This award is funded under the American Recovery and Reinvestment Act of 2009 Public Law 111-5).
This SBIR Phase II project will provide technology to perform remote nucleic acid testing (NAT) in any location. The combination of (a) our Probe-Target-Reporter (PTR) assay which allows the detection of unlabeled Target DNA, (b) our Parallume optical encoding technology which provides the ability to multiplex a large number of samples in each assay and (c) an inexpensive, battery-powered imaging system, based on a $500 commercial CMOS that is completely portable, will be used to build an autonomous NAT platform. This system will be used to detect and defend against the imminent invasion of California's citrus crop by the Liberibacter pathogen and its insect vector/host which causes the 100% fatal and incurable Citrus Greening disease of citrus. Collaborator Isca Technologies will provide a "Front End" instrument which can selectively identify an insect by measuring its wing beat frequency as it flies through a laser curtain. This selective insect trap will provide a filtered homogenate of primarily the desired insect vector and pathogen which will be analyzed with our PTR in the field. The data will be relayed to a central database which can provide a real time assessment of the location and bacterial load of the insect vector.
The Nucleic Acid Testing (NAT) technology under development as part of this project represents a substantial advance in the ability to perform assays outside of the traditional laboratory or clinical setting. This technology can be used to detect Target DNA sequences without the need to chemically label the sample thereby allowing the NAT analysis to be performed in any location. This NAT technology can be combined with our Parallume optical encoding technology which allows many sample to be measured simultaneously. The ability to analyze many DNA simultaneously without access to a laboratory on a completely portable system will allow NAT to be performed in Low Resource Settings or for Agricultural applications around the world.
This project focused on the development of a low cost, multiplexed nucleic acid-based test to detect various strains of a pathological bacteria (Liberobacter) which attacks and kills citrus trees. The disease, which spread from southern China in the early 20th century, is carried by an insect host which feeds on the sap of citrus trees. Once infected with the bacteria, there is no cure and the tree eventually dies. For this Grant, we proposed to build an autonomous insect trap which would, by use of a test we developed to detect and quantitate the presence of DNA from the Liberobacter, determine if the citrus tree or orchard was affected by the pathogen. The first part of the Grant performance period was used to develop the DNA test in which we were able to measure the Liberobacter DNA without labeling after PCR amplification. The DNA assay was successful and we were able to detect 10-20 copies of unlabeled bacterial DNA by this method. Using this knowledge we designed and built a machine which automatically amplifies and detects the pathogen DNA by using the developed DNA assay. The assay can detect both the insect host and identify individual Liberobacter strains. The optical system for the instrument which detects the multiplexed DNA assays was designed, built and successfully tested. The motion control system that loads the samples into the thermal cycler and transfers them to the optical analyzer was designed, built, programmed and successfully tested. The integrated system was completed and we will test the entire system shortly.
