Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women.

BACKGROUND: An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women. OBJECTIVE: Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection. METHODS: This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10-1 to 10-9. RESULTS: An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 µl input, 76.4 copies/10 µl input, and 34.4 copies/10 µl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 µl input, 166 copies/10 µl input, and 43.7 copies/10 µl input, respectively. CONCLUSION: TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

Anti-bacterial and anti-biofilm properties of green synthesized copper nanoparticles from Cardiospermum halicacabum leaf extract.

In the present study, a copper nanoparticle (Cu NPs) was synthesized by a green synthesis method with Cardiospermum halicacabum leaf extract. The surface area of Cu NPs was measured with dynamic light scattering (DLS). UV-Vis spectrum clearly illustrates the typical absorption peak of Cu NPs. The crystalline property of Cu NPs was confirmed from the XRD pattern. TEM analysis clearly indicates the average particle size of synthesized Cu NPs was in the range of 30-40 nm with hexagonal shape. Energy-dispersive spectroscopy confirms the major strong peaks of Cu NPs. FTIR analysis confirms the existence of various functional biomolecules over the metal nanoparticles and they are playing an important role in the formation of Cu NPs. The antibacterial and anti-biofilm analyses were carried out to confirm their aptitude for biomedical applications. Interestingly, Cu NPs control the development of biofilm by attaching over the cell wall and disturb their growth and development.

Facile synthesis of reduced graphene oxide using Acalypha indica and Raphanus sativus extracts and their in vitro cytotoxicity activity against human breast (MCF-7) and lung (A549) cancer cell lines.

In the present study, an eco-friendly approach is adapted for the synthesis of reduced graphene oxide (rGO's) by a simple hydrothermal reaction using two plant extracts namely Acalypha indica and Raphanus sativus. After the hydrothermal reaction, GO turns into a black color from brown color, which indicates the successful reduction of graphene oxide. Further, various characterization techniques such as UV-Vis spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction is used to confirm the physicochemical properties of synthesized rGO's. Raman analysis confirms the reduction of GO by noticing an increase in the ID/IG ratio significantly. Field emission scanning electron microscopy and transmission electron microscopy clearly show the morphology and crystalline nature of rGO's. FT-IR spectrum confirms that the bioactive molecules of the plant extract (i.e. polyphenols, flavonoids, terpenoids, etc.) playing a key role in the elimination of oxygen groups from the GO surface. Further, the synthesized rGO's are tested for their potential against human lung and breast cancer cell lines. A significant cancer cell inhibition activity is obtained even in the less concentration of rGO's with IC50 values for lung cancer cell lines are 38.46 µg/mL and 26.69 µg/mL for AIrGO and RSrGO, respectively. Similarly, IC50 values for breast cancer cell lines are 35.97 µg/mL and 33.22 µg/mL for AIrGO and RSrGO, respectively.

A statistical approach of zinc remediation using acidophilic bacterium via an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology.

The aim of the present study was to isolate an indigenous acidophilic bacterium from tannery effluent contaminated sludge (TECS) sample and evaluate its potentiality towards the removal of zinc using an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology in zinc spiked soil at an initial concentration of 1000 mg/kg. The isolated acidophilic bacterium was characterized by biochemical and 16S rRNA molecular identification and was named as Serratia marcescens SMAR1 bearing an accession no. MG742410 in NCBI database. The effect of pH and inoculum dosage of SMAR 1 strain showed an optimal growth at pH 5.0 and 4% (v/v) respectively. Based on these experimental data, a statistical analysis was done using Design Expert computer software, v11 to study the interaction between the process parameters with respect to zinc reduction as an output response. Electrokinetic experiments were conducted in a customised EK cell under optimised process conditions, employing titanium electrodes. Experiments for zinc removal were demonstrated for bioleaching, electrokinetic (EK) and BEER technology. On comparing, the integrated process was found to evidence as an excellent metal remediation option with a maximum zinc removal of 93.08% in 72 h than plain bioleaching (72.86%) and EK (56.67%) in 96 h. This is the first report of zinc removal in a short period of time using Serratia marcescens. It is therefore concluded that the BEER approach can be regarded as an effective technology in cleaning up the metal contaminated environment with an easy recovery and reuse option within short period of time.

Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301µg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496µg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of V. pachynema extract was 58.1µg/ml (CQ-s) and 71.46µg/ml (CQ-r), while nano-CdS IC50 was 76.14µg/ml (CQ-s) and 89.21µg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8µg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of CdS nanoparticles and Cd ions in aqueous solution was also assessed in mud crabs, showing higher toxicity of aqueous Cd ions if compared to nano-CdS. Overall, our results underlined the efficacy of green-synthesized CdS nanoparticles in malaria vector control, outlining also significant impacts on the enzymatic activity of non-target aquatic organisms, with special reference to mud crabs.