Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinaseﾠpolymeraseﾠamplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sampleﾠconcentrationﾠusing aﾠstandard curveﾠIn this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user

Multimodal optical imaging platform for the early diagnosis for oral neoplasia

Early diagnosis is critical to reducing the global burden of oral cancer. In the US, 65% of oral cancer patients are diagnosed after regional metastasis; these patients have a 50% five-year mortality compared to 17% for those with localized disease. A major reason for late diagnosis is that clinicians are unable to accurately distinguish neoplastic lesions, which require treatment, from benign lesions. Furthermore, clinicians are unable to accurately select to biopsy the site with the worst diagnosis within a larger lesion. Please download the file below for full content

Spectroscopic probe for in vivo measurement of raman signals

An optical probe is disclosed which is suitable for rapidly measuring Raman spectra in vivo. The probe is designed to minimize interfering Raman and fluorescence signals generated within the probe itself. In addition, the probe design is compact, making it particularly suited for use in confined spaces such as body cavities. In one embodiment, the probe is employed to detect tissue abnormalities such as cervical cancers and precancers.Board of Regents, University of Texas Syste

Real-time video mosaicing with a high-resolution microendoscope

Microendoscopes allow clinicians to view subcellular features in vivo and in real-time, but their field-of-view is inherently limited by the small size of the probe's distal end. Video mosaicing has emerged as an effective technique to increase the acquired image size. Current implementations are performed post-procedure, which removes the benefits of live imaging. In this manuscript we present an algorithm for real-time video mosaicing using a low-cost high-resolution microendoscope. We present algorithm execution times and show image results obtained from in vivo tissue

Aptamer-Targeted Gold Nanoparticles As Molecular-Specific Contrast Agents for Reflectance Imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer−gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(−) cell lines treated with the anti-PSMA aptamer−gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications

A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA

Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA), this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings

Development of a multimodal foveated endomicroscope for the detection of oral cancer

A multimodal endomicroscope was developed for cancer detection that combines hyperspectral and confocal imaging through a single foveated objective and a vibrating optical fiber bundle. Standard clinical examination has a limited ability to identify early stage oral cancer. Optical detection methods are typically restricted by either achievable resolution or a small field-of-view. By combining high resolution and widefield spectral imaging into a single probe, a device was developed that provides spectral and spatial information over a 5 mm field to locate suspicious lesions that can then be inspected in high resolution mode. The device was evaluated on ex vivo biopsies of human oral tumors

Development of a single-board computer high-resolution microendoscope (PiHRME) to increase access to cervical cancer screening in underserved areas

Over 85% of cervical cancer deaths occur in developing countries.1 Even though the early detection and treatment of cervical precancerous lesions has been shown to prevent invasive cervical cancer, limited resources make it difficult to implement standard cervical cancer screening methods, such as the Pap Smear, in low-resource areas. Instead, many developing countries rely on the visual inspection of the cervix with acetic acid (VIA) to help identify precancerous and cancerous lesions. While VIA has a high sensitivity (82.14%), it has a poor specificity (50.00%), resulting in the overtreatment of women and misallocation of limited resources.2 Recent studies have shown that combining VIA with high-resolution microendoscope (HRME) imaging increases the specificity of cervical cancer screening.3-4 The HRME is a low-cost imaging system (~$2,100) that allows the user to image epithelial tissue in vivo at sub-cellular resolutions at the point-of-care. The current HRME imaging system is also accompanied with automatic image analysis software to distinguish normal and low-grade lesions from high-grade precancerous and cancerous lesions of the cervix. Please click Additional Files below to see the full abstract