DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.     

Biotinylated DNA probe for detection of streptotricin acetylation genes

The biotin-labelled DNA probe for identification of functioning and silent genes for streptotricin acetylation has been constructed. The probe is homologous to sat1 gene of the movable genetic element Tn1825. The simplified modification of the hybridization technique using the biotin-labelled DNA probe is described.     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

TaqMan probe array for quantitative detection of DNA targets

To date real-time quantitative PCR and gene expression microarrays are the methods of choice for quantification of nucleic acids. Herein, we described a unique fluorescence resonance energy transfer-based microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 3′ amino-modified TaqMan probes were designed and immobilized on a glass slide composing a regular microarray pattern, and used as probes in the consecutive PCR carried out on the surface. During the extension step of the PCR, 5′ nuclease activity of DNA polymerase will cleave quencher dyes of the immobilized probe in the presence of nucleic acids targets. The increase of fluorescence intensities generated by the change in physical distance between reporter fluorophore and quencher moiety of the probes were collected by a confocal scanner. Using this new approach we successfully monitored five different pathogenic genomic DNAs and analyzed the dynamic characteristics of fluorescence intensity changes on the TaqMan probe array. The results indicate that the TaqMan probe array on a planar glass slide monitors DNA targets with excellent specificity as well as high sensitivity. This set-up offers the great advantage of real-time quantitative detection of DNA targets in a parallel array format.     

Synthesis of a novel fluorescent probe useful for DNA detection

In this paper, a novel fluorescent probe 2-methylbenzo[b][1,10] phenanthrolin-7(12H)-one (m-BPO) is synthesized, and its molecular structure has been characterized by IR, UV, MS, (1)H-NMR and elements analysis. The fluorescent characteristics of m-BPO were investigated in detail. It was found that DNA had the ability to quench the fluorescence of m-BPO at 411 nm (lambda(ex)=286 nm), and the quenched intensity of fluorescence was proportional to the concentration of DNA. Based on this fact, m-BPO has been used as the fluorescent probe for detection of calf thymus DNA (ctDNA) and fish semen DNA (fsDNA). Under the optimal conditions, the calibration curves are linear up to 15.0 microg/ml for both ctDNA and fsDNA. The corresponding detection limits are 3.6 ng/ml for ctDNA and 5.5 ng/ml for fsDNA, respectively. The interaction mechanism for the binding of m-BPO to ctDNA was studied in detail, and the results suggested that the interaction mode between m-BPO and ctDNA was groove binding.     

Sensitive and specific DNA probe for detection of Plasmodium falciparum.

The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.     

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.     

Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition

Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.     

Polycation-assisted DNA detection by reduction triggered fluorescence amplification probe

We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection.     

Evaluation of a DNA probe test kit for detection of Salmonellae in biosolids

AIMS: Current US regulations (40 CFR 503) for 'Class A' biosolids (treated sewage sludge) requires use of multiple-tube fermentation techniques for fecal coliform or multiple tube enrichment techniques for Salmonella spp. followed by isolation and biochemical and serologic confirmation. The technical difficulties and the time required to complete the procedure for enumeration of Salmonellae in biosolids and sludges has limited the use of this assay. This study was conducted to determine if a commercially available molecular probe system could be used to isolate and enumerate Salmonella spp. in biosolids or sludges in less time than cultural techniques with biochemical confirmation. METHODS AND RESULTS: Several types of treated and untreated municipal sludges were assayed for Salmonellae using a cultural technique with biochemical and serologic confirmation and a DNA probe diagnostic test kit. The results indicate that the molecular probe and the conventional fermentation tube technique yielded equivalent results. Interestingly, the probe technique yielded results within 52 h following initiation of sample analysis compared with the conventional fermentation tube technique with confirmation which required approx. 120 h. CONCLUSIONS: These results suggest that the molecular probe system used for this work may be used to determine the presence or absence of Salmonella spp. in biosolids within a relatively short time frame. SIGNIFICANCE AND IMPACT OF THE STUDY: The ease of using the DNA probe test kit, along with its ability to produce results in less than half the time of conventional culture techniques, suggests that this assay is useful for determining the presence or absence of Salmonellae in biosolids samples.     

A non-radioactive DNA probe for the detection of Mycoplasma pulmonis in murine mycoplasmosis

A non-radioactive DNA probe for the detection of Mycoplasma pulmonis was developed by using photobiotin. The probe detected 0.3 ng chromosomal DNA of M. pulmonis. No cross-hybridization was observed between the probe and the other murine mycoplasma species, M. arthritidis and M. neurolyticum.     

A visual DNA probe for detection of enterotoxigenic Escherichia coli by colony hybridization

A colony hybridization method for visual detection of heat-labile enterotoxin (LT)-producing Escherichia coli has been developed. The interchangeability of probe DNA in the system offers the advantage of a more definitive indicator and detection procedure over current plating procedures. Preliminary studies have demonstrated complete specificity of the visual probe for target DNA. This model system has potential for use as a more definitive indicator method for the examination of foods and wastewater for LT toxin genes.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.     

Bis-pyrene-labeled molecular beacon: a monomer-excimer switching probe for the detection of DNA base alteration

A new bis-pyrene-labeled oligonucleotide probe (BP-probe) has been designed for the detection of a single base mismatch in single strand (ss) DNA as a target. The sequence of BP-probe was chosen to form stem-loop structure similar to a molecular beacon (MB-probe), yielding bis-pyrene-labeled molecular beacon (BP-MB-probe). Partially double stranded (ds) BP-MB-probes were prepared by complexation with oligonucleotides whose sequences are complementary to the loop segment but not to the stem and exchangeable with the target DNA. The partially ds BP-MB-probes were shown to exhibit monomer fluorescence as major fluorescence, while the ss BP-MB-probe in the stem-loop form displays strong excimer fluorescence. The strand exchange reactions between partially ds BP-MB-probe and target ss DNA in the presence of cationic comb-type copolymer as a catalyst were monitored by the excimer fluorescence changes. The existence of a mismatched base can be determined by the slower PASE rates compared with fully matched DNA.