Nucleic acid tests for the diagnosis of sexually transmitted diseases.

Nucleic acid (NA) assays have been developed and commercialized for many sexually transmitted diseases (STDs). Solid phase, liquid phase or in situ hybridization of nucleic acids without amplification procedures have been successfully used for diagnosing Chlamydia trachomatis, Neisseria gonorrhoeae and human papillomaviruses. Tests which use amplification procedures have provided better sensitivity and specificity than traditional tests. With special temperatures and enzymes, the new tests are designed to amplify either the target nucleic acid or the probe after annealing to the target. A third approach uses signal amplification. This article discusses the technology, specimen requirements and the current status of NA assay performance for diagnosing STDs and HIV by traditional and non-invasive clinical specimens.     

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.     

DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease

A sequence-specific electrochemical sexually transmitted disease (STD) sensor based on self-assembled monolayer of thiolated DNA probe specific to target opa gene for detection of Gonorrhoea, a sexually transmitted disease has been fabricated. 6-Mercapto-1-hexanol (MCH) has been used as a blocking agent to facilitate oligos "stand" up at the surface, a configuration favoring subsequent DNA hybridization and to repel non-specific adsorption of undesired DNA. The results of differential pulse voltammetric studies of this STD sensor reveal low detection limit (1.0 × 10(-18)M) and a wide dynamic range (from 1.0 × 10(-6)M to 0.5 × 10(-18)M) arising due to the stable hybridization using methylene blue as an electro-active DNA hybridization indicator. The experimental results with genomic DNA, clinical patient sample of Neisseria gonorrhoeae, culture of non-N. gonorrhoeae Neisseria species (NgNS) and gram negative bacteria indicate that the fabricated sensor is specific to this STD.     

Fabrication and optimization of the multiplex PCR-based oligonucleotide microarray for detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum

To detect and identify the pathogens responsible for sexually transmitted diseases (STDs) at the early stage of infection and with a high throughput, a new microarray with a bifunctional probe modification was prepared using Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum as a model system. During the fabrication of the microarray, an asymmetric fluorescently labeled multiplex PCR was introduced. The fabrication optimization proved that the best hybridization results would be obtained by spotting N. gonorrhoeae probe at a position near the side of the fluorescently labeled reverse primer within its target gene and spotting each probe at a concentration of 50 microM onto the aldehyde-derived glass slides using spotting solution S1 and using hybridization solution H2 for hybridization. The probes designed by our laboratory could specifically discriminate the pathogens of N. gonorrhoeae, C. trachomatis and U. urealyticum in the presence of the internal control on the microarray simultaneously and separately. By incorporating the key features of DNA microarray with those of multiplex PCR, the microarray provides a fast high throughput platform for multiple infections and multiple samples to be detected and identified simultaneously for STD clinics. It also provides a new platform for other diseases and gene mutations to be detected and identified at a high throughput.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

PNA and LNA throw light on DNA

In some aspects, homogeneous (all-in-solution) nucleic acid hybridization assays are superior to the traditionally used heterogeneous (solution-to-surface) alternatives. Profluorescent probes, which reveal fluorescence enhancement or fluorescence polarization upon their binding to DNA and RNA targets, are a paradigm for the real-time sequence-specific homogeneous detection of nucleic acids. A variety of such DNA or RNA-derived probes of different constructs has already been developed with numerous applications. However, the recent additions to the field - locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) - significantly increase the potential of profluorescent probes and provide a robust impulse for their new uses.     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

Detection of Chlamydia trachomatis in clinical specimens by nucleic acid spot hybridization

A nucleic acid spot hybridization assay was used to detect Chlamydia trachomatis DNA. The hybridization probes included DNA isolated from elementary bodies of lymphogranuloma venereum (LGV) strains and cloned fragments of both chromosomal and plasmid DNA. The sensitivity of the test was in the range 10 to 100 pg homologous DNA and 10 in vitro infected cells. Cross-reactivity with bacterial DNA was avoided when purified chlamydia-specific DNA fragments were used as probes. C. trachomatis was detectable in most of the clinical specimens with large amounts of infectious particles. Also some isolation-negative specimens gave a positive signal in the test.     

Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of Legionellae in biofilms

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.     

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose.

We have developed a general procedure for the rapid and efficient enrichment of specific DNA, RNA or cDNA sequences. Biotinylated DNA or RNA is used as a hybridization probe in solution, avidin is then added to label both the probe and hybrid molecules, and the hybridization mixture chromatographed over cupric-iminodiacetic acid agarose beads. Avidin-probe and avidin-hybrid molecules are selectively retained on the column; non-hybridized sequences are contained in the flow-through fraction. Sequences retained on the column are recovered in high yield by the addition of ethylenediamine tetracetic acid in the buffer. The method can be used in both subtractive enrichment and positive selection protocols. Here we report its application to the isolation of Neisseria gonorrhoeae specific genomic DNA clones and the purification of a cDNA subpopulation representing mRNA sequences that are over-expressed in murine Friend cells after dimethylsulfoxide induction.     

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

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.     

Molecular beacons: nucleic acid hybridization and emerging applications.

Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.     

Deoxyribonucleic Acid Homologies Among Species of the Genus Neisseria

Eleven aerobic species of Neisseria, a Mima sp., and a Herellea sp. were tested for deoxyribonucleic acid (DNA) homology in direct hybridization experiments. DNA labeled with either (14)C or (32)P was prepared from five species of Neisseria. Unlabeled DNA from the various microorganisms was immobilized on membrane filters, which, after pretreatment, were incubated with labeled DNA (4,000 counts per min per filter) for 14 hr at 67 C. The measure of relatedness was expressed as the relative percentage of direct binding compared to that obtained with homologous DNA. All serological types of N. meningitidis, including the newly proposed types, were homologous to the standard strain of N. meningitidis with one possible exception, type Z. The genus Neisseria is heterogeneous in nature, forming at least three distinct groups: first, N. meningitidis and N. gonorrhoeae; second, N. perflava, N. subflava, N. sicca, N. flavescens, and N. flava; third, N. catarrhalis and N. caviae. Mima and Herellea species show no significant homology with the Neisseria.