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The Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+) on the Verigene® System is a qualitative nucleic acid multiplex test intended to simultaneously detect and identify multiple respiratory virus nucleic acids in nasopharyngeal (NP) swab specimens from individuals with signs and symptoms of respiratory tract infection. The following virus types and subtypes are identified using the RV+: Influenza A, Influenza A subtype H1, Influenza A subtype H3, 2009 H1N1, Influenza B, Respiratory Syncytial Virus (RSV) subtype A, and RSV subtype B. The test is not intended to detect Influenza C virus. Detecting and identifying specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection, if used in conjunction with other clinical and laboratory findings. Negative results for Influenza A, Influenza B, or RSV do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis, treatment, or patient management decisions. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection. Performance characteristics for Influenza A Virus were established when Influenza A/H3, A/H1, and 2009 H1N1 were the predominant Influenza A viruses circulating. These characteristics may vary when other Influenza A viruses are emerging. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions used specifically for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The entire RV+ test is performed on the Verigene® System, which is a bench-top molecular diagnostics workstation that consists of two instruments, the Verigene Processor SP and the Verigene Reader. The Verigene Processor SP performs the assay steps on each sample by using a robotic pipettor to transfer and mix reagents within and between separate testing modules designed for nucleic acid extraction, target amplification, and the Verigene Hybridization Test. The Verigene Hybridization Test module is the same as in the original Verigene System with added modules for nucleic acid extraction and RT-PCR target amplification. Key functions of the Verigene Processor SP include: 1) Reading of the barcode identification label on inserted Test Consumables to maintain positive identification of patient samples throughout processing. 2) Facilitation of nucleic acid extraction, multiplex RT-PCR target amplification, and the Verigene Hybridization Test. 3) Real-time communication of test processing status to the Reader. The Verigene Reader is the same instrument as in the FDA-cleared RVNATSP. It is a free-standing instrument with a touch screen control panel and a wand-based barcode scanner. It utilizes a graphical user interface to guide the user through the process of ordering tests and reporting results. There are no serviceable parts and no user calibration is required. Interaction with the touch screen is minimized through barcode use. This instrument also serves as the reader of the Test Cartridges using advanced optics. The key functions of the Verigene Reader include: 1) Entry and tracking of specimen identification numbers via manual keyboard input or via barcode-reader wand. 2) Test selection for each specimen. 3) Automated transfer of specimen processing instructions on Test Cartridge-specific basis to linked Processor SP unit(s). A single Reader unit can control up to 32 Processor units. 4) Automated imaging and analysis of Test Cartridges. 5) Results display. 6) Results report generation. RV+ consumables within each single-use disposable test kit include: (i) Tip Holder Assembly; (ii) Extraction Tray; (iii) Amplification Tray; and (iv) RV+ Test Cartridge. The kit components are inserted into the corresponding module of the Verigene Processor SP prior to each test, and the sample is added to the Extraction Tray. Patient information is entered into the Reader to initiate the test procedure. 1) Tip Holder Assembly - The robotic pipettor picks up pipettes from the Tip Holder Assembly. The pipettes are used for mixing and transferring reagents within the test procedure. 2) Extraction Tray – Nucleic acids are extracted from the sample by using magnetic bead-based methods within the Extraction Tray. Each Tray contains reagents for a single extraction procedure. A robotic pipette transfers reagents to designated wells within the Extraction Tray to affect the steps of lysis, capture of nucleic acids onto the magnetic beads, washing, and eluting the isolated nucleic acids from the magnetic beads. 3) Amplification Tray – The isolated nucleic acids are amplified by using multiplex RT-PCR within the Amplification Tray. Each Tray contains reagents for a single multiplex RT-PCR procedure. A robotic pipette transfers the reagents to a specific well within the Amplification Tray. A set thermal profile is then initiated to perform all of the amplification related steps including UDG-based decontamination, reverse transcription, and multiplex PCR in a single tube. Upon completion, an aliquot of the amplified sample is mixed with hybridization buffer containing the virus specific mediator probes. The sample is then transferred to the Test Cartridge. 4) RV+ Test Cartridge for Verigene Hybridization Test – The virus-specific and subtype-specific amplicons are detected and identified within a Test Cartridge by using specific nucleic acid probes in conjunction with gold nanoparticle probe-based detection technology. Each Test Cartridge is a self-contained, laboratory consumable that consists of two parts. The upper housing of each cartridge is called the "reagent pack" and contains reservoirs filled with the detection reagents. When in place with the 'substrate holder', the reagent pack creates an air-tight hybridization chamber surrounding the region of the substrate containing a target-specific capture array. As each step of the test is completed, old reagents are moved out of the hybridization chamber and new reagents are added from the reagent pack via microfluidic channels and pumps. Once the test is complete, the Test Cartridge is removed from the Verigene Processor SP unit and the reagent pack is snapped off and discarded. The remaining slide is now ready for imaging and analysis in the Verigene Reader. 5) End-point detection on the Verigene Reader: The test slide is inserted into the Verigene Reader wherein it is illuminated along its side. The gold-silver aggregates at the test sites scatter the light, which is in turn captured by a photosensor. The relative intensity arising from each arrayed test site is tabulated. Net signals, defined as the absolute signal intensities with background signals subtracted, are compared with thresholds determined by negative controls within the slide in order to arrive at a decision regarding the presence or absence of target. These results are linked to the test and patient information entered at the beginning of each test session to provide a comprehensive results file.