Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.     

Development of a chromosomal DNA probe for the laboratory diagnosis of aspergillosis

Chromosomal DNA was extracted from clinical isolates of Aspergillus fumigatus of human and animal origin using the protoplast lysate method. The probe was developed by the nick translation of the chromosomal DNA genome fraction with p³² as the radiolabel. Hybridization of the probe with endonuclease-cleaved DNA of the same species resulted in a pattern of recognition sites specific for the species. The latter was not seen in other species encountered in clinical specimens. Trials were carried out on sputum experimentally inoculated with the fungus where crude DNA was directly extracted, treated with the endonuclease and hybridized with the probe. The efficacy of the probe was as good with the crude as the purified DNA. The specificity of the probe was determined by testing it against single and mixed DNA populations extracted from different species of several fungal and bacterial genera isolated from and/or known to occur in clinical specimens of respiratory infection origin. The sensitivity of the probe was assessed by detecting a DNA concentration in the specimen equivalent to 3 C.F.U.This research project was supported by the Mayo Visiting Clinician Program, Mayo Medical Center, Rochester, MN 55905, USA.     

Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces

pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces.

pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormalities of the human growth hormone gene and protooncogenes in some human pituitary adenomas

Hypersecretion of human GH (hGH) or PRL by human pituitary adenomas is not under normal homeostatic control despite normal receptor function mediating the regulatory effects of hypothalamic peptides for these trophic hormones. This implies that the defects underlying hormonal hypersecretion may not reside at the plasma membrane of the adenoma cell; instead, dysregulation may reside at the hormone gene level. To investigate this, genomic DNA derived from a prolactinoma and a hGH-secreting adenoma were digested with the restriction endonuclease EcoRI and the methylation sensitive restriction endonuclease HpaII and hybridized with the 32P-labeled genomic hGH (2.6 kilobase) probe. Our data revealed hypomethylation of genes of the hGH family (hGH and chorionic somatomammotropin) in the absence of gross abnormalities such as gene translocation. In a similar analysis using a 32P-labeled probe consisting of the EcoRI-BamHI (500 base pair) fragment in the 5'-flanking region upstream of the first exon of the hGH gene, hypomethylation of this specific site of the hGH genes was observed. These results are consistent with the concept that hypomethylation of genes is involved in gene expression. At the same time, protooncogene abnormalities in these adenomas were investigated to delineate any genetic basis for their neoplastic growth. Genomic DNA of adenomas were subjected to Southern blotting analysis using a panel of protooncogene probes. Amplification of the v-fos gene was observed in one prolactinoma. The significance of this observation is discussed.     

酶标探针在转基因植物检测中的应用

采用碱性磷酸酶标记DNA制备分子探针, 并首次在植物中应用。酶在苯醌作用下与单链DNA联结, 形成DNA和酶的共价复合物即酶标探针。此探针通过分子杂交与待测DNA结合, 再与酶的底物作用显色, 3～6h 内可观察结果。用此探针检测转基因植物中的UidA基因, 点杂交和Southern杂交结果表明, 所合成的酶标探针具有快速、准确、安全而经济的优点。点杂交证明外源UidA基因被成功转化到受体植物中, Southern 杂交对转基因的材料检测的结果证明, 该材料包含多个外源UidA基因拷贝, 初步确定其外源UidA基因拷贝数在5个以上。     

Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen

A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

The use of 2-hydroperoxytetrahydrofuran as a reagent to sequence cytosine and to probe non-Watson-Crick DNA structures.

2-Hydroperoxytetrahydrofuran (THF-OOH) can be employed to sequence cytosine (C) and to probe for non-canonical DNA structures involving C. Using 32P-labeled oligomers and a DNA restriction fragment, it is demonstrated that THF-OOH has a strong preference for Cs in single-stranded (s-s) DNA regions, and in bulges, loops and mismatches. The reactivity of C is diminished below pH 6.0, but is not affected by substitution of 5-methylcytosine. To demonstrate the utility of the reagent, it is directly compared to methoxylamine and chloroacetaldehyde, two other reagents commonly used to chemically probe C residues in non-Watson-Crick DNA structures.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.