PubMed Health⌕ Search

Biomedical subjects

A K Bej

Publications and source records attributed to A K Bej.

At least 37 records · Page 2Linked to original sources
PubMed1992

Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes.

Giardia spp. are waterborne organisms that are the most commonly identified pathogenic intestinal protozoans in the United States. Current detection techniques for Giardia species in water include microscopy and immunofluorescence techniques. Species of the genus Giardia are classified on the basis of taxonomic criteria, such as cell morphology, and on host specificity. We have developed a polymerase chain reaction- and gene probe-based detection system specific for Giardia spp., which can discriminate between the relevant species of the G. duodenalis type pathogenic to humans and other Giardia species that are not human pathogens. This method can detect a single Giardia cyst and is therefore sensitive enough for environmental monitoring.

Animals↗
PubMed1991

Acquisition of mitochondrial DNA by a transformation vector for Ustilago violacea.

Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus. Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid. In addition to multiple integrations of plasmid sequences into U. violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe. Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria [mitochondrial (mt) DNA preparations] of transformed U. violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well. The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure. Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx. 8.0-kb plasmid. A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1. This unique fragment did hybridize strongly with mt DNA from untransformed U. violacea. This derivative thus appears to have acquired mt sequences from U. violacea.

Blotting, Southern↗
PubMed1991

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.

Animals↗
PubMed1991

Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples.

To detect low levels of microorganisms in environmental samples by using polymerase chain reaction (PCR)-gene probe detection, samples were concentrated by filtration. Fluoropore (Millipore Corp.) filters were compatible with PCR DNA amplification, whereas various other filters including nitrocellulose and cellulose acetate filters inhibited PCR amplification. By concentrating cells on Fluoropore filters and releasing the DNA by freeze-thaw cycling, PCR DNA amplification could be performed without removing the filter. Concentration with Fluoropore FHLP and FGLP filters permitted the detection of single cells of microorganisms in 100-ml samples by PCR-gene probes.

Bacteria↗
PubMed1991

Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods.

Methods using polymerase chain reaction (PCR) and gene probes to detect viable Legionella pneumophila were investigated with cells exposed to biocide or elevated temperature. Exposure to hypochlorite caused viable nonculturable cells to form. Culturable and viable nonculturable cells showed positive PCR amplification, whereas nonviable cells did not. Viable cells were also specifically detected with mip mRNA as the target, reverse transcription (to form cDNA), and PCR amplification. After exposure to elevated temperature, only viable culturable cells were detected, which corresponded with positive PCR amplification.

Base Sequence↗
PubMed1991

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid.

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.

Base Sequence↗
PubMed1991

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.

Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli.

Base Sequence↗
PubMed1991

Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications.

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies--examining the critical parameters and variations and their widespread applications--giving the strengths and limitations of these methodologies.

Bacterial Infections↗
PubMed1991

Detection of bacterial mRNA using polymerase chain reaction.

A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern blot hybridization. Detection of mRNAs by reverse transcription-PCR-Southern blot analysis is orders of magnitude more sensitive than Northern blot hybridization.

Blotting, Southern↗
PubMed1990

Introduction and maintenance of prokaryotic DNA in Ustilago violacea.

A strain of the basidiomycete, Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin. U. violacea transformants were selected at a frequency of 5 per microgram pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipient U. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction of U. violacea transformants failed to produce E. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction in U. violacea transformants produced E. coli transformants resistant to neomycin. The E. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harbor U. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryote U. violacea and acquire endogenous sequences from this organism.

Blotting, Southern↗
PubMed1990

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water.

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.

Bacteriological Techniques↗
PubMed1990

Detection of Legionella with polymerase chain reaction and gene probe methods.

Methods were developed for the detection of Legionella in environmental water sources, based upon the polymerase chain reaction (PCR) and gene probes. All species of Legionella, including all 15 serogroups of L. pneumophila tested, were detected by PCR amplification of a 104 bp DNA sequence that codes for a region of 5S rRNA followed by radiolabelled oligoprobe hybridization to an internal region of the amplified DNA. Strains of L. pneumophila (all serogroups) were specifically detected based upon amplification of a portion of the coding region of the macrophage infectivity potentiator (mip) gene. Pseudomonas spp. that exhibit antigenic cross-reactivity in serological detection methods did not produce positive signals in the PCR-gene probe method using Southern blot analyses. Single cell, single gene Legionella detection was achieved with the PCR-gene probe methods.

Base Sequence↗
PubMed1990

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.

Base Sequence↗
PubMed1990

Microbial degradation of quinoline and methylquinolines.

Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and P. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.

Biodegradation, Environmental↗
PubMed1989

A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment.

A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus. After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U. violacea transformants appeared on Hyg-agar at a frequency of 60-80 per microgram pUCH1 DNA. The Hyg phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions. Southern DNA-DNA hybridization revealed multiple integrations of the pUCH1 plasmid into the U. violacea nuclear DNA. In addition, Escherichia coli transformants appeared at a frequency of 12 per microgram nuclear fraction DNA from Hyg U. violacea transformants; these E. coli consistently contained a deleted pUCH1 plasmid. This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences.

Anti-Bacterial Agents↗
PubMed1988

Model suicide vector for containment of genetically engineered microorganisms.

A model suicide vector (pBAP19h), designed for the potential containment of genetically engineered microorganisms, was made by constructing a plasmid with the hok gene, which codes for a lethal polypeptide, under the control of the lac promoter. The vector plasmid also codes for carbenicillin resistance. In the absence of carbenicillin, induction of the hok gene in vitro caused elimination of all detectable cells containing the suicide vector; pBAP19h-free cells of the culture survived and grew exponentially. In the presence of carbenicillin, however, the number of cells containing pBAP19h initially declined after induction of hok but then multiplied exponentially. The surviving cells still had a fully functional hok gene and had apparently developed resistance to the action of the Hok polypeptide. Thus, high selective pressure against the loss of the suicide vector led to a failure of the system. Soil microcosm experiments confirmed the ability of a suicide vector to restrict the growth of a genetically engineered microorganism in the absence of selective pressure against the loss of the plasmid, with 90 to 99% elimination of hok-bearing cells within 24 h of hok induction. However, some pBAP19h-bearing cells survived in the soil microcosms after hok induction. The surviving cells contained an active hok gene but were not capable of normal growth even after elimination of the hok gene; it appears that a mutation that made them Hok resistant also reduced their capacity for membrane functions needed for energy generation and exponential cell growth. Thus, the model suicide vector was shown to be functional in soil as well as in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗