically significant DNA.

Bacterial Toxins↗
PubMedDate not supplied

Direct detection of diarrheagenic Aeromonas from faeces by polymerase chain reaction (PCR) targeting aerolysin toxin gene.

Detection of diarrheagenic Aeromonas specific aerolysin toxin (Aer) gene by PCR based assay and isolation, identification of diarrhea causing Aeromonas from faeces by culture methods were carried out in the Division of Active Surveillance, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India for a period of 12 months. Out of 602 faecal samples collected from patients with acute diarrhea admitted in Infectious Diseases (ID) Hospital, Kolkata, 68 (11.29%) samples were found to be possessing Aer gene by PCR technique. The conventional culture methods using selective media yielded only 64 (10.6%) Aeromonas strains from the same faecal samples. The different Aeromonas species possessing Aer gene identified by PCR based technique include A. hydrophila (55.8%), A. caviae (17.6%), A. veronii (10.2%), A. schubertii (4.4%), A. jandaei (2.9%) and A. trota (8.8%). The isolation and identification of Aeromonas by routine culture did not detect enterotoxigenic A. trota present in four diarrheal faecal samples. The failure of the growth of enterotoxigenic A. trota on selective media may be attributed to the ampicillin susceptibility of those strains. The quality control studies revealed that PCR method for the direct detection of Aer gene from the faeces has the sensitivity of 100% and specificity of 98%.

Adolescent↗
PubMed1998

[Direct detection of mutations in the LDL receptor gene in patients with familial hypercholesterolemia].

BACKGROUND: Familial hypercholesterolemia is one of the most frequent hereditary metabolic diseases. As a result of the functional disorder of the molecule of the LDL receptor LDL cholesterol is not sufficiently eliminated from the blood stream and exerts an atherogenic effect. The objective of the study was to introduce direct detection of mutations in the gene for the LDL receptor and characterize the spectrum of mutations in the Czech population. METHODS AND RESULTS: The authors analyzed a group of 84 unrelated patients where on the basis of clinical and biochemical criteria the diagnosis of FH was established. From the group 12 patients were eliminated (14.3%) where a mutation 3500 in the gene for apolipoprotein (apo) B-100 was detected. This mutation is most frequently the cause of a familial defect of apo B-100 (FDB), which cannot be differentiated clinically or biochemically from FH. In the LDL receptor gene a total of 11 mutations were found in 14 unrelated patients (16.7%), incl. 7 mutations not described hitherto. CONCLUSIONS: This is the first systematic characteristic of the spectrum of mutations in the LDL receptor gene in the Czech population. Molecular genetic analysis of the gene for the LDL receptor in affected families can contribute towards early assessment of the diagnosis of FH and thus to prevention of life threatening cardiovascular episodes in asymptomatic subjects.

Apolipoprotein B-100↗
PubMed2006

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA.

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.

Base Sequence↗
PubMed2000

Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences.

Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.

Animals↗
PubMed2003

Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.

SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.

Fluorometry↗
PubMed2001

Direct detection of cellular immune responses to cancer vaccines.

The evaluation of cancer immunotherapy is predicated on the hypothesis that markers of tumor antigen-specific T-cell immunity will cone-late with clinical efficacy. Establishing which candidate vaccines should enter large-scale clinical trials will necessitate optimal application of immunologic monitoring assays. Evidence suggests that available techniques are adequate for the direct detection of clinically significant antigen-specific T-cell responses from tissue specimens. To achieve this goal, it is important to have an understanding of individual methods and their limitations, to include appropriate control antigens in the monitoring strategy, and to incorporate statistical considerations into the design and analysis of such studies.

Cancer Vaccines↗
PubMed1992

[Rapid and direct detection of Mycobacterium tuberculosis complex and mycobacteria in sputum by advanced method, PCR].

We developed two PCR methods, which amplify bovine tuberculous MPB70 gene and mycobacterial 16S rRNA gene, for detection of tubercle bacilli and mycobacteria in sputum, respectively. Among 27 Mycobacterium species and 57 species of 30 genuses other than Mycobacterium, only M. tuberculosis (TB) complex, i.e., M. tuberculosis, M. bovis, M. africanum, M. microti showed DNA amplification by PCR for MPB70, and amplification of 16S rRNA gene were observed specific in Mycobacterium species. A combination of these PCR abilities were available to differentiate the TB complex and nontuberculous mycobacteria (NTM). We investigated the correlation between these methods and conventional methods with 311 sputa that were suspected mycobacteriosis. The PCR method could detect 12 cases of TB complex and 4 cases of NTM in 17 specimens, which were positive by conventional methods, but could not for one specimen. Among 294 specimens that were negative with conventional methods, the PCR method detected 13 and 8 cases of TB complex and NTM, respectively. These results were confirmed by commercial tuberculous specific DNA probe or investigation of the clinical background of the patients. On the other hand, 273 specimens showed negative result either PCR nor conventional methods. The PCR method did not detect tuberculous DNA in normal 197 sputa, which were not suspected mycobacteriosis. These results indicate that each one of these PCR methods is highly specific to TB complex or Mycobacterium species. We concluded that these PCR methods are useful and advanced methods for rapid and direct detection of tuberculosis and mycobacteriosis.

DNA, Bacterial↗
PubMed2003

Direct detection of the Yarkovsky effect by radar ranging to asteroid 6489 Golevka.

Radar ranging from Arecibo, Puerto Rico, to the 0.5-kilometer near-Earth asteroid 6489 Golevka unambiguously reveals a small nongravitational acceleration caused by the anisotropic thermal emission of absorbed sunlight. The magnitude of this perturbation, known as the Yarkovsky effect, is a function of the asteroid's mass and surface thermal characteristics. Direct detection of the Yarkovsky effect on asteroids will help constrain their physical properties, such as bulk density, and refine their orbital paths. Based on the strength of the detected perturbation, we estimate the bulk density of Golevka to be 2.7(+0.4)(-0.6) grams per cubic centimeter.

Journal Article↗