Scientists report on the development of a hybridizing magnetic relaxation nanosensor (hMRS) technology that they claim can rapidly detect the presence of otherwise hard-to-diagnose intracellular pathogens in minimally processed clinical samples such as peripheral blood and tissue. The hMRS platform uses ligand-coated magnetic nanoparticles to detect specific genomic sequences in the target pathogens, and is at least as sensitive and specific as PCR analysis of pure DNA. However, claims the University of Florida-based developers, is much faster, can be carried out on crude DNA, and requires no amplification. In fact, they maintain, their studies indicate that hMRS can detect a single genome copy of an intracellular pathogen in a sample of peripheral blood or tissue.
Moreover, the nanospheres are relatively cheap to produce and stable under different environmental conditions, and detection is carried out using a easy-to-use table-top instrument. These features could facilitate the development of a multiplexed system for identifying microbial pathogens or other disease-related biomarkers in both clinical and field-based settings. J. Manuel Perez, Ph.D. and colleagues describe their platform in PLoS One in a paper titled “Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors.”
Immunoassay techniques can’t effectively detect intracellular pathogens such as Mycobacterium tuberculosis in biological fluids, because the organisms are hidden in their host cells. Diagnosis therefore requires the isolation of infected cells, extraction of DNA, and detection of specific bacterial genomic markers using PCR. It may also be necessary to carry out potentially lengthy culturing processes to generate enough DNA prior to extraction and purification for subsequent PCR. And while these methods are effective, they are also time-consuming, labor-intensive—culturing of slow-growing bacteria can take weeks—and require homogeneous and pure DNA for PCR analysis.
In contrast, the approach developed by the University of Central Florida investigators hinges on the use of magnetic relaxation nanosensors (MRS). These are polymer-coated iron oxide nanoparticles onto which affinity ligands are conjugated. When the target DNA in a sample binds to the ligand on the nanoparticle, it causes a change in the sample’s magnetic resonance signal, which is effectively amplified by the water molecules surrounding the particle itself, dramatically increasing sensitivity. This enables the direct detection of low levels of pathogen in crude DNA, the investigators claim.
In their published paper, Dr. Perez and colleagues report on studies with hMRS nanoparticles designed to bind to a genomic marker previously identified as unique to Mycobacterium avium spp. Paratuberculosis (MAP). This intracellular pathogen causes Johne disease in cattle, and has been implicated in Crohn disease in humans. Previous work that has successfully identified MAP in human peripheral blood from Crohn disease patients has taken months to complete, and required culturing of the very slow growing bactrium, followed by nested PCR (nPCR). “Thus contemporary diagnostic methodologies rely on cell isolation, cell lysis, and nucleic acid isolation, in order to yield high-purity DNA,” the investigators write.
In their reported studies the team demonstrated that the hMRS technology was as specific and sensitive as direct nPCR at detecting MAP in DNA extracted from cultures of clinical isolates from Crohn disease patients. However, while nPCR required pure DNA, the hMRS technique could be carried out on crude DNA derived from the samples in a one-step process. The investigators then successfully screened ileal biopsies from Crohn disease patients to detect MAP, both at the genomic level using the hMRS nanoparticle, but also at the epitope levels using an MAP-epitope-sensing MRS designed to detect a conserved MAP bacterial surface protein antigen. Independent confirmation of the MAP MRS data was achieved by carrying out DNA extraction, purification, and nPCR analysis. Both the hMRS and epitope MRS nanoparticles were subsequently used to detect MAP, again at the genomic and protein epitope level, respectively, in tissue samples from cattle infected with Johne disease.
And in a final set of tests, the team used the genomic marker-targeting hMRS particles to screen blood samples from both healthy individuals and Crohn disease patients. Peripheral blood was collected and DNA was isolated from white blood cells either via a 30-minute crude DNA extraction protocol for the hMRS procedure, or using a multistep procedure to isolate high-purity and -quality DNA for direct nPCR screening.
Again, the hMRS results were in complete agreement with nPCR results for a cohort of samples that had generated conclusive positive or negative data using the amplification method. hMRS was also capable of providing conclusive data on each of a second cohort of blood samples that had generated inconclusive direct nPCR results due to smeared or faint bands. For this cohort, hMRS screening on crude DNA was just as sensitive and specific as carrying out culture-based nPCR, a process that first required 12 weeks of incubation of samples in specialized mycobacterial media.
“Comparing both PCR setups with the crude DNA hMRS methodology, we found that the nanoparticle-based method outperformed both PCR-based methods in correlating MAP’s presence and disease state,” the team writes. “As a matter of fact, hMRS is a better predictor of the state of the disease, correlating the clinical state of a patient with the presence of MAP in blood samples.”
The researchers claim their studies indicate that hMRS and the magnetic relaxation technology can reliably identify the presence of an intracellular pathogen in clinical samples in a fast, cost-effective, and highly sensitive way. “The use of hMRS could be a powerful new tool in the study of the mechanism of infection of intracellular pathogens and its relation to disease state, as well as in elucidating intracellular pathogens’ adaptation strategies,” they add. “This enhanced detection translates into a more sensitive pathogen detection and more accurate diagnosis of the disease, providing important clinical information for successful treatment and therapeutic interventions due to early diagnosis.”