A Novel Amplification Method Permits Pathogens to be Detected with Microarrays
Description of Invention:
Available for licensing and commercial development is a high throughput, microarray-based multiplex method of detecting target nucleic acids in a sample. In particular, PCR is coupled with microarrays for the qualitative identification of multiple target nucleic acids, with primers specific for a target sequence, and used to detect genomic nucleic acids of pathogens of interest, or transcripts derived therefrom. Also claimed are oligonucleotide microarrays for use in such methods.
The present method is distinguished from other multiplex PCR assays by the additional steps to ensure specificity and sensitivity, so that a larger number of probes can be detected simultaneously in each single reaction. An important application of this method, for which it was developed, is the detection of multiple "Category A List" agents for the purpose of differential diagnosis in case of bioterrorism attacks. The method comprises: a) screening the genomes of the desired infectious agents to find sequences specific for each of them and distinct from human sequences; b) designing 60 base long oligonucleotide targets, to print on microarrays; and c) including in the microarrays both sense and antisense versions of each, as well multiple targets per virus, to increase reliability.
Other methods, such as PCR amplification followed by separation and characterization of DNA products by gel electrophoresis, are simple and sensitive, but they have a number of inherent shortcomings. Highly sensitive PCR amplification tends to generate nonspecific DNA products, which complicate interpretation of the results. Additionally, in a typical method for detecting pathogens in a sample, PCR reactions for each pathogen must be run separately from one another due to differences in amplification conditions. Furthermore, in cases where multiplex PCR coupled with a microarray is used for the qualitative detection of several pathogens, the generation of nonspecific DNA products can be a significant problem. The current method is a rapid, high-throughput method for qualitative identification of multiple target nucleic acids that is sensitive, highly discriminating and robust.
Inventors:
Michael J. Brownstein (NIMH) Charles Xiang (NIMH) and Zhi-Qing Qi (NIMH)
Patent Status:
DHHS Reference No. E-184-2004/0 --
U.S. Provisional Application No. 60/635,239 filed 09 Dec 2004
U.S. Patent Application No. 11/299,025 filed 09 Dec 2005
For Additional Information Please Contact: Cristina Thalhammer-Reyero PhD MBA
NIH Office of Technology Transfer
6011 Executive Blvd, Suite 325
Rockville, MD 20852-3804
Phone: (301) 435-4507
Email: thalhamc@mail.nih.gov
Fax: (301) 402-0220
