FLUXERGY ANALYZER BETA A MODULAR PLATFORM FOR POINT-OF-CARE PCR

Transcription

1 ß BETA FLUXERGY ANALYZER BETA A MODULAR PLATFORM FOR POINT-OF-CARE PCR Fluxergy LLC Alton Parkway Suite 15 Irvine, CA USA (949) 5-41 inquire@fluxergy.com

2 OBJECTIVE To introduce the Fluxergy point-of-use platform and illustrate its flexibility and capability for conducting gold standard DNA and RNA detection via polymerase chain reaction (PCR) in varying sample types. Using real-time PCR on the Fluxergy Analyzer Beta, Fluxergy has detected a variety of nucleic acid biomarkers with applications ranging from veterinary medicine to quality assurance. The Fluxergy device and overall system serves to spread distributed molecular testing by providing a cost-effective, portable and sensitive solution for testing at the source of sample collection. BACKGROUND In a point-of-care setting, sample preparation becomes a bottleneck in PCR testing due to several limitations. Namely, lengthy processes are needed to extract and purify the specified target from raw sample. This extraction process can take upwards of a few hours and involves many labor-intensive handling steps that require a highly trained technician. Moreover, molecular testing thus requires outsourcing to 3rd party laboratories often increasing complexity due to logistics (i.e. shipping, maintaining sample integrity) and further exaggerating the turnaround time to result. This delay can be a critical period for an infectious patient or high-valued sample potentially prohibiting immediate and appropriate therapy or action. Fluxergy combats this by automating the sample preparation process via fluid and chemical manipulations to allow for direct seeding of crude samples (e.g. plasma, whole blood, feces, etc.) and detection of biomarkers in a disposable microfluidic cassette, the Fluxergy Card. Fluxergy has developed a modular biochemistry and microfluidic interface that accepts a variety of sample types from various species for biological target detection. Species Sample Matrices Targets Equine Canine Feline Bovine Porcine Human è è Whole blood Plasma Feces Respiratory swab Saliva Urine Bacterial Viral Fungal è Endogenous è Figure 1. Capability of the Fluxergy Analyzer Beta for POC PCR. The Fluxergy Analyzer Beta allows for detection of bacterial, viral, fungal and endogenous biological targets in multiple sample types for multiple species. The Fluxergy process overcomes inhibition via automated sample handling. Fluxergy s Applications lab is in constant development of novel matrix workflows. FLUXERGY S POINT-OF-CARE DIAGNOSTIC SYSTEM The Fluxergy real-time qpcr device can be broken into two primary parts: a disposable test card, the Fluxergy Card which accepts input sample, and a device that powers and monitors the test card. Specifically, when a test card is inserted into the device, the device controls and monitors the test card through optical, fluidic, and electronic subsystems. The device acts as a general sensor system for a modular test card allowing for fluorescent, colorimetric, image-based, and electrochemical measurements. The overall testing workflow is designed for a user with minimal training. Raw patient sample is collected, mixed with the target specific buffer, and dispensed into the test card loading port using a capillary loading transfer pipette. The test card is then placed into the device and results are exported within 35 minutes depending on the assay. Each test card and associated PCR is self-contained and is a closed system from the device preventing sample, device, and user cross-contamination.

3 DISPENSE INSERT TEST Figure 2. Workflow. (A) Mix raw sample with target specific buffer and dispense into test card. (B) Insert test card into device. (C) Run test and export results in < 35 minutes. FLUXERGY CARD Each self-contained test card has a highly integrated electro-fluidic environment that interfaces with the device. The test card channels and embedded electronics allow for easily adaptable designs to accommodate assay specific reaction volumes and requirements. This capability allows for multiplexing of biological targets as multiple assay chemistries are lyophilized and stored on a single test card allowing for multi-target detection and quantitation. The Fluxergy Analyzer Beta currently supports detection of 6 individual PCR targets. Figure 3. The Fluxergy Analyzer Beta set-up. Taking up less than 1 ft 2, the Fluxergy Analyzer Beta requires a minimal amount of bench space. The platform is also portable and can be battery powered for field usage. The software is capable of being employed on a desktop, laptop, tablet or phone. FLUXERGY ANALYZER BETA The Fluxergy qpcr device has an integrated optics and computer system with zero moving parts. The optical sensor system captures the entire microfluidic test card in real-time and monitors the intercalating or probe-based dye reaction(s) with high sensitivity. In addition, Fluxergy improves test accuracy by applying advanced image-processing algorithms to the reaction zones(s). This yields highly-accurate and consistent Ct values suitable for real-time qpcrs based on titer value, such as HIV viral load or copy number detection. Once the PCR is complete, two-channel fluorescent detection enables positive control implementation for error checking and erroneous result prevention. Finally, the Fluxergy software user interface serves to interpret test data into a result for the user. The software is modular in nature and can be used from a laptop, tablet, or phone with the capability of controlling up to 255 devices from one UI. Lastly, the software was designed to disperse data appropriately and can integrate into LIMS, EMR and other cloud based data management systems.

4 DATA The following data is shown as a snapshot of applications for the Fluxergy Analyzer Beta. Specific pathogens detected include HIV-1, Clostridium difficile, FeLV, Salmonella and Streptococcus equi subsp. equi in human, feline and equine samples , Viral Particles/mL 447, Viral Particles/mL 7,78, Viral Particles/mL Detection of HIV-1 viral RNA in human blood plasma The above graph shows amplification of HIV-1 in HIV-1 positive EDTA treated blood plasma on the Fluxergy Analyzer Beta. Samples were acquired from acquired and previously categorized by a 3rd party centralized laboratory for viral RNA. Direct PCRs were performed using reactions of sample with primers and probe specific to a conserved region of the gag gene. Target was amplified using Fluxergy s HIV-1 Viral RNA Buffer. A linear relationship can be seen between resultant cycle threshold and titer concentration Clinical Sample, Unknown Pathogenic Load Synthetic Template, 4 copies/ml of Feces Detection of Clostridium difficile A and B in human feces A 6 sterile rayon swab was inserted into Clostridium difficile toxin A and B positive human feces. Feces was acquired from and previously categorized by ispecimen via PCR. The swab was then inserted into 1mL of Fluxergy Hydration Buffer for 5 seconds. The swab was removed and discarded. After agitating the sample, PCR was conducted using the Fluxergy Analyzer Beta with % sample and Fluxergy PCR Clostridium difficile A/B Buffer. As a positive control, synthetic template at a concentration of 4 cp/ml was spiked into C. difficile toxin A/B negative feces. The resultant PCR was performed with the same process as the clinical sample. Further, additional positive and negative samples were tested showing % sensitivity and specificity. See Fluxergy s Clostridium difficile A/B Assay Spec Sheet.

5 copies/ml 5 copies/ml 6 copies/ml Detection of Feline leukemia virus in feline whole blood Feline leukemia virus (FeLV) synthetic template was spiked in negative feline whole blood. Feline whole blood was acquired from BioreclamationIVT. PCR reactions consisted of spiked whole bloodand a Fluxergy FeLV Buffer consisting of FeLV provirus specific primers and probe. Concentrations of 4, 5 and 6 NA cp/ml of blood were run on the Fluxergy Analyzer Beta Synthetic Template Clinical Sample Detection of Salmonella in equine feces PCR was conducted using the Fluxergy Analyzer Beta with clinical salmonella positive equine feces and a Fluxergy buffer with salmonella specific primers and probes. As a positive control, synthetic template at a concentration of 9 cp/mlwas spiked into salmonella negative equine feces. Negative sample showed no amplification. Positive pathogen specific detection can be seen for both clinical and spiked samples. 35 Detection of Streptoccocus equi subsp. equi in equine guttural pouch lavage Clinical sample, specifically equine guttural pouch lavage positive for S. equi equi, was tested using the Fluxergy Analyzer Beta and S. equi equi assay. Sample was and previously categorized by UC Davis. Results showed amplification and positive agreement with the reference laboratory, standard of care assay.

6 NOT JUST FOR PCR, BUT ALSO FOR OTHER ASSAYS By reconfiguring the sub-systems and various sensors in different combinations along with changing the modular dimension and electronic requirements on the test card, the Fluxergy Analyzer Beta can accommodate assay types outside of PCR including chemistry, immunochemistry and cytometry. The platform is designed to consolidate an entire laboratory into a single device thus negating the need for multiple devices, manuals, consumables and other sources of overhead. Become a partner in making laboratory testing accessible to all. BECOME A DEVELOPER Fluxergy is committing to translating benchtop innovations to a format that is easy-to-use and built for the field where they can have the most impact. The Fluxergy Applications Lab is open for collaboration and currently testing new biomarkers in novel sample matrices such as soil, plant lysate and tissue. The Fluxergy Analyzer Beta accommodates pre-validated 3rd party assays for crude sample applications and also serves as a cost-effective real-time thermocycler for research use. APPLICATIONS ü Veterinary ü Medical ü Research ü Companion Diagnostics ü Agriculture ü Food Inspection ü Forensics ü Defense ü Space