Target-enrichment technologies offer increasingly powerful and cost-effective ways to detect various pathogens, disease biomarkers, and toxins by molecular amplification or direct capture in clinical diagnostics, basic research, and industrial applications.
The latest developments in the field were discussed earlier this year at two conferences—CHI’s “Sample Prep and Target Enrichment in Molecular Diagnostics” and the Knowledge Foundation’s “Sample Prep”.
Mark Eshoo, Ph.D., director of new technology at Ibis Biosciences, a subsidiary of Abbott Molecular, talked about an isothermal amplification, broad-range PCR, electrospray ionization mass spectrometry (IA/PCR/ESI-MS) assay that allows early diagnosis of Lyme disease. The assay works by detecting the tick-borne pathogen Borrelia burgdorferi directly from whole blood, cerebrospinal fluid, or other clinical specimens. To achieve the necessary sensitivity, the assay selectively enriches the target Borrelia sequences in large sample volumes.
“The Centers for Disease Control and Prevention reports that there are an estimated 300,000 new Lyme disease cases a year in the United States. If left untreated, this pathogen can lead to chronic long-term infection,” said Dr. Eshoo.
“The optimal time to treat Lyme disease is at the onset of symptoms,” said Dr. Eshoo. “Currently, serological tests have low sensitivity early in the infection due to the biologically delayed immune response, and as a result, the tests cannot distinguish active infections from prior exposures.”
“Abbott has been working to develop a method for the direct detection of the bacteria that causes Lyme disease, Borrelia burgdorferi. To detect the bacteria, Abbott has developed methods for using large volumes of blood and an isothermal amplification technique that we used to increase our assay sensitivity,” added Dr. Eshoo.
The isothermal amplification, an effective and simple non-PCR DNA enrichment technique, was added to the existing PCR/ESI-MS assay. The study targeted eight Borrelia loci by using 50 primers per target (400 primers in total) in conjunction with a strand-displacing DNA polymerase to increase the target enrichment efficiency in the sample DNA extracted from 1.25 mL of whole blood.
The IA/PCR/ESI-MS assay takes eight hours and can detect Borrelia burgdorferi prior to an active infection. These capabilities allow doctors to diagnose erythema migrans and acute Lyme disease early in disease progression before seroconversion.