NEW YORK (GenomeWeb) – Researchers at the University of Maryland and Canon US Life Sciences have developed a sample preparation method using chitosan microparticles, claiming to be the first to show that amplifying human genomic and viral DNA directly from the beads provides similar PCR results as standard methods on whole blood samples.
Sample prep is increasingly being called the biggest bottleneck in molecular diagnostics, in terms of cost, time, and number of steps. So far, a number of commercially available point-of-care tests that detect nucleic acids get around this bottleneck by using samples that are easier to process, like nasopharyngeal swabs. Whole blood, on the other hand, can be much trickier to work with as it contains inhibitors.
In a study published last month in Analytical Chemistry and led by Ian White, a bioengineer at U Maryland, researchers showed that chitosan can be coated onto magnetic beads which are also used to perform bead-based lysis. Chitosan eliminates the chaotropic salt treatment, washing, and elution steps, and the magnetic beads can then easily be transferred to a PCR reaction. In the study, the group showed that whole blood spiked with plasmid DNA and treated with this prep method yielded comparable cycle thresholds to standard sample prep techniques.
Chitosan is a polysaccharide that can be derived from chitin, like bug or crab shell, White said in an interview. It is useful for DNA sample preparation due to its charge switching behavior. Namely, it is positively charged at slightly low pH, and neutral at moderate and slightly elevated pH. Therefore, negatively charged DNA can electrostatically bind to chitosan beads and be unbound with pH elevation that is biologically relevant, White said.
The chitosan method holds the potential for reducing the number of steps and cost of PCR and sequencing sample prep compared to chaotropic salts, solid-phase absorption, and centrifuge-based methods.
And White noted that there appears to be a growing trend toward getting the pipettes, and manual steps, out of sample prep. "Looking back 20 years, a lot of researchers, and some companies, were saying 'we are going to put PCR in microfluidics and that is going to make it automated,' kind of ignoring the sample prep piece," he said. But, history seems to have shown that "just making PCR smaller doesn't necessarily change the workflow." This is true with respect to timing, but also in terms of US Food and Drug Administration regulation, White said. "If there are pipettes involved, then it is not point of care."
Other work published in Analytical Chemistry by a group in Beijing the same week as the White lab's work showed chitosan-modified glass fiber membrane filter paper could be used for nucleic acid capture in a microfluidic. That group also "showed efficient PCR without having to elute the DNA out," White said.
An author of that study, Peng Liu, a biomedical engineering at the Tsinghua University School of Medicine, said in an email that the method has three unique advantages. By using pH changes instead of salts, the group did not need to use guanidinium or sodium iodide salts, which are known PCR inhibitors. A "bind-wash-elute" protocol can also dilute the extracted nucleic acids during elution, while the chitosan method facilitates the use of all the captured DNA for PCR without any loss, leading to an enhanced sensitivity.
And the method can also be easily integrated into a microchip, enabling the group's ultimate goal: to develop a fully integrated "sample-in-answer-out" system for genetic analysis. "In the future, our group will develop several microsystems based on this technology for forensic DNA typing from low-copy-number DNA samples and pathogen detection in the early infection period," Liu said.
Commercially for the point-of-care or near-patient setting, sample prep is typically done with the standard salt and filter methods, but incorporated into a cartridge. "Our view of it is, the cartridges are still a little bit more complicated and expensive than having [testing] done in a central lab, or just running cultures," White said.
Yet Chitosan has not yet been adopted commercially, to White's knowledge. This may be because, up until the past year or two, the method was not refined enough to take out more than a few steps. "Our goal is to get rid of all manual steps ... but also to have very few automated steps" to keep costs low, White said.
The lab has been collaborating with Canon Life Sciences for a number of years on a long-term project to develop a diagnostics platform, and one of the study authors is a researcher at that company. A representative from Canon noted in an email that the firm collaborates with investigators around the world on various research projects and works to effectively promote their advancements, but Canon does not comment on ongoing internal product development projects or on how its collaborative efforts impact product development.
White noted that the researchers have worked with Canon to patent their work, and the company will have access to the technology via the collaboration. He said a fair amount of the work his lab has done with the firm has been around finding new ways to do sample prep.
Meanwhile, White's lab also remains interested in point-of-care testing. It has a long-standing focus on fungal and bacterial infections in blood, "A difficult problem and a complicated sample prep scenario," White said. One of the follow-ups to the Analytical Chemistry work will involve probing bound DNA repeatedly on the chitosan bead, and taking advantage of the fact that amplicons also bind to the beads and can be subsequently probed. This could be particularly useful in species identification and drug resistance testing in the same sample, White said.
The research team also examines isothermal methods and White said his group sees that as being very relevant toward developing easy-to-use point of care tests.
Overall, "We hope that people will recognize this as a universal sample prep, and that we did PCR but it should be applicable to any nucleic acid tests — even sequencing," White said.