Chitosan-iron oxide nano-composite platform for mismatch-discriminating DNA hybridization for Neisseria gonorrhoeae detection causing sexually transmitted disease

Electrochemically fabricated nano-composite film of chitosan (CH)-iron oxide (Fe(3)O(4)) has been used to detect gonorrhoea, a sexually transmitted disease (STD) via immobilization of biotinylated probe DNA (BDNA) using avidin-biotin coupling for rapid and specific (mismatch-discriminating) DNA hybridization. The presence of Fe(3)O(4) nanoparticles (～18nm) increases the electro-active surface area of the nano-biocomposite that provides desirable environment for loading of DNA with better conformation leading to increased electron transfer kinetics between the medium and electrode. The differential pulse voltammetric (DPV) studies have been conducted using BDNA/avidin/CH-Fe(3)O(4)/ITO electrode owing to the reduction of the methylene blue (MB) indicator and investigate electron transfer between MB moieties and electrode for one and two-bases mismatch. This STD biosensor is found to have a detection limit (1 × 10(-15)M) and a wide dynamic range (from 1 × 10(-16)M to 1 × 10(-6)M) using the complementary target DNA. In addition, the sensing system can be utilized to accurately discriminate complementary sequence from mismatch sequences.     

High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode

A novel enzyme-linked DNA hybridization assay on an interdigitated array (IDA) microelectrode integrated into a microfluidic channel is demonstrated with sub-nM detection limit. To improve the detection limit as compared to conventional electrochemical biosensors, a recyclable redox product, 4-aminophenol (PAP) is used with an IDA microelectrode. The IDA has a modest and easily fabricated inter-digit spacing of 10 μm, yet we were able to demonstrate 97% recycling efficiency of PAP due to the integration in a microfluidic channel. With a 70 nL sample volume, the characterized detection limit for PAP of 1.0 × 10?1? M is achieved, with a linear dynamic range that extends from 1.0 × 10?? to 1.0 × 10?? M. This detection limit, which is the lowest reported detection limit for PAP, is due to the increased sensitivity provided by the sample confinement in the microfluidic channel, as well as the increased repeatability due to perfectly static flow in the microchannel and an additional anti-fouling step in the protocol. DNA sequence detection is achieved through a hybridization sandwich of an immobilized complementary probe, the target DNA sequence, and a second complementary probe labeled with β-galactosidase (β-GAL); the β-GAL converts its substrate, 4-aminophenyl-d-galactopyranoside (PAPG), into PAP. In this report we present the lowest reported observed detection limit (1.0 × 10?1? M) for an enzyme-linked DNA hybridization assay using an IDA microelectrode and a redox signaling paradigm. Thus, we have demonstrated highly sensitive detection of a targeted DNA sequence using a low-cost easily fabricated electrochemical biosensor integrated into a microfluidic channel.     

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.     

Lactobacillus crispatus and its enolase and glutamine synthetase influence interactions between Neisseria gonorrhoeae and human epithelial cells

Journal of Microbiology - Neisseria gonorrhoeae, an obligatory human pathogen causes the sexually transmitted disease gonorrhea, which remains a global health problem. N. gonorrhoeae primarily...     

Rapid one step detection of pathogenic bacteria in urine with sexually transmitted disease (STD) and prostatitis patient by multiplex PCR assay (mPCR)

We developed a multiplex PCR (mPCR) assay to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Corynebacterium spp. and seudomona aeruginosa. This method employs a single tube and multiple specific primers which yield 200, 281, 346, 423, 542, and 1,427 bp PCR products, respectively. All the PCR products were easily detected by agarose gel electrophoresis and were sequenced to confirm the specificity of the reactions. To test this method, DNA extracted from urine samples was collected from 96 sexually transmitted disease or prostatitis patients at a local hospital clinical center, and were subjected to the mPCR assay. The resulting amplicons were cloned and sequenced to exactly match the sequences of known pathogenic isolates. N. gonorrhoeae and Corynebacterium spp. were the most frequently observed pathogens found in the STDs and prostatitis patients, respectively. Unexpectedly, P. aeruginosa was also detected in some of the STD and prostatitis samples. More than one pathogen species was found in 10% and 80.7% of STD and prostatitis samples, respectively, indicating that STD and prostatitis patients may have other undiagnosed and associates. The sensitivity of the assay was determined by sing purified DNA from six pathogenic laboratory strains and revealed that this technique could detect pathogenic DNA at concentrations ranging from 0.018 to 1.899 pg/ul. Moreover, the specificities of this assay were found to be highly efficient. Thus, this mPCR assay may be useful for the rapid diagnosis of causative infectious STDs and prostatitis. useful for the infectious STDs and prostatitis.     

A urea electrochemical sensor based on molecularly imprinted chitosan film doping with CdS quantum dots

An improved imprinted film-based electrochemical sensor for urea recognition was developed using CdS quantum dots (QDs) doped chitosan as the functional matrix. The microstructure and composition of the imprinted films depicted by scanning electron microscopy (SEM), attenuated total reflection infrared (ATR-IR), X-ray diffraction (XRD), and electrochemical impedance spectroscopy (EIS) indicated the fabricated feasibility of the nanoparticle doped films via in situ electrodeposition. Differential pulse voltammetric responses under the optimal fabrication conditions showed that the sensitivity of CdS QDs-MIP (molecularly imprinted polymer) electrochemical sensor was enhanced from the favorable electron transfer and magnified surface area of CdS QDs with a short adsorption equilibrium time (7 min), wide linear range (5.0 × 10(-12) to 4.0 × 10(-10) M and 5.0 × 10(-10) to 7.0 × 10(-8) M), and low detection limit (1.0 × 10(-12) M). Meanwhile, the fabricated sensor showed excellent specific recognition to template molecule among the structural similarities and coexistence substances. Furthermore, the proposed sensor was applied to determine the urea in human blood serum samples based on its good reproducibility and stability, and the acceptable recovery implied its feasibility for practical application.     

Construction of DNA biosensor at glassy carbon surface modified with 4-aminoethylbenzenediazonium salt

A simple, label-free electrochemical impedance-spectroscopy method for sequence-specific detection of DNA using a 4-aminoethylbenzenediazonium (AEBD) salt as a binder for amino-modified probe DNA is reported. This novel method simplifies the anchoring of DNA at the GC surface and opens new ways for the detection of hybridization. The hybridization of target DNA, without and with mismatches, with the probe DNA anchored at the GC surface modified with AEBD, greatly increases the interfacial electron transfer resistance at the double-stranded DNA modified electrodes for the redox couple Fe(CN)(6)(3-/4-). The resistance was measured using electrochemical impedance spectroscopy. The sensor response increased linearly with logarithm of concentration of target DNA in the range 2×10(-12)÷2×10(-6) M. The obtained quantification limit was circa 6.5×10(-17) mole in a 7 μL droplet and corresponded to a concentration of 9.2×10(-12) M of target DNA in the sample. This limit is equivalent to the detection of circa 4×10(7) copies of DNA in a 7 μL droplet or circa 5.7×10(12) DNA copies in one litre of sample.     

Gonococcal infection and human fertility in sub-Saharan Africa.

An analysis is presented of the influence of Neisseria gonorrhoeae on human population growth in regions of sub-Saharan Africa where gonococcal infections are prevalent in sexually active adults. Combining epidemiological and demographic data within the framework of a mathematical model, we show that gonorrhoea has a major impact on fertility and, concomitantly, on net population growth in areas with a high prevalence of untreated infections. Specifically, a 20% prevalence in sexually active adults is predicted to induce a 50% reduction in net population growth. Model predictions are in good agreement with observed data from Uganda, and the sensitivity of the prediction to various complications, such as heterogeneity in sexual behaviour, is assessed. The analysis suggests that the predicted increase in fertility arising from expanded sexually transmitted disease (STD) control programmes in Africa to help combat the spread of human immunodeficiency viruses (HIV-1 and HIV-2) will help to offset the predicted demographic impact of AIDS in the worst afflicted areas. In other areas the rise in fertility associated with effective STD control will need to be countered by the linkage of STD control programmes with family planning initiatives.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

Draft genome sequence of a dominant, multidrug-resistant Neisseria gonorrhoeae strain, TCDC-NG08107, from a sexual group at high risk of acquiring human immunodeficiency virus infection and syphilis

Neisseria gonorrhoeae infection is the second major cause of sexually transmitted diseases worldwide. Development of resistance to multiple classes of antimicrobials in N. gonorrhoeae has compromised treatment and disease control. Herein, we report the availability of the draft genome sequence of a multidrug-resistant N. gonorrhoeae isolate, TCDC-NG08107, which spread in groups of men who have sex with men (MSM) in Taiwan.     

Design of a "microbicide" for prevention of sexually transmitted diseases using "inactive" pharmaceutical excipients.

The human immunodeficiency virus (HIV-1) pandemic has been driven primarily by the sexual transmission of the virus, and facilitated by prior infections with other sexually transmitted disease (STD) pathogens. Although treatment of these STDs has been proposed as a means to decrease the rate of HIV-1 sexual transmission, preventive measures effective against both HIV-1 and other STD pathogens are expected to have a larger impact. These measures include topically applied mechanical and chemical (i.e. microbicidal) barriers. Microbicides of preference should have a broad specificity against diverse STD pathogens and a well established safety record, considering their repeated use over decades. Here, we report that cellulose acetate phthalate (CAP), an "inactive" pharmaceutical excipient, commonly used in the production of enteric tablets and capsules: (1) has antiviral activity against HIV-1 and several herpesviruses (HSV); and (2) when appropriately formulated, in micronized form, inactivates HIV-1, HSV-1, HSV-2, cytomegalovirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Haemophilus ducreyi and Chlamydia trachomatis but does not affect Lactobacilli, components of the natural vaginal flora contributing to resistance against STDs. Thus, the CAP formulations meet the criteria for preferred microbicides and warrant further evaluation in vivo in humans.     

Asymptomatic gonorrhoea and chlamydial infection in rural Tanzanian men.

OBJECTIVE: To measure the prevalence of urethritis due to Neisseria gonorrhoeae and Chlamydial infection trachomatis in rural Tanzanian men DESIGN: About 500 men aged 15-54 years were selected from each of 12 rural communities by random cluster sampling; interviewed concerning past or present symptoms of sexually transmitted diseases; and asked to provide a first catch urine specimen, which was tested for pyuria with a leucocyte esterase dipstick test. Subjects with symptoms or with a positive result on testing were examined, and urethral swabs were taken for detection of N gonorrhoeae by gram stain and of C trachomatis by antigen detection immunoassay. SETTING: Mwanza region, north western Tanzania. SUBJECTS: 5876 men aged 15-54 years. MAIN OUTCOME MEASURES: Prevalence of urethral symptoms, observed urethral discharge, pyuria, urethritis ( > 4 pus cells per high power field on urethral smear), N gonorrhoeae infection (intracellular gram negative diplococci), and C trachomatis infection (IDEIA antigen detection assay). RESULTS: 1618 (28%) subjects reported ever having a urethral discharge. Current discharge was reported by 149 (2.5%) and observed on examination in 207 (3.5%). Gonorrhoea was found in 128 subjects (2.2%) and chlamydial infection in 39 (0.7%). Only 24 of 158 infected subjects complained of urethral discharge at the time of interview (15%). CONCLUSION: Infection with N gonorrhoeae and C trachomatis is commonly asymptomatic among men in this rural African population. This has important implications for the design of control programmes for sexually transmitted disease.     

Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens

Aims:  To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma genitalium , Mycoplasma hominis , Ureaplasma urealyticum and Trichomonas vaginalis .
Methods and Results:  Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 μg ml⁻¹ of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay.
Conclusions:  The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples.
Significance and Impact of the Study:  It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.     

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.     

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved.     

A selective novel non-enzyme glucose amperometric biosensor based on lectin-sugar binding on thionine modified electrode

A novel non-enzyme glucose amperometric biosensor was fabricated based on biospecific binding affinity of concanavalin A (Con A) for D-glucose on thionine (TH) modified electrode. TH can be covalently immobilized on potentiostatically activated glassy carbon electrode through Schiff-base reaction. Subsequently, the surface-adherent polydopamine film formed by self-polymerization of dopamine attached to TH and afforded binding sites for the subsequent immobilization of Con A molecules via Michael addition and/or Schiff-base reaction with high stability. Thus, a sensing platform for specific detection towards D-glucose was established. The binding of Con A towards D-glucose can be monitored through the decrease of the electrode response of the TH moiety. Due to the high affinity of Con A for D-glucose and high stability of the resulting sensing platform, the fabricated biosensor exhibited high selectivity, good sensitivity, and wide linear range from 1.0×10(-6) to 1.0×10(-4) M with a low detection limit of 7.5×10(-7) M towards D-glucose.     

Label-free electrochemical detection of short sequences related to the hepatitis B virus using 4,4'-diaminoazobenzene based on multiwalled carbon nanotube-modified GCE

A novel label-free electrochemical DNA biosensor based on 4,4'-diaminoazobenzene (4,4'-DAAB) and multiwalled carbon nanotube (MWNT)-modified glassy carbon electrode (GCE) for short DNA sequences related to the hepatitis B virus (HBV) hybridization detection was presented. Differential pulse voltammetry (DPV) was used to investigate hybridization event. The decrease in the peak current of 4,4'-DAAB was observed on hybridization of probe with the target. This electrochemical approach was sequence specific as indicated by the control experiments, in which no peak current change was observed when a noncomplementary DNA sequence was used. Numerous factors affecting the target hybridization were optimized to maximize the sensitivity. Under optimal conditions, this sensor showed a good calibration range between 7.94 x 10(-8) M and 1.58 x 10(-6) M, with HBV DNA sequence detection limit of 1.1 x 10(-8) M.     

DNA electrochemical biosensor based on thionine-graphene nanocomposite

A novel protocol for development of DNA electrochemical biosensor based on thionine-graphene nanocomposite modified gold electrode was presented. The thionine-graphene nanocomposite layer with highly conductive property was characterized by scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. An amino-substituted oligonucleotide probe was covalently grafted onto the surface of the thionine-graphene nanocomposite by the cross-linker glutaraldehyde. The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis using an electroactive intercalator daunomycin as the indicator. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The complementary oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-7)M with a good linearity (R(2)=0.9976) and a low detection limit of 1.26 × 10(-13)M (S/N=3). In addition, the biosensor was highly selective to discriminate one-base or two-base mismatched sequences.     

A proposed feedback loop of sexually transmitted diseases and sexual behavior: the Red Queen's Dilemma

(Micro)organisms, such as bacteria, which cause sexually transmitted diseases (STDs) in humans are presented with an interesting ecological challenge. These microorganisms need humans to have sexual contact with each other in order for the microorganisms to spread to other hosts as well as to have subsequent generations of descendants. However, diseases tend to lower the sex drive and to render the host less sexually attractive. It is argued that, over time, selective advantages sculpted organisms which cause STDs to be minimally symptomatic and to indirectly increase the number of sexual partners of the host. Neisseria gonorrhoeae which cause the STD gonorrhea are used as a prototype for these putative sexual dynamics. As a counter to the (micro)organisms' biological adaptations, human cultural innovations emerged and became integrated into the various traditions of social structures.