BEVERLY, MA, United States
BEVERLY, MA, United States

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Tong Y.,Biohelix Corporation | McCarthy K.,Biohelix Corporation | Kong H.,Biohelix Corporation | Lemieux B.,Biohelix Corporation
Journal of Molecular Diagnostics | Year: 2012

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology.


Lemieux B.,Biohelix Corporation | Li Y.,Biohelix Corporation | Kong H.,Biohelix Corporation | Tang Y.-W.,Sloan Kettering Cancer Center
Expert Review of Molecular Diagnostics | Year: 2012

The first near instrument-free, inexpensive and simple molecular diagnostic device (IsoAmp® HSV, BioHelix Corp., MA, USA) recently received US FDA clearance for use in the detection of herpes simplex viruses (HSV) in genital and oral lesion specimens. The IsoAmp HSV assay uses isothermal helicase-dependent amplification in combination with a disposable, hermetically-sealed, vertical-flow strip identification. The IsoAmp HSV assay has a total test-to-result time of less than 1.5 h by omitting the time-consuming nucleic acid extraction. The diagnostic sensitivity and specificity are comparable to PCR and are superior to culture-based methods. The near instrument-free, rapid and simple characteristics of the IsoAmp HSV assay make it potentially suitable for point-of-care testing. © 2012 Expert Reviews Ltd.


Motre A.,Biohelix Corporation | Kong R.,Biohelix Corporation | Li Y.,Biohelix Corporation
Journal of Microbiological Methods | Year: 2011

In this study, we report the development of a helicase-dependent amplification assay for rapid detection of Mycobacterium tuberculosis (MTB). By applying a step-by-step optimization method, the amplification time from an input of 2-copy MTB genomic DNA was reduced from about 60. min to less than 30. min. © 2010 Elsevier B.V.


Tong Y.,Biohelix Corporation | Lemieux B.,Biohelix Corporation | Kong H.,Biohelix Corporation
BMC Biotechnology | Year: 2011

Background: In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).Results: Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BESt™ cassette).Conclusions: The strategies addressed in this proof-of-concept study are independent of expensive equipments, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics. © 2011 Tong et al; licensee BioMed Central Ltd.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 600.00K | Year: 2012

DESCRIPTION (provided by applicant): In this Phase I project, we propose to investigate a novel primer chemistry and probe detection system called the Zip nucleic acids (ZNA ). ZNAs are oligonucleotides conjugated to a number of cationic spermine moietiesthat enhance the effective concentration of primers and probes near nucleic acid targets. This property has been reported to enhance the speed and sensitivity of RT-PCR (Moreau et al. 2009). ZNAs are compatible with Taqman detection formats (Paris et al. 2010). We expect this detection technology will further accelerate our RNA detection assays as well as increase our accuracy. We will compare the performance of ZNA to LNA DNA dual labeled (Tong et al. 2008, Li et al. 2010) and CPT probes. By the endof the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. Our phase I research plan includes 4 aims: 1. Design and test ZNA primers targeting all subtypes of HIV-1, 2.Design and test with both ZNA taqman and ZNA cycling probes for the HIV assay, 3. Develop a simple work flow for extraction of RNA from dry blood spots (DBS) and dry plasma spots (DPS), and 4. Test the sensitivity and specificity of assays using theZNA technology in combination with IsoAmp in a panel of HIV-1 isolates. At the conclusion of Phase I, we will be ready to identify the best probe technology to develop a commercial IsoAmp HIV-1 quantitative assay for commercial distribution in the US and abroad. We will also have a clear indication of the type of sample extraction method that best suits HDA viral load testing. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US. We would also explore commercial release in the rest of the World. PUBLIC HEALTH RELEVANCE: Human immune-deficiency virus (HIV) viral load testing is the standard of care for monitoring anti-retroviral therapy in the United States and Europe. HIV quantitative tests rely of high throughput systems that use the polymerase chain reaction (PCR) and all suffer one major limitation: a need for expensive instrumentation, and skilled personnel to operate the equipment (Fiscus et al. 2006). In the developing World, low-cost CD4+ monitoring tests (Rodriguez et al. 2005) are used because of economic drivers, despite the fact that CD4+ monitoring lags a rise in viral RNA load in cases of therapeutic failure (Vaidya et al. 2010). BioHelix has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that can solve this problem. This technology has 4 advantages over PCR methods of viral load testing: 1) it relies on a low-cost instrument (1/10 the cost of PCR machines), 2) it can amplify RNA faster than DNA and can match the fastest PCR assays (Goldmeyer et al. 2007), 3) it is more tolerant of base variations in primers and probes than PCR, and 4) it is more tolerant to amplification inhibitors found in clinical samples than PCR. In this Phase I project, we will explore the potential application of Zip nucleic acids (ZNA) to enhance the performance of our HIV assays (Tang et al. 2010). By the end of the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US.


Patent
Biohelix Corporation | Date: 2016-02-25

The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.


Patent
Biohelix Corporation | Date: 2014-03-04

The invention provides methods and compositions for enhancing the speed and sensitivity of helicase-dependent amplification through the use of an endonuclease.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 546.87K | Year: 2010

DESCRIPTION (provided by applicant): This SBIR-AT-NIAID Phase I application addresses the problem of malaria diagnostics in resource- limited settings. BioHelix, has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that is tolerant of the DNA amplification inhibitors present in whole blood. This proposal will focus on the application of this technology to the detection of malarial asexual parasites without nucleic acid extraction. Based on our preliminary results, the expected sensitivity of the species-specific molecular test (~100 nucleic acid copies/5L blood) better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). Our specific objectives for this 2-year-long project are: 1. Develop an HDA assay to detect P. falciparum, P. vivax, P. ovale, and P. malariae. 2. Develop competitive internal controls (CIC) for the species-specific assay, as well as a 2-plex assay to detect the CIC with the targets in whole blood. 3. Develop manufacturing processes for the proposed assay kit that will enable field-use of the technology. 4. Evaluate the performance of the assay relative to the gold-standard tests. 5. Obtain CE marking for the assay. 6. Submit a pre-IDE to FDA as a preliminary step to seeking FDA clearance for the test. PUBLIC HEALTH RELEVANCE: As malaria is the leading cause death in endemic regions, the current treatment approach is not based on laboratory, or microscopy testing because establishing quality-assured microscopy in rural and resource- poor settings is difficult. The liberal use of anti-malarial drugs that results from such practices presents a problem in that resistance to first line drug treatments is wide-spread. This SBIR-AT-NIAID proposal focuses on the development of a species-specific for the detection of Plasmodium in blood. More sensitive, species- specific, and stage-specific diagnostic tests will help focus drug treatment, and may slow the spread of anti- malarial drug resistance in the pathogen population. Based on our preliminary results, the expected sensitivity of the species-specific DNA test (~100 nucleic acid copies/5L blood) will be better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). A more sensitive test that can be performed in the field near patients would greatly enhance malaria fighting activities. We anticipate tests in the range of 7- 10 for the HDA tests should be possible, if total test volumes reach 200,000.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 482.81K | Year: 2011

DESCRIPTION (provided by applicant): This STTR-AT-NIAID seeks to develop an integrated nucleic acid system based on research done by Catherine Klapperich's laboratory at Boston University. The BU lab-on-a-chip includes a micro solid phase extraction (lt SPE) column, flap valves and hydrophobic vents to gate fluid movements through micro channels, and multiple reaction chambers for experimental replicates and control reactions. Dr. Klapperich's laboratory has successfully performed nucleic acid amplifications in the chip using BioHelix's isothermal amplification process. Biohelix's proprietary technology is called helicase-dependent amplification (HDA). It uses DNA helicases to separate DNA strands during exponential amplification at a constant temperature of65:C. Like the polymerase chain reaction (PCR), HDA assays use a competitive internal control (i.e., a template DNA of known concentration spiked into the raw sample) that can be amplified by the same primers as the analyte, but detected separately; henceallowing us to detect amplification inhibitors in direct clinical samples. The objectives of Phase I are to: 1) modify the BU microfluidic device design to incorporate a lateral flow strip as a means of detecting amplification products using the naked eye; and 2) demonstrate the feasibility of performing integrated tests using the device from aim 1 using CT and NG spiked at 104 in 1 mL urine to establish proof of concept for integrated assays performed in a modified BU chip that includes a lateral flow strip. Our specific aims for Phase II will be to develop a lateral flow reader sub-system, integrate it with the BU instrument to build a pre-commercial looks-like / works-like prototypes, simplify chip design and fabrication to allow for pilot scale manufacturing, and implement a pilot scale manufacturing for the disposable at BioHelix / Quidel. At this stage Qiagen is the most likely manufacturer for the reader. At the conclusion of Phase II, we should be ready for clinical validation of the new assay system. PUBLIC HEALTH RELEVANCE: This Phase I STTR-AT-NIAID project seeks proof-of-concept project will focus on the most abundant sexually transmitted disease (STD) pathogens: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). The scientific literature clearly shows that molecular testing is the most sensitive means of detecting CT and NG and the molecular CT/NG high throughput screening market is currently valued at over 300M/year. Moreover, CDC urges STD clinics to test patients with POC tests if health care workers suspect these patients are unlikely to return to the STD clinic to learn the results of the test. Unfortunately, there are no point-of-care (POC) CT NG molecular tests, and existing POC molecular testing systems like the GeneXpertare too costly for use in STD clinics. This proposed project would seek to remedy to this short coming. We propose to develop a low-cost POC molecular diagnostic system using a design developed by Dr. Catherine Klapperich's laboratory at Boston University(BU). Although the current BU disposable can perform our proprietary isothermal amplification reactions, it does not allow for low cost, instrument-free detection of amplification products; i.e., a fluorescence microscope can be used to detect product formation but this is not a commercially viable option. The device we envisage for Phase I will incorporate a lateral flow strip as a means of detecting the presence or absence of nucleic acid amplification products by simple visual inspection. BioHelix has experience in developing molecular tests using lateral flow based detection.


Compositions and methods useful in nucleic acid assays are provided. The invention permits detection of multiple target sequences and control nucleic acids using isothermal nucleic acid amplification methods and subsequent detection of amplification products at different temperature steps by at least two probes with different annealing temperatures. This method can be used in isothermal nucleic acid amplification reactions to detect multiple targets of interest. In a particular example, cycling hybridization probes with different spectral and hybridization temperatures are used to detect different target sequences. Probes become fluorescent when they are cleaved by a thermostable ribonuclease, which only acts when the probes are hybridized to their respective templates.

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