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Background & Objectives: Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs) and has significant health implications in women. The use of nucleic acid amplification tests (NAATs) has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals., Methods: Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods., Results: Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture) whereas 17 patients were found to be positive based on PCR results., Interpretation & Conclusions: The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene-based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection., (© 2013.)

The nanocomposite based on polyaniline (PANI)-iron oxide nanoparticles (nFe(3)O(4)) and multi walled carbon-nanotubes (CNT) has been fabricated onto indium tin oxide (ITO) coated glass plate via facile electrochemical synthesis of polyaniline in presence of nFe(3)O(4) (~20 nm) and CNT (20-80 nm in diameter). The results of transmission electron microscopic studies show evidence of coating of PANI and nFe(3)O(4) onto the CNT. The PANI-nFe(3)O(4)-CNT/ITO nanoelectrode has been characterized by Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy studies. The biotinylated nucleic acid probe sequence consisting of 20 bases has been immobilized onto PANI-nFe(3)O(4)-CNT/ITO nanoelectrode using biotin-avidin coupling. It is shown that the PANI-nFe(3)O(4)-CNT platform based biosensor can be used to specifically detect bacteria (N. gonorrhoeae) at minute concentration as low as (1×10(-19) M) indicating high sensitivity within 45 s of hybridization time at 298 K by differential pulse voltammetry using methylene blue as electroactive indicator. This bacterial sensor has also been tested with 4 positive and 4 negative PCR amplicons of gonorrhoea affected patient samples. The results of these studies have implications towards the fabrication of a handheld device for Neisseria gonorrhoeae detection that may perhaps result in a decrease in the human immunodeficiency virus infections., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Polyaniline/carbon nanotubes composite (PANI-CNT) electrochemically deposited onto indium-tin-oxide (ITO) coated glass plate has been utilized for Neisseria gonorrhoeae detection by immobilizing 5'-amino-labeled Neisseria gonorrhoeae probe (aDNA) using glutaraldehyde as a cross-linker. PANI-CNT/ITO and aDNA-Glu-PANI-CNT/ITO electrodes have been characterized using scanning electron microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), and differential pulse voltammetry (DPV). This bioelectrode can be used to detect N. gonorrhoeae using methylene blue (MB) as redox indicator with response time of 60 s and stability of about 75 days when stored under refrigerated conditions. DPV studies reveal that this bioelectrode can detect complementary DNA concentration from 1 x 10(-6) M to 1 x 10(-17) M with detection limit of 1.2 x 10(-17) M. Further, this bioelectrode (aDNA-Glu-PANI-CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non-Neisseria gonorrhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB).

Objectives In the absence of a single nucleic acid amplification test (NAAT) that is both highly specific and sensitive for gonorrhoea, many have put forward the 16S-based assay as a confirmatory test for . This study was undertaken to evaluate the performance of PCR based on 16S ribosomal gene in comparison with a pseudogene-based assay. Methods The specificity of both the pseudogene-based PCR and 16S ribosomal gene PCR was checked against a panel of strains comprising of non sp (NgNS) and other gram-negative and gram-positive bacteria. The sensitivity studies were performed using different dilutions of DNA. PCRs were also done on endocervical and urethral swab samples collected from a total of 100 female and 50 male patients presenting to sexually transmitted disease clinics, Dermatology OPD of AIIMS and Safdarjang Hospital, New Delhi, India, recruited as per inclusion criteria. Results PCR assay based on 16S ribosomal gene showed cross-reactivity with three of six strains of . The pseudogene-based PCR was highly specific. Analytical sensitivity of 16S-based ribosomal assay was more than that of pseudogene-based assay. In clinical samples, for female patients, sensitivity, specificity, positive predictive value and negative predictive value of 16S ribosomal assay was 100% (95% CI 51.7% to 100%), 91.5% (95% CI 83.4% to 96%), 42.9% (95% CI 18.8% to 70.4%) and 100% (95% CI 94.7% to 100%), respectively, while for the male patients it was 100% (95% CI 85% to 100%), 95.5% (95% CI 75.1% to 99.8%), 96.6% (95% CI 80.4% to 99.8%) and 100% (95% CI 80.8% to 100%), respectively. Conclusions The data presented in this report supports use of 16S ribosomal assay as a screening assay only. The pseudogene target is highly specific for and may be used as a supplemental assay. [ABSTRACT FROM PUBLISHER]

Abstract: We report results of studies relating to preparation and characterization of DNA electrodes based on the chitosan–MWCNT/ITO nanobiocomposite platform for electrochemical detection of gonorrhoea, the most common bacterial sexually transmitted disease. (STD). This biosensing electrode has been characterized using FT-IR, SEM, cyclic voltammetry and differential pulse voltammetry, etc. The sensing characteristics have been investigated in phosphate buffer saline using methylene blue as the hybridization indicator. This DNA biosensor has a response time of 60s and is found to be stable for about 4months when stored at 4°C. The results of DPV studies reveal that this bioelectrode can detect complementary DNA concentration in a wide range of 1×10−6 M to 1×10−17 M with a detection limit of 1×10−16 M. [Copyright &y& Elsevier]