Strain Specific Detection of Influenza at the Point-of-Care 1. PROJECT SUMMARY Influenza viruses cause significant morbidity and mortality worldwide;World Health Organization (WHO) estimates 250,000 ~ 500,000 yearly deaths. In particular, a highly pathogenic strain - avian influenza virus H5N1 (15, 56) - poses a threat of a possible pandemic because it can rapidly mutate to acquire the ability to transmit among humans (23, 49). The detection of minimal human infectious dose (~100 viral particles) (65) of Influenza A from patient samples (e.g. nasal swabs or mucus) is a significant analytical challenge. Detection by viral culture is sensitive and specific;however, this method is slow (3-10 days) and cannot be performed at POC. Surface marker-based detection (e.g. ELISA) provide a simple and rapid testing, however, it suffers from a poor limit of detection (~ 105 viral particles/ml) (9, 21, 59). Currently, polymerase chain reaction (PCR) offers the highest sensitivity in a much shorter turn-around time (2-4 hours) (29, 61, 67). Nevertheless, due to the difficulties in POC operation, patient samples are currently analyzed at central laboratories which typically requires ~2-3 days. Currently in the field, there is no automated technology solution that can perform specific influenza detection directly from patient samples with high sensitivity and specificity, and the WHO has specifically emphasized the critical need to fill this important technological gap (75). Towards a solution to this important problem, this group of three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop a powerful, field- portable, microfluidic platform for Influenza detection at POC. This effort is truly unique in many facets;first, to circumvent the problem posed by rapid mutation of the coat protein, we propose to generate novel DNA aptamer reagents which will specifically bind to the nucleoprotein complex of the virus. Currently, there are no reported aptamers for the nucleoprotein complex, and due to the fact that the nucleoprotein is conserved, it will serve as a universal tag to label Influenza A. Secondly, the aptamer reagent will be conjugated to magnetic beads to purify the nucleoprotein complex from nasal swab samples using our unique micromagnetics technology. Thirdly, by leveraging our expertise in miniaturized genetic analysis systems, we will develop the IMED chip capable of 1) integrated micromagnetic sample purification, 2) reverse transcription-PCR amplification and 3) sequence-specific electrochemical detection in a single monolithic device. The successful completion of this project will allow a quantitative """"""""sample-to-answer"""""""" capability - the input into the system will be unprocessed patient samples, and the output will be an electrochemical signal that will be directly proportional to the number of copies of specific influenza RNA sequences in the sample. Such powerful combination of novel affinity reagents with highly integrated disposable devices will enable the development of critically needed effective POC diagnostics systems.
Strain Specific Detection of Influenza at the Point-of-Care Due to the fact that the envelope of influenza A virus differs among subtypes and evolves continuously, there is an urgent need for a field-portable genetic detection platform that can rapidly identify pathogenic strain of viral pathogens from unprocessed samples with high sensitivity and specificity. Towards an effective solution, the three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop an effective point-of-care diagnostic system for influenza by 1) generating specific, high affinity DNA aptamers to tag the conserved regions of Influenza A, and 2) developing a highly integrated microfluidic system capable of magnetic sample preparation, RT-PCR amplification and sequence specific electrochemical detection in a single chip. By integrating the functions in a monolithic chip, the success of this project will yield a novel POC analysis system with unprecedented capabilities which will have a significant impact for Influenza A detection as well as many other applications in food safety, environmental monitoring and homeland security. 1
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