Matching Items (3)

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Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes

Description

Background
Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and

Background
Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems.
Methods
We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe.
Results
Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA.
Conclusions
Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Contributors

Agent

Created

Date Created
  • 2017-10-13

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Feasibility Study of an Optical Caustic Plasmonic Light Scattering Sensor for Human Serum Anti-Dengue Protein E Antibody Detection

Description

Antibody detection and accurate diagnosis of tropical diseases is essential to help prevent the spread of disease. However, most detection methods lack cost-effectiveness and field portability, which are essential features

Antibody detection and accurate diagnosis of tropical diseases is essential to help prevent the spread of disease. However, most detection methods lack cost-effectiveness and field portability, which are essential features for achieving diagnosis in a timely manner. To address this, 3D-printed oblate spheroid sample chambers were fabricated to measure green light scattering of gold nanoparticles using an optical caustic focus to detect antibodies. Scattering signals of 20–200 nm gold nanoparticles using a green laser were compared to green light emitting diode (LED) light source signals and to Mie theory. The change in signal from 60 to 120 nm decreased in the order of Mie Theory > optical caustic scattering > 90° scattering. These results suggested that conjugating 60 nm gold nanoparticles and using an optical caustic system to detect plasmonic light scattering, would result in a sensitive test for detecting human antibodies in serum. Therefore, we studied the light scattering response of conjugated gold nanoparticles exposed to different concentrations of anti-protein E antibody, and a feasibility study of 10 human serum samples using dot blot and a handheld optical caustic-based sensor device. The overall agreement between detection methods suggests that the new sensor concept shows promise to detect gold nanoparticle aggregation in a homogeneous assay. Further testing and protocol optimization is needed to draw conclusions on the positive and negative predictive values for this new testing system.

Contributors

Created

Date Created
  • 2017-08-17

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Identifying Cell Death Pathways Activated by Myxoma virus in Melanoma

Description

Melanoma is a type of skin cancer that can metastasize in advanced stages to other organs such as the brain, lymph nodes, lungs and liver. Current standard treatment options include

Melanoma is a type of skin cancer that can metastasize in advanced stages to other organs such as the brain, lymph nodes, lungs and liver. Current standard treatment options include surgery, radiation therapy, chemotherapy, and immunotherapy. More recently, oncolytic virotherapy is being studied as a new strategy to fight cancer. Specifically, for melanoma, a herpes virus (T-VEC) was approved by the U.S Food and Drug Administration in 2015 to treat advanced disease. Oncolytic viruses have the capacity to replicate mostly in cancer cells while leaving healthy somatic cells free from infection. Additionally, most of these viruses have the ability to induce an immune response against the cancer as well. Myxoma virus (MYXV) causes myxomatosis in European rabbits but not in any other mammal. In humans, MYXV can infect and kill cancer cells acting as an oncolytic virus. However, the mechanisms behind how myxoma kills cancerous cells are not completely known. To investigate this, we treated melanoma murine cancer cells (B16F10) in vitro with different genetically modified myxoma virus mutants, as well as with a novel second mitochondria-derived activator of caspase mimicking drug SMAC-LCL161, to understand the mechanisms by which MYXV induces cell death. In parallel, B16F10 lacZ cells were subcutaneously injected into mice to engraft melanoma tumors. These tumors were treated with intratumoral injections of different viral mutants or armed viruses derived from MYXV along with SMAC-L61. After a period of treatment, the tumors were isolated. Cell death pathways in both cell culture and in tumors obtained from subcutaneous pathways were identified using different techniques. The study showed an increase in activated caspase 3 and cleaved PARP-1 activity in B16F10 lacZ cells from cell culture when compared to cells in vivo however the two apoptosis markers did not track with each other consistently.

Contributors

Agent

Created

Date Created
  • 2019-12