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Garber E.A.E.,U.S. Food and Drug Administration | Venkateswaran K.V.,Radix Biosolutions, Ltd | O'brien T.W.,Tetracore Inc.
Journal of Agricultural and Food Chemistry | Year: 2010

Detection of proteinaceous toxins in complex heterogeneous mixtures requires highly specific and sensitive methods. Multiplex technology employing multiple antibodies that recognize different epitopes on a toxin provides built-in confirmatory analysis as part of the initial screen and thereby increases the reliability associated with both presumptive positive and negative results. Polyclonal and monoclonal antibodies were obtained for abrin, botulinum toxins, ricin, and Staphylococcus enterotoxins A, B, and C (SEA, SEB, and SEC). Food samples were spiked with the toxins either individually or mixed and analyzed following 40-fold dilution. Abrin, botulinum toxin A complex, ricin, and SEB displayed limits of detection in the original food samples ranging from 0.03 to 1.3 μg/mL, from 0.03 to 0.07 μg/mL, from 0.01 to 0.1 μg/mL, and from <0.01 to 0.03 μg/mL, respectively. Redundancy, that is, multiple antibodies for each toxin, some recognizing different epitopes or displaying different binding affinities, provided a "fingerprint" for the presence of the toxins and built-in confirmation, thus reducing the likelihood of false-positive and false-negative results. Inclusion of internal controls, including a unique protein, helped control for variations in dilution. Paramagnetic microspheres facilitated the detection of analyte in foods containing particulate matter incompatible with the use of filter plates normally used in the wash steps of assays employing standard polystyrene microspheres. © 2010 American Chemical Society.


Goldman E.R.,U.S. Navy | Anderson G.P.,U.S. Navy | Zabetakis D.,U.S. Navy | Walper S.,National Research Council Postdoctoral Fellow resident | And 7 more authors.
Toxins | Year: 2011

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicit), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. © 2011 by the authors; licensee MDPI, Basel, Switzerland.


Hestekin C.N.,Northwestern University | Hestekin C.N.,University of Arkansas | Lin J.S.,Northwestern University | Lin J.S.,Stanford University | And 7 more authors.
Electrophoresis | Year: 2011

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10min. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Almassian D.R.,Tetracore Inc. | Cockrell L.M.,Tetracore Inc. | Nelson W.M.,Tetracore Inc.
Chemical Society Reviews | Year: 2013

A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies. © 2013 The Royal Society of Chemistry.


Basile A.J.,Centers for Disease Control | Horiuchi K.,Centers for Disease Control | Panella A.J.,Centers for Disease Control | Laven J.,Centers for Disease Control | And 5 more authors.
PLoS ONE | Year: 2013

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic cross-reactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression "Logitboost" as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 169.40K | Year: 2015

DESCRIPTION provided by applicant Increased human travel urbanization and climate changes have resulted in a wide geographic spread of febrile illness caused by both Dengue Virus DENV and Chikungunya Virus CHIKV These two infections show a substantial overlap in clinical presentation In addition both are also co prevalent in many countries This poses a significant challenge to clinicians as the two illnesses require different clinical management A recent infection can only be confirmed by detection of the virus in a blood sample as positive results in serological assays may instead be indicative of prior infection However as antibodies develop in response to the infection the viral load in the blood decreases Thus sensitive and specific detection of these viruses is imperative for an accurate diagnosis Typically molecular methods that employ reverse transcriptase real time polymerase chain reaction rtRT PCR based detection of viral RNA require nucleic acid extraction of whole blood samples In this Phase I plan we propose to develop a multiplexed molecular assay for the direct detection of either DENV or CHIKV in blood samples and then to incorporate this multiplexed assay into a Collect to Test C T system in order to overcome the need for complex sample processing The C T system includes a simple blood collection and processing method that requires no pipetting or sample manipulation combined with a cartridge containing the dried down assay reagents for simultaneous sample preparation and template amplification The sample preparation and cartridge loading are equivalent to methods currently used to run lateral flow devices This system will allow for simultaneous detection of DENV and CHIKV by rtRT PCR from a minimal volume of whole blood i e a finger prick without the need for centrifugation or time consuming extraction methods Critical to this is the development of a rtRT PCR based assay optimized for the detection of viral RNA directly from blood samples and requiring no further sample extraction These reagents together with the C T system will be compatible with the T CORandquot portable thermocycler Tetracore has extensive experience in the development of highly sensitive and specific dried down room temperature stable assays for real time PCR including a DENV specific assay previously developed with Naval Medical Research Center This assay was systematically evaluated and successfully tested using clinical samples on a commercial laboratory rtRT PCR platform Based on our preliminary data and experience we are confident to propose this plan to develop a multiplex rtRT PCR assay for differential detection of DENV and CHIKV that can be combined for use with the C T cartridge and the T COR portable thermocycler to create an integrated point of care system that can be equally effectively in both public health labs and low resource settings PUBLIC HEALTH RELEVANCE Dengue and Chikungunya fevers are both mosquito borne viral diseases with similar early clinical presentations but requiring distinctly different medical management Thus accurate diagnosis is critical to ensure optimal patient outcomes In Phase I of this project we propose to develop a rapid molecular diagnostic multiplex assay for direct detection and differentiation of Dengue and Chikungunya virus in whole blood compatible with a point of care POC device designed for use in low resource settings In Phase II we plan to further validate this assay in public health labs endemic areas and low resource diagnostic settings such as remote test labs and physiciansandapos offices using real world samples


PubMed | Swine Vet Consulting L.L.C., University of Veterinary Medicine, Poldanor SA, Warsaw University of Life Sciences and 3 more.
Type: | Journal: Research in veterinary science | Year: 2016

Recently oral fluid has become a novel sample type for pathogen nucleic acid and antibody detection, as it is easy to obtain with non-invasive procedures. The objective of the study was to analyze porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV) circulation in growing pigs from three Polish production farms, using Real Time PCR and ELISA testing of oral fluid and serum. Oral fluids were collected every 2weeks, in the same 3-4 pens of pigs aged between 5 and 17weeks. Additionally, blood samples were collected every 4weeks from 4 pigs corresponding to the same pens as oral fluid and tested for the presence of PRRSV nucleic acid (pooled by 4) and antibodies. In farm A no PRRSV circulation was detected and only maternal antibodies were present. In farm B and farm C antibodies to PRRSV in serum and oral fluid were detected in most samples. In farm B PRRSV Type 1 was detected in 80.9% of oral fluid samples and in 58.3% of serum pools, and in farm C in 92.8% of oral fluid samples and 75% serum pools. Striking differences were observed between different pens in PRRSV detection patterns. In farms B and C ORF5 sequence analysis showed the presence of wild type strains which were about 84-85% identical to the modified live vaccine used. In all three farms two waves of IAV shedding with oral fluid were detected, in weaners and fatteners.


Trademark
Tetracore Inc. | Date: 2012-06-14

Diagnostic kits consisting primarily of real-time nucleic acid detection and/or diagnostic reagents, testing device, disposable sample vials, and associated instructional materials provided therewith, for the detection and/or diagnosis of harmful microorganisms or biological contaminants.


Carrillo C.,U.S. Department of Agriculture | Prarat M.,U.S. Department of Agriculture | Vagnozzi A.,U.S. Department of Agriculture | Calahan J.D.,Tetracore Inc. | And 3 more authors.
Journal of Clinical Microbiology | Year: 2010

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID 50) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as post-mortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


PubMed | Tetracore Inc.
Type: Journal Article | Journal: Chemical Society reviews | Year: 2013

A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies.

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