Jan 10th, 2018 | By John Gilmore
NEW YORK (GenomeWeb) – Researchers at Harvard University and non-profit Diagnostics for All have combined electrochemical detection with recombinase polymerase amplification (RPA) on a portable device to improve detection of genetic material from multiple tuberculosis strains.
In the study, published last month in Analytical Biochemistry, the team aimed to simplify the amplification and electrochemical detection of genetic material at the point of care, showing that coupling isothermal RPA assays with an electrochemical readout allows accurate and rapid detection of genes. The study was partly funded by the Gates Foundation and the Defense Threat Reduction Agency of the US Department of Defense.
Originally developed by TwistDx, now part of Alere, and soon to be part of Abbott, RPA is a DNA/RNA amplification technology that can act as an alternative to PCR. It is especially attractive in low-resource settings because it does not require a relatively expense thermal cycler.
The RPA reaction uses enzymes called recombinases that form complexes with oligonucleotide primers and pair the primers with homologous sequences in DNA. A single-stranded DNA binding protein binds to the displaced DNA strand and stabilizes the resulting loop. The primer then initiates DNA amplification by a polymerase, but only if the target DNA sequence is present.
According to Tsaloglou, running the assay on the inexpensive portable device in the developing world will allow accurate tuberculosis detection with little sacrifice to sensitivity or accuracy.
According to the study, the team's platform uses disposable, paper-based strips that integrate three screen-printed carbon electrodes and accomplish thermoregulation with +/-0.1 ºC temperature accuracy. The universal mobile electrochemical detector (uMEDNA), described in a paper published in 2014 in the Proceedings of the National Academy of Sciences, enables electrochemical detection of the amplified DNA using a variety of pulse sequences, and is easy enough to use with minimal training and supervision. The strips can be used with the UMEDNA or electrochemical analyzers, both benchtop or portable.
Maria-Nefeli Tsaloglou, then a postdoc at Harvard funded by Marie Curie Actions, and senior author of the latest study explained that the researchers have been developing the basic diagnostic device since 2010. Her team is working to commercialize both the paper strip, electrochemical detection technology, and proprietary uMEDNA device that performs the RPA assay. Roughly the size of a smartphone, the device provides components and algorithms to control the DNA amplification and perform electrochemical detection at the same time.
To detect DNA, the team first prepares the paper test strip that includes the blood sample and primers, in addition to integrated electrodes that contain the reagents for RPA. The test strips allows the team to cut down on reaction volume, reducing reagent cost and blood
sample size. After identifying a 213-bp region common to both Mycobacterium tuberculosis and Mycobacterium smegmatis, the team designed appropriate primers for the RPA assay to amplify the specific sequence.
Performed at 65 ºC, the assay combined isothermal amplification with electrochemical readout of redox-active hexa-amine ruthenium (III) (Ru(NH3)6]3+) as an electroactive mediator for the electrochemical detection of DNA.
Tsaloglou and her colleagues also performed the reaction with varying levels of initial concentrations of the M. smegmatis target DNA in order to demonstrate the assay's sensitivity. According to the study, the assay's limit of detection is 0.04 nanograms/microliter, equating to 11 CFU/ml of M. tuberculosis.
Because RPA assays do not need additional sample prep time, the assay in the study required 20 minutes to identify the biosignal. Tsaloglou emphasized that the RPA assay could potentially identify the signal faster depending on the type of primer and target sequence.
Data that the uMEDNA device collects can be transmitted by plugging it into a cell phone's headphone jack. Tsaloglou explained that the uMEDNA device also communicates with any bluetooth-enabled device and can link to any computer, tablet, or smartphone. At the moment, the researchers have developed software for Apple devices and are currently working on adapting the program for Android devices.
While the team used M. smegmatis as a surrogate strain for M. tuberculosis, the benchtop RPA assay Tsaloglou and her colleagues developed can detect up to 19 Mycobacterium species. In addition, the team also carried out experiments using samples spiked with M. tuberculosis DNA that highlights that the electrochemical method also works with the specific bacterial strain.
The Harvard researchers have filed a joint patent with Diagnostics for All for the electrochemical detection of nucleic acids on a portable reader using paper-based disposable strips. Harvard chemistry professor George Whitesides, corresponding author on the new study, founded Salem, Massachusetts-based Diagnostics for All in 2014 to order to focus on healthcare and diagnostics in the developing world. The patent describes use of electrochemistry for detection RPA or other isothermal methods.
The non-profit has partnered with the Harvard lab to explore low-cost applications for the paper-based uMEDNAdevice.
In terms of commercialization, Tsaloglou explained that the lab is working on a prototype reader, which is currently in alpha stage. Tsaloglou's team has partnered with undisclosed experts in the electronics industry to move to beta and commercial rollout, and is seeking additional partners. She foresees research use within a year, and commercial use by 2020, provided the team is able to secure additional necessary partners and move the platform through regulatory approval as planned.
Detecting pathogens using genetic biomarkers is a well known method employed by multiple companies, with Cepheid being one of the most well-known examples for tuberculosis detection. Cepheid's GeneXpert MTB/RIFassay has been endorsed by the World Health Organization for diagnosing active tuberculosis, and demonstrates strong performance in detection of rifampin susceptibility and resistance.
The researchers noted, though, that GeneExpert is limited to the lab and is too expensive for most applications in the developing world and at the point of care.
Tsaloglou said that the uMEDNA device will only cost the end user about $30. Introducing the DNA detection paper-based strips will keep keep the assay price relatively low, as well.
"Electrochemical analyzers in the lab are bulky, expensive, almost in the range of $300,000 to $1 million," she said. "In the paper, we proved that the size and electronics within the analyzer can be cheaper."
Tsaloglou believes that the RPA assay could perform well in resource-limited areas due to its isothermal nature. According to Tsaloglou, running the assay on the inexpensive portable device in the developing world will allow accurate tuberculosis detection with little sacrifice to sensitivity or accuracy. The device could also potentially be used to identify a variety of infectious diseases in rural clinics, country borders or ports, and eventually at home for lifestyle monitoring, she noted.