Nucleic Acid Detection System and Method for Detecting Influenza

Abstract

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Description

RELATED APPLICATIONS[0001]This patent application is a divisional application of U.S. patent application Ser. No. 11 / 894,908, entitled “Nucleic Acid Detection System and Method for Detecting Influenza”, filed on Aug. 22, 2007 and issuing on Mar. 17, 2015 as U.S. Pat. No. 8,980,561, which application claims the benefit of the filing date of U.S. Provisional patent application No. 60 / 839,537 filed Aug. 22, 2006 under 35 U.S.C. 119(e).STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under Contract No. DE-AC52-06NA25396, awarded by the United States Department of Energy. The government has certain rights in this invention.BACKGROUND OF THE INVENTIONI. Influenza Viruses:[0003]Influenza viruses have a segmented genome of single-stranded negative-sense RNA and belong to the family Orthomyxoviridae. They have been isolated from a variety of animals, including humans, pigs, horses, sea mammals, an...

AI Technical Summary

Benefits of technology

[0027]The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

[0032]The isolation of influenza virus particles may be achieved by first incubating the clinical sample with magnetic beads functionalized with at least one affinity ligand capable of binding to the influenza A virus particles, followed by separating magnetic bead-bound cells and / or particles from other elements present in the clinical sample by applying a magnetic field to the sample. Various affinity ligands are known and may be used in the practice of the method of the invention. Typically, an antibody is used. One or more wash steps may be added following the separation step. Total nucleic acid from the influenza virus particles is preferably achieved by alkaline lysis followed by neutralization, or by heating to release nucleic acid. This provides an initial gateway for establishing the specificity of the detection system, in that RNA extracted from isolated virus substantially eliminates contamination by genomic RNA (and DNA) from host cells or other organisms which may be present in the clinical specimen. In addition, these methods eliminate the need for centrifugation, which typical in nucleic acid extraction methodologies, thereby enabling highly simplified, downstream nucleic acid amplifications. In order to improve the efficiency and yields of the amplification reaction, in some embodiments primers incorporating a poly dA or poly dT sequence of between 5 and 20 bases in length are be used.

[0037]In another alternative procedure, bead-functionalized detection oligonucleotides are added to the RT / HDA reaction prior to termination of the HDA reaction. This approach provides the advantage of presenting the detection oligonucleotides to the amplified target DNA as the latter is being produced, thereby minimizing the extent to which amplified DNA can form duplexes or secondary structures that could interfere with hybridization to the detection oligo. Accordingly, this approach eliminates the need for a separate denaturation step prior to hybridization to detection oligo, and thus may be preferred for POC and field applications in which performing a denaturation step is impractical.

[0038]The system of the invention provides unprecedented assay speed and simplicity. The system neither requires thermocycling equipment for the amplification of DNA, nor the use of sophisticated instrumentation for obtaining assay results. The amplification component of the system is run at constant temperature and does not require small volume handling or technical sophistication. The detection component runs automatically following sample administration, and produces a visual result that can be seen by the naked eye in a matter of minutes.

Problems solved by technology

In both situations, prior immunity to influenza might not prevent infection with the new type, leading to localized epidemics or, in the case of genetic shift, a global pandemic of influenza.

In early 2003, an outbreak of highly pathogenic H7N7 in the Netherlands (spreading to Belgium and Germany) resulted in the death or culling of about 30 million birds which caused significant economic loss and social panic.

Minor genetic drift occurring in the circulating viruses can reduce the effectiveness of the vaccine in preventing illness but, even then, partial immunity afforded by the vaccine will often attenuate the infection, reducing the occurrence of severe illness and complications (Kilbourne et al., 2002, Proc. Natl. Acad. Sci.

However, these drugs may not always be effective, as influenza virus strains can become resistant to one or more of these medications.

Unfortunately, these techniques often take days or weeks to complete, which is often far too late for therapeutic intervention.

Nucleic acid-based assays for pathogen detection and identification are unparalleled with respect to providing sensitivity, specificity and resolution.

Nonetheless, technologies for nucleic acid detection continue to be relatively elaborate and often costly, limiting their utility for point of care diagnostics and deployment under field conditions where a supporting laboratory infrastructure is absent.

Reliance on polymerase chain reaction (PCR) and fluorescent detection of amplified nucleic acids has contributed significantly to the complexity and cost of nucleic acid diagnostics.

Retaining assay sensitivity while circumventing requirements for thermocyclers and fluorescence detection hardware remains a significant challenge.

The appeal of lateral flow detection in the context of a PCR-based assay is limited by the fact that real-time PCR detection would offer similar hardware complexity compared to post-thermocycling introduction of PCR reactions onto a lateral flow detector with single amplicon detection capacity.

In addition to the hardware requirements of PCR, these devices have employed schemes poorly suited to multiplexed detection, further limiting their utility to single-plex PCR assays.

However, PCR presents inherent limitations for applications in which simple, rapid nucleic acid assays are desired.

PCR requires the use of a thermocycler, an expensive, electrified machine that is not easily adapted to environments outside of laboratories with technically trained personnel.

While eliminating the need for thermocycling, currently-available isothermal amplification technologies require varying degrees of technical expertise and some rely on multiple primers and other factors which negatively effect assay time, expense, sensitivity and specificity.

Many of the isothermal amplification technologies described to date are slow, insensitive, and unreliable.

In addition, many of the viable isothermal amplification technologies are run at temperatures far higher than ambient temperatures, and therefore require heating.

Although SDA has been improved to work well at 60° C., the reaction requires several hours to perform at ambient temperatures.

Although this technology initially held promise, it is extremely unreliable, nonspecific, and difficult to use.

Relying on thermophilic polymerases and reaction conditions that must be perfectly controlled, this technology is hampered by problems including spontaneous primer-independent polymerization of non-specific amplification products.

Rarely is this the case in the context of pathogen target detection, and therefore this technology is fundamentally limited.

However, RT-PCR is burdened by all of the limitations inherent to PCR, including the requirement for thermocycling and the double-stranded nature of the resulting amplification product.

For example, as previously mentioned, infections by the highly virulent strains of the avian influenza virus must be treated within 48 hours of initial infection, thereby rendering useless any assay technology that cannot be performed rapidly and with reliable specificity and sensitivity.

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