Comparison of culture, enzyme immunoassay and nucleic acid sandwich hybridization in detecting Chlamydia trachomatis in genital tract infections

Abstract Culture, enzyme immunoassay (Chlamydiazyme™) and nucleic acid sandwich hybridization were compared in detecting Chlamydia trachomatis in uncomplicated genital tract infections. Urethral and cervical specimens were collected from 100 males and 100 females attending a sexually transmitted disease clinic. Chlamydial culture was performed under optimal conditions (duplicate inoculation within the day specimen was collected, culture in vials, monoclonal antibody staining of inclusions, blind passage for negative samples). Here the sensitivity of culture exceeded that of the rapid methods. The sensitivity of a chlamydial antigen detection method (Chlamydiazyme™) was 68% in male and 86% in female specimens, when compared with culture, and the specificity was 100% and 97%, respectively. Acinetobacter calcoace9icus present in clinical specimens did not interfere with Chlamydiazyme™. The sensitivity of the nucleic acid sandwich hybridization was 53% of that of the culture, and specificity 100%. By comparing the three methods it was apparent that the rapid methods did not reveal chlamydial infections not detectable by culture. Thus, if performed carefully, culture is the most sensitive diagnostic method in acute genital infections due to C. trachomatis .     

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.     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

肽核酸相关技术在杂交检测中的应用

肽核酸是一种寡核苷酸的类似物,它是由丹麦哥本哈根大学的Ｎｉｅｌｓｅｎ、Ｅｇｈｏｌｍ等人首先发明合成的。肽核酸与传统的寡核苷酸相比,骨架结构发生了根要变化。肽核酸的电中性骨架有许多ＤＮＡ所不具备的性质,例舅高灵敏度、高特异性、非盐依赖性等,从而使它成为一种优良的寡核苷酸的取代物,尤其是杂交检测领域。     

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.     

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO₂) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO₂ nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO₂ nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO₂ nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO₂-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.     

New,sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D -luciferin (Photinus pyralis) from D -luciferin-O-phosphate. Liberated D -luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 × 10^?21 mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.     

Nucleic acid spot hybridization for detection of Chlamydia trachomatis

Abstract The TMAO reductase activity of Escherichia coli grown anaerobically in the presence or absence of TMAO was analysed on linear sucrose gradients and on non-denaturing polyacrylamide gels. The results, together with those obtained by analysis of some other properties of TMAO reductase, showed that there are significant differences between the enzyme synthesized in the absence of TMAO ("constitutive" enzyme) and that synthesized in its presence ("inducible" enzyme).
A similar study of a tor mutant specifically altered in the structural gene for TMAO reductase, showed that the enzymes synthesized under the 2 growth conditions are probably 2 distinct enzymes encoded by different genes.     

Chitosan scaffolds for biomolecular assembly: coupling nucleic acid probes for detecting hybridization

Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single-stranded DNA oligonucleotide probes in a fluorescence-based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96-well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK-specific oligonucleotide from 0.73 microM to 6.6 microM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique.     

A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 10² – 4.39 × 10³ s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8–5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.     

Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets

Chlamydia trachomatis (Ct) is a Gram‐negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild‐type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.     

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis

With an estimated 3–4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively while organisms known to co-inhabit the human urogenital tract react negatively.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

Making contacts on a nucleic acid polymer

The interaction of proteins bound at distant sites on a nucleic acid chain plays an important role in many molecular biological processes. Contact between the proteins is established by looping of the intervening polymer, which can comprise either double- or single-stranded DNA or RNA, or interphase or metaphase chromatin. The effectiveness of this process, as well as the optimal separation distance, is highly dependent on the flexibility and conformation of the linker. This article reviews how the probability of looping-mediated interactions is calculated for different nucleic acid polymers. In addition, the application of the equations to the analysis of experimental data is illustrated.