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Researchers Boost Sensitivity of HDA Isothermal Method, Develop TB Test Rivaling PCR

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NEW YORK (GenomeWeb) – Researchers at the University of Oviedo in Spain have developed an isothermal assay for Mycobacterium tuberculosis that is as sensitive and specific as a PCR-based assay, and could potentially enable rapid testing for the infection in low-resource settings.

Tuberculosis caused 1.5 million deaths and 6 million newly diagnosed infections in 2014. Cultures and microscopy are problematic diagnostic methods because they can take a long time or have low sensitivity, while existing molecular testing is sometimes too complex and expensive.

The isothermal test, which was described earlier this month in Analytical and Bioanalytical Chemistry, uses helicase-dependent amplification to detect an 84 base-pair-long region in the TB genome. It was shown to be as specific and sensitive as a standard PCR assay.

The test also has the ability to detect attomolar quantities of nucleic acids, as also demonstrated in a prior study by the researchers published in Biosensors and Bioelectronics.  In the current work, the assay detected as few as 15 copies of M. tuberculosis genomic DNA. This sensitivity was accomplished in part through enhanced primer design, as well as the use of magnetic-based purification and electrochemical detection via a technique called chronoamperometry.

The group has been working for more than a decade to develop simple, sensitive, and reliable methods for clinical diagnostics and food safety monitoring, Maria Jesús Lobo-Castañón, corresponding author on the study, told GenomeWeb in an email.  

Initially, her team experimented with synthetic short oligonucleotides, using hybridization to specifically recognize targets. But with genomic DNA, they realized that sample pretreatment to restrict the size of the target would be essential. "At that point, we turned our attention to PCR, which could restrict the size of the target by specifically amplifying it," Lobo-Castañón said.

However, the need for thermal cycling complicates miniaturization, which is a key to subsequent adaptation of testing to a point-of-care setting.

"HDA is an isothermal amplification process with a simple design similar to the well-known PCR, giving rise to amplification products usually shorter than PCR, which we think [are] ideal for coupling to the surface-hybridization process," she told GenomeWeb.

The HDA reaction, however, struggles with selectivity, and this may be the reason it has not blossomed into a commonly used diagnostic method, Lobo-Castañón said. Its isothermal character makes the method more prone to unspecific products. But specifically entrapping the desired products through hybridization onto magnetic beads allowed the group to create a robust system that can detect very low amounts of pathogen.

"This is the main novelty of our proposal," Lobo-Castañón said. Using chronoamperometry for detection, meanwhile, also enables the system to be simple and low cost.

After demonstrating the parity with PCR, the researchers tested the method on a small number of clinical samples, showing its effectiveness on sputum, urine, and pleural fluid samples. They further showed it was selective by testing it with Legionella pneumophila, another respiratory pathogen.

With further miniaturization and simplification, the assay might now be developed into a more portable version. "When combined with magnetic beads, which can be easily manipulated using a magnetic field, the assay protocols are greatly facilitated, [and] we are thinking of a microfluidic system combining magnetic manipulation with electrochemical detection," which, Lobo-Castañón noted, is very amenable to miniaturization at low cost.

The group is not planning to patent the method, "but it will be nice to find a partner in industry that can help us to integrate amplification and detection in a simple platform," said Lobo-Castañón.

For now, the group is also testing new surfaces to entrap the amplification products. "We have very promising results using indium-tin-oxide (ITO) surfaces, which are electrically conductive surfaces that can be modified with DNA, entrapping the amplification products for subsequent electrochemical detection."

The electrochemical genomagentic HDA technology is also generally applicable, Lobo-Castañón said. It fulfills "the ever-increasing demands of not only bedside analysis, but also food safety and environmental monitoring, and it would be particularly suitable in developing countries where access to large facilities to carry out these analyses is not always achievable."

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