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Researchers Pair Acoustic Detection with PCR for Label-Free SNP Genotyping, Gene Expression Assays

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Scientists from the University of Crete and the Foundation for Research & Technology-Hellas, or FORTH, have developed a method of using an acoustic biosensor to directly detect and quantify PCR amplicons in a solution based on their size and shape, according to a recently published study.

Using the method, the researchers were able to genotype SNPs of an insecticide resistance-related gene in mosquitoes and quantify expression of a gene that produces an important liver protein in mice, demonstrating the technique's applicability to biological assays typically conducted with real-time PCR or hybridization assays.

In addition, the researchers are now working to commercialize a dedicated bioassay platform based on the technology for use in a variety of genetic testing applications, Electra Gizeli, an associate professor of biology at the University of Crete and FORTH's Institute of Molecular Biology & Biotechnology, told PCR Insider.

Gizeli and colleagues detailed their method in a paper published last week in the open-access journal Scientific Reports.

Gizeli's lab has been working with acoustic wave sensors for several years, she explained in an email to PCR Insider, and recently decided to pair the methodology with PCR in an attempt to create a fast, simple, and cost-effective alternative to real-time PCR or hybridization assays, which necessitate fluorescent labels and complex optical equipment and are difficult to multiplex.

In acoustic biosensing, the presence of an analyte at a sensor's surface affects the velocity and energy of an acoustic wave. This in turn can be monitored as changes in frequency, which reflects the amount of adsorbed mass, and energy dissipation, which reflects the viscoelastic properties of the bound molecules.

The researchers found in previous work that energy dissipation per unit mass — also called the acoustic ratio — can be used to directly measure the intrinsic viscosity of surface-attached DNA molecules, which in turn is directly related to the hydrodynamic volume — essentially the size and shape — of the attached analyte.

As such, different DNA molecules have unique "acoustic signatures" that can be used to distinguish them from one another. This feature allowed the researchers to develop genetic assays where various double-stranded DNA molecules, produced in the same PCR reaction, can be loaded on the device surface and characterized solely by the measured acoustic ratio, the Scientific Reports paper explains.

Gizeli and colleagues first tested their technology's applicability to genetic testing by creating a SNP genotyping assay for the ace-1 gene in the mosquito species Anopheles gambiae. A glycine-to-serine substitution at position 119 of the ace-1 gene confers high levels of resistance to certain insecticides — a mutation that has been spreading in populations of this mosquito in West Africa, thus potentially thwarting insect population and disease control.

The researchers developed a two-step assay to detect this substitution in the ace-1 gene in genomic DNA. The first step involved a bidirectional PCR amplification reaction of specific alleles with genomic DNA used as the template. In the second step, the researchers directly measured the PCR products with the acoustic sensor technology.

Using this approach, they were able to distinguish wild-type samples from those containing SNPs based solely on the acoustic ratios of the PCR amplicons that resulted following a standard thermal cycling procedure. Their data jibed with both TaqMan-based PCR assays and gel analysis of the SNP.

In another assessment of their technology, Gizeli and colleagues measured differences in the expression of the ABCA1 gene in mice. The membrane protein ABCA1 plays an important role in the biogenesis of high-density lipoproteins in the liver, and the expression of the gene can be induced by synthetic ligands of certain liver receptors in many cell types.

The researchers conducted PCR and real-time PCR gene expression assays on the ABCA1 genes of mice that were treated with some of these synthetic ligands and mice that were not, revealing that the gene is overexpressed approximately two-fold in treated samples.

By designing PCR assays for specific varying lengths of DNA in both ABCA1 and the housekeeping gene GAPDH, then using the acoustic biosensing assay to detect and quantify the resulting amplicons of both, the researchers were able to obtain data that agreed closely with their previously run real-time PCR assays.

Currently, real-time PCR gene expression quantification would generally involve constructing standard curves for the two genes being compared in separate tubes containing one fluorescent dye.

"Our method has a major advantage over this practice since we measure the relative expression ratio of two genes in two samples … and the DNA fragments are produced within the same PCR reaction," the researchers wrote.

They further noted that "while it is possible to have real-time measurements using two fluorescent dyes for simultaneous measurements of two genes in one tube, this would increase the assay complexity and cost significantly. The quantification accuracy of the acoustic method was found to be comparable to … real-time PCR," but is superior in terms of cost, speed, and simplicity, they wrote.

Another potential advantage of the new method is its multiplexing capability. "So far we have managed to detect up to three different PCR products, without the use of labels, produced in the same PCR," Gizeli said. "We are confident that with the current setup we can go up to seven products; theoretically this can be extended up to 10." Currently, the practical multiplexing limit of real-time PCR based on fluorescent labels is five or six targets.

The method requires only a standard thermal cycler, found in almost every laboratory, and an acoustic wave device, which can be obtained from several manufacturers. In their work, Giseli and colleagues used a quartz crystal microbalance, which is meant for use in laboratories.

Giseli told PCR Insider that the group is currently "building a purpose-made system that will be portable and stable to temperature variations and other environmental factors," which would allow the technology to be used in field-based assays.

She also noted that the assay method has the greatest near-term potential for applications such as screening for mutations of hereditary diseases like cancer and cystic fibrosis; detecting infectious diseases, such as malaria, HIV, and diarrhea-causing microorganisms in patient samples; and detecting food pathogens and plant microbes.

Longer term, the technology is "applicable as the detection element in a [lab-on-a-chip] platform ... for the development of a portable, low-cost platform for pathogen detection in developing countries," Giseli said.

The researchers have been granted a patent by the US Patent and Trademark Office and are currently waiting for approval from the European patent office for a corresponding patent. Giseli said that the researchers are "proceeding in the commercial exploitation of this technology with the help of our technology transfer office," but offered no further details.

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