Program Director/Principal Investigator (Last, First, Middle): ANUBHAV TRIPATHI, 1 R21 AI 73808-01A1 In this collaborative program, we will develop an integrated Influenza Detection Microchip capable of performing identification of sequence-specific influenza subtypes (H1, H3, and H5). Our device accepts clinical specimens from mucosal swabs and isolates influenza virus RNA (vRNA) based on conserved consensus sequences to complementary DNA oligonucleotides immobilized on microbeads. These sequences are used as templates for reverse transcription utilizing Nucleic Acid Sequence Based Amplification (NASBA) mechanism inside the chamber of a packed bed microchip. The transcription products of the continuous flow reaction are sequentially mixed with flow stream containing dual probes (also called molecular beacons) specific for one of each of the repertoire of possible H sequences. The dual probes fluoresce only when specifically hybridized with the complementary viral sequence. A low cost camera records the down stream detection area to collect the transient signal to identify the viral subtype;the results are then compared to the positive and negative controls. The proposed program will significantly advance fundamental understanding of physical &chemical cues necessary in an isothermal and exponential continuous flow amplification and detection of vRNA microreactor. The proposed device has significant advantages over current methods of detection and genetic analysis of infections outside of core lab facilities. The main advantages include rapid identification, high sensitivity, accuracy, minimal. sample preparation time and high information content. The combination of this experienced team of infectious disease physicians (Artenstein and Opal) and microfluidic bioengineers (Tripathi, IVIcCalia &Sarma) provides a unique advantage to completion of this goal. This research team has extensive experience with microfluidics, infectious diseases and advanced molecular biology techniques, which are all critical to the success of this project. The outcome of the program has a potential to provide public health laboratories, hospital clinical laboratories, and point-of-care facilities for highly sensitive and specific influenza diagnostic tests that rapidly generate a virologic diagnosis, subtype-specific information and epidemiologic tracking of influenza.
