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PubMed | AI Biosciences Inc. and University of Texas Medical Branch
Type: | Journal: Scientific reports | Year: 2016

Zika virus (ZIKV) has gained global attention as an etiologic agent of fetal microcephaly and Guillain-Barr syndrome. Existing immuno-based rapid tests often fail to distinguish between Zika and related flaviviruses that are common in affected regions of Central and South Americas and the Caribbean. The US CDC and qualified state health department laboratories can perform the reverse transcription polymerase chain reaction (RT-PCR) ZIKV test using highly sophisticated instruments with long turnaround times. The preliminary results of a portable and low-cost molecular diagnostics system for ZIKV infection are reported here. In less than 15minutes, this low-cost platform can automatically perform high quality RNA extraction from up to 12 ZIKV-spiked urine samples simultaneously. It can also perform reverse transcription recombinase polymerase amplification reaction (RT-RPA) in 15minutes. The fluorescent signal produced from probe-based RT-RPA or RT-PCR assays can be monitored using LEDs and a smartphone camera. In addition, the RT-RPA and RT-PCR assays do not cross-react with dengue and chikungunya viral RNA. This low-cost system lacks complicated, sensitive and high cost components, making it suitable for resource-limited settings. It has the potential to offer simple sample-to-answer molecular diagnostics and can inform healthcare workers of patients diagnosis promptly.


Chan K.,AI Biosciences Inc. | Wong P.-Y.,AI Biosciences Inc. | Yu P.,AI Biosciences Inc. | Hardick J.,Johns Hopkins University | And 6 more authors.
PLoS ONE | Year: 2016

The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC's ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC's performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings. © 2016 Chan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Chan K.,AI Biosciences Inc. | Coen M.,AI Biosciences Inc. | Hardick J.,Johns Hopkins University | Gaydos C.A.,Johns Hopkins University | And 5 more authors.
PLoS ONE | Year: 2016

Most molecular diagnostic assays require upfront sample preparation steps to isolate the target's nucleic acids, followed by its amplification and detection using various nucleic acid amplification techniques. Because molecular diagnostic methods are generally rather difficult to perform manually without highly trained users, automated and integrated systems are highly desirable but too costly for use at point-of-care or low-resource settings. Here, we showcase the development of a low-cost and rapid nucleic acid isolation and amplification platform by modifying entry-level 3D printers that cost between $400 and $750. Our modifications consisted of replacing the extruder with a tip-comb attachment that houses magnets to conduct magnetic particle-based nucleic acid extraction. We then programmed the 3D printer to conduct motions that can perform high-quality extraction protocols. Up to 12 samples can be processed simultaneously in under 13 minutes and the efficiency of nucleic acid isolation matches well against gold-standard spin-column-based extraction technology. Additionally, we used the 3D printer's heated bed to supply heat to perform water bathbased polymerase chain reactions (PCRs). Using another attachment to hold PCR tubes, the 3D printer was programmed to automate the process of shuttling PCR tubes between water baths. By eliminating the temperature ramping needed in most commercial thermal cyclers, the run time of a 35-cycle PCR protocol was shortened by 33%. This article demonstrates that for applications in resource-limited settings, expensive nucleic acid extraction devices and thermal cyclers that are used in many central laboratories can be potentially replaced by a device modified from inexpensive entry-level 3D printers. © 2016 Chan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Priye A.,Texas A&M University | Wong S.,AI Biosciences Inc. | Ugaz V.M.,Texas A&M University
MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences | Year: 2015

We describe an ultra-portable bioanalysis platform for rapid nucleic acid-based diagnostics using consumer-class quadcopter drones. Our approach exploits the ability to isothermally actuate the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices. Time-resolved fluorescence detection and quantification are achieved using a smartphone camera and integrated image analysis app. Functionality is demonstrated in the context of infectious disease diagnostics (Ebola, Staphylococcus aureus, and Dengue). These advancements make it possible to overcome existing price, performance, portability, and ruggedization. © 15CBMS-0001.


Priye A.,Texas A&M University | Wong S.,AI Biosciences Inc. | Bi Y.,Texas A&M University | Carpio M.,Texas A&M University | And 14 more authors.
Analytical Chemistry | Year: 2016

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone's motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging. © 2016 American Chemical Society.


Wong G.,AI Biosciences Inc. | Wong I.,AI Biosciences Inc. | Chan K.,AI Biosciences Inc. | Hsieh Y.,AI Biosciences Inc. | Wong S.,AI Biosciences Inc.
PLoS ONE | Year: 2015

Background Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. Methodology/Principal Findings In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths. Conclusions/Significance We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings. © 2015 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 209.30K | Year: 2015

DESCRIPTION provided by applicant The goal of this Phase I application is to develop a nanoparticle and surface enhanced Raman spectroscopy SERS based molecular detection system to directly detect norovirus gastroenteritis without the use of target amplification techniques such as polymerase chain reaction or isothermal amplification Noroviruses are the most common cause of epidemic gastroenteritis According to the CDC noroviruses are the most common cause of foodborne disease outbreaks in the United States Norovirus outbreaks can affect people in a variety of settings For example norovirus outbreaks in military forces are regularly reported The virus affects around million people and causes over deaths each year these deaths are usually in less developed countries and in the very young elderly and immuno suppressed Norovirus infections are highly contagious as the infectious dose is less than virus particles and the virus is shed to high titers of particles per gram in the feces We propose to use multifunctional gold coated magnetic nanoparticle AuMNPs and SERS based molecular detection system to capture and detect norovirus RNA in stool samples After sample lysing the capture purification and detection of norovirus RNA will be achieved using AuMNPs that have been functionalized with positively charged peptide nucleic acid capture probes ve PNA and intrinsically strong Raman labels These multifunctional nano sized AuMNPs nm provide for a more rapid and efficient capture of RNA than those offered by micron size particles or dimensional sensing surfaces The use of Raman labels on the AuMNPs as a single tag takes advantage of the well established surface enhancing characteristics of Raman reporter molecules coated on gold nanoparticles Working with nanotechnology expert Chuan Jian Zhong SUNY Binghamton we will develop and optimize the AuMNPs to capture RNA and to form a Raman tag for the direct identification of RNA for norovirus genogroup typing We will carry out AuMNPs separation and magnetic focusing for SERS nanoparticle detection using microchannel electrophoresis with the electroosmotic flow suppressed Working with Professor Robert Gilman of Johns Hopkins University we will carry out proof of principle work to detect genogroups I and II noroviruses in about previously collected and de identified stool samples that have been characterized by real time reverse transcription PCR qRT PCR These samples contain low medium and high concentration of virus as well as different genotypes under each group Our platform is unique as it uses multifunctional AuMNPs to provide RNA capture magnetic manipulation and SERS detection Our Phase I goal is to complete a sensitive and specific assay in min using low viral load samples Our eventual goal is to perform all the assay steps in a closed system when we integrate the detection scheme into a sample preparation cartridge that weandapos re already developing The assay when fully commercialized will be operated in automated manner PUBLIC HEALTH RELEVANCE Noroviruses are the most common cause of epidemic gastroenteritis The virus affects around million people and causes over deaths each year The viruses are transmitted by fecally contaminated food or water by person to person contact and via aerosolization of the virus and subsequent contamination of surfaces A deployable and easy to use molecular diagnostics system that provides rapid accurate and early detection of norovirus infection by public health officials private industries e g cruise ships companies and even the military will promptly alert the authorities to implement sanitizing and precaution efforts to minimize the spread of this illness and its adverse effect on the citizens customers and military readiness


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.90K | Year: 2014

DESCRIPTION (provided by applicant): The aim of this application is to develop a rapid, easy-to-use, and inexpensive multi-sample diagnostic system to identify sexually transmitted infection (STI) pathogens, such as Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG) in non-traditional healthcare settings. According to the CDC, chlamydia and gonorrhea, caused by C. trachomatis and N. gonorrhoeae, respectively, are the first and second most frequently reported STIs in the US and likely the rest of theworld. These STIs are often under-diagnosed, leading to delayed treatment, continued spread and higher public healthcare costs (~ 16 billion annually). As a result of their prevalence and long term health consequences, there is a need for rapid, sensitivemethods of detecting STIs in order for patients to get results and treatment immediately. It is important that patients receive results and initial consultation or treatment during the first patient visit, as they rarely return for a second visit. Poi


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 199.86K | Year: 2014

DESCRIPTION (provided by applicant): Dengue is transmitted mainly by the Aedes aegypti mosquitoes which inhabit the tropics, making Dengue endemic to these areas. It is caused by four genetically and serologically related viruses, termed DENV1, DENV2, DENV3, and DENV4. Dengue infection is a leading cause of illness and death in the tropics and subtropics, including Puerto Rico and the U.S. Virgin Islands, where thousands of U.S. citizens develop dengue fever every year. With three billion of the world's population at risk from dengue, an estimated 50-100 million cases of Dengue Fever (DF), and hundreds of thousands of cases of severe dengue (previously known as Dengue Hemorrhagic Fever or DHF) that occur every year, the demand for a rapid, sensitive, and serotype- specific dengue diagnostics test is high. Patients would be diagnosed sooner to receive treatment, and public health laboratories would have a clearer picture of the true number of dengue cases. Dengue is an acute illness where most patients ar


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 209.72K | Year: 2014

DESCRIPTION (provided by applicant): This SBIR Phase I proposal aims to develop and commercialize a portable, easy-to-use system for nucleic acid preparation for effective molecular diagnostics in low-resource settings (LRS). The technology will also aid in the monitoring of treatment response for patients of global diseases such as AIDS, malaria, and tuberculosis. Because a reliable and reproducible high-quality sample preparation process for NA is critical to the success of most molecular diagnostic assays and the subsequent treatment and monitoring of recovery progress, the availability of an effective sample preparation technology is especially important if the tests are performed in non-traditional or low-resource health care settings. Most processes incurrent commercial sample preparation methods are labor intensive and require special instrumentation. Although commercially available automated systems can perform extensive sample processing, they are expensive for use in non-central laboratory set

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