Rapid nucleic acid assay protocol shows strong potential for use in diagnostics and surveillance

Researchers detect three biothreat agents

Mar. 9, 2010—A Lab research team has developed a rapid, nucleic acid assay protocol for detecting disease-causing pathogens using a multiplex oligonucleotide ligation-polymerase chain reaction (MOL-PCR). Unlike other ligation-based assays that require multiple steps, this protocol consists of a single tube reaction, followed by hybridization to a microsphere array for detection. Members of the team are Alina Deshpande of D-3; Jason Gans, Laura Taylor, Yuliya Kunde, Po-E Li, Jacob Mark, Jian Song, Momchilo Vuyisich, and Scott White of B-7; Lance Green of B-6; Heung Bok Kim of B-9; and Steve Graves, formerly of B-9.

Rapid diagnosis of disease is essential for timely mitigation and treatment. An ideal diagnostic assay should

• be rapid,
• not require multiple rounds of confirmatory testing,
• have low costs,
• identify the cause of disease, and
• allow additional characterization.

Although assays can be made using many types of molecules, nucleic acid-based assays are ideal for diagnostics due to their high specificity and the minimal sample size that is required. In addition, the assays readily can be configured to detect almost any target, and automation of the assay is simple. Performing the assays on inactivated or sterilized samples is an important safety advantage compared to culture-based or immunoassays. The acceptance of nucleic acid-based assays for diagnostics has brought a need for more robust, cost-effective, simple, and rapid assays that provide high confidence results.

MOL-PCR enables direct detection of multiple nucleic acid signatures, such as unique sequences, single nucleotide polymorphisms, insertions, deletions, and repeats from complex mixtures. Therefore, MOL-PCR can simultaneously detect a disease-causing pathogen using one type of signature and characterize its strain or determine antibiotic resistance using a different type of signature. DNA targets of only 40 to 50 nucleotides are needed for the assay. Therefore, detection of degraded targets, which are common in environmental, forensic, and clinical samples, is possible. Combined with the ease and robustness of this assay, MOL-PCR shows strong potential for use in diagnostics and surveillance.

The team recently collaborated with the University of New Mexico and the New Mexico Department of Health to detect three biothreat agents: B. anthracis (anthrax), Y. pestis (plague), and F. tularensis (tularemia). Their work was published in the Journal of Microbiological Methods.

The Department of Homeland Security (Greg Van Tuyle, LANL program director for Countering Weapons of Mass Effect), the National Flow Cytometry Resource (under the direction of Jim Freyer and funded by the National Institutes of Health), and LANL's Laboratory Directed Research and Development program supported the research.