Rachna Verma

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.

Nanobiocomposite platform based on polyaniline-iron oxide-carbon nanotubes for bacterial detection.

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.

Polyaniline/carbon nanotubes platform for sexually transmitted disease detection.

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 × 10−6 M to 1 × 10−17 M with detection limit of 1.2 × 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). Copyright

Diagnostic implications of 16S ribosomal assay for gonorrhoea

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 Neisseria gonorrhoeae. This study was undertaken to evaluate the performance of PCR based on 16S ribosomal gene in comparison with a porA pseudogene-based assay. Methods The specificity of both the porA pseudogene-based PCR and 16S ribosomal gene PCR was checked against a panel of strains comprising of non N gonorrhoeae Neisseria sp (NgNS) and other gram-negative and gram-positive bacteria. The sensitivity studies were performed using different dilutions of N gonorrhoeae 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 N sicca. The porA pseudogene-based PCR was highly specific. Analytical sensitivity of 16S-based ribosomal assay was more than that of porA 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 porA pseudogene target is highly specific for N gonorrhoeae and may be used as a supplemental assay.

Coupling electrochemical response of a DNA biosensor with PCR for Neisseria gonorrhoeae detection.

Early diagnosis of gonococcal infections is important with regard to a patients 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.

P2.008 Mistaken Case of Child Abuse: A Case Report

Introduction The diagnosis of child abuse is based on a combination of child’s history, physical findings, and when appropriate, laboratory and other tests. Overall, the diagnosis is often complicated but suspicion should always be followed by further investigations. Formulating a conclusion and reaching a diagnosis of child abuse may require the assistance and coming together of different specialities in the hospital. Case history: A seven year old girl presented to the Dermatology and Venereology OPD of a tertiary hospital in New Delhi, India with chief complaints of vaginal discharge for last 4 years. The vaginal swab(s) on Gram stain revealed numerous pus cells with GNDC, intracellular as well as extracellular. The child was treated based on clinical suspicion of gonorrhoea. However, RCUT put up from suspected colonies on modified Thayer Martin medium was positive for Neisseria meningitidis. In addition, crgA gene PCR from DNA extracted from the swab as well as the isolate was positive for N. meningitidis while opa-gene PCR for N. gonorrhoeae was negative. Although she was initially treated for suspected gonococcal infection, the clinical diagnosis was refuted by the results of culture and PCR. Discussion & conclusion: The findings of the present case emphasise the importance of careful culture techniques for isolation of organisms & their correct identification which is the cornerstone of appropriate therapy. It also drives home the necessity of using lactose in Rapid Carbohydrate Utilization Test (RCUT), which is crucial to differentiate between N. meningitidis and N. lactamica. It is also important for the laboratory (especially one that is considered a referral laboratory) to have capacity to perform molecular tests to confirm or refute presumptive findings, as was done in the present case. This observation stresses that an interdisciplinary approach appears to be a valuable tool for evaluating such children.

P4-S1.02 Coupling of electrochemical detection with PCR amplification for sensitive detection of Neisseria gonorrhoeae

Background Despite the recent development of different detection methods, better diagnostic tools are required for quick and reliable detection of pathogens. Use of biosensors for detection of pathogens is gradually gaining momentum. However, PCR amplification of DNA target is still necessary for application on biosensor for accurate detection of the pathogen. An in-house PCR using self-designed primers targeting the opa gene (GenBank accession no. PUID 9716120 SNUM 2706 Ng_opa) was performed. The generated amplicons were used to evaluate the DNA biosensor utilising a 19-mer oligonucleotide sequence (GenBank PUID SNUM: 9716119 2705 Ng_opa) as probe. Methods In-house PCR was standardised and an amplified product (amplicons) of 188 bps were obtained for positive samples. Fabrication of bioelectrode was performed by immobilising the activated probe onto pre-treated screen-printed gold electrodes. The fabricated nucleic acid functionalised gold electrode was characterised using, SEM, CV, DPV techniques. The presence of target DNA was detected electrochemically by monitoring the redox peak of methylene blue indicator. Standardisation of working conditions was done using complementary, non-complementary, one base mismatch DNA, and amplicons from standard strain of N. gonorrhoeae (ATCC 49226) & 16 clinical isolates. In addition, DPV measurements of hybridised bioelectrode with amplicons of 26 clinical samples of which 10 were culture positive, was done. A cut-off value for positives was determined by using the software STATA (version 9). Results The analytical sensitivity of PCR was 10–17 M of DNA and the bioelectrode could detect up to 1.0×10–20 M of the DNA amplicons. An 11.49% decrease in signal intensity was taken as the cut-off. Samples giving an equivalent or more decrease than this value were considered as positives. Conclusions The coupling of electrochemical detection with PCR amplification showed the advantage of higher sensitivity and increased specificity for detection of N gonorrhoeae. This may prove to be particularly valuable for the identification of asymptomatic infections and could greatly improve gonorrhoea control.