SAN FRANCISCO — An executive from Danish biotech firm Anapa Biotech this week disclosed new data demonstrating how its proprietary twisted intercalating nucleic acid (TINA) molecules for modifying real-time PCR primers increased the efficiency and analytical sensitivity of a multiplexed real-time PCR assay for the detection of Staphylococcus aureus and methicillin-resistant S. aureus.
In addition, the executive, CSO Uffe Vest Schneider, disclosed that the company is developing a next-generation molecule called stacking nucleic acids, and demonstrated how SNA-modified primers can be used to develop highly specific real-time PCR assays using SYBR Green detection technology.
Schneider discussed these developments at a presentation during the "PCR for Molecular Medicine" track at Cambridge Healthcare Institute's Molecular Medicine Tri-Conference, held here this week.
Last summer, researchers from Anapa — which in December changed its name from QuantiBact and named a new CEO — published a paper in PLOS One describing how its TINA molecules can be used to modify PCR primers to increase the efficiency, specificity, and sensitivity of quantitative and multiplex endpoint PCR assays.
Specifically, the researchers demonstrated that adding so-called ortho-TINA molecules — one of several configurations of the molecule — at the 5' position to standard primers in a qPCR experiment enabled 100 percent efficiency in significantly stressed reaction conditions, including lower primer concentrations and increased annealing temperatures.
At the time, researchers from the company noted that the properties of primers modified with TINA molecules could be attractive to molecular diagnostic developers, and to that end they demonstrated proof of principle for using such primers in a multiplexed Escherichia coli assay.
This week, Schneider provided Tri-Con attendees a glimpse of data from the company's collaboration with Denmark's Hvidovre University Hospital to apply TINA-modified primers to a four-plex SA and MRSA assay that targets the femA and nuc genes, which are specific to SA; and the mecA and mecC genes, which confer methicillin resistance in more than 95 percent and less than 5 percent, respectively, of all MRSA strains.
According to unpublished data, the researchers found that the TINA-modified primers provided more balanced PCR performance between the different targets than with unmodified primers, and significantly improved PCR for two out of four targets, Schneider noted.
In general, Schneider said, the TINA molecules worked well with all of the primers used in the assay, which indicates that the molecules will confer increased freedom in primer design. In addition, the company has determined that TINA-modified primers can provide real-time PCR assays with up to 64-fold increased analytical sensitivity.
TINA-modified oligonucleotides are commercially available through Eurofins MWG in Europe and TriLink Biotechnologies in the US.
During his Tri-Con presentation, Schneider also discussed the company's latest technology, the so-called stacking nucleic acid (SNA) molecules.
Because of the early development stage of this technology, Schneider noted that he was only providing a preview. He described SNAs as "a novel intercalating molecule [that] allows Taq DNA polymerase to read through it when placed internally in oligonucleotides."
He also compared the molecules to Exiqon's locked nucleic acid technology, but noted that the latter product does not always work well with primers, while Anapa believes SNAs will be compatible with a broad range of primers.
Schneider provided a glimpse of how SNA-modified primers could be used in a real-time PCR assay employing SYBR Green detection as opposed to TaqMan probes, and noted that the company has demonstrated internally that PCR primers modified with SNA molecules at their 3’ ends "work well for real-time PCR."
Further, the company has showed that SNA molecules increase amplicon melting temperature in PCR reactions and are able to produce specific amplicons, which has been verified by gel electrophoresis. The new molecules may also be used to modify primers and further increase the efficiency of PCR reactions by decreasing Cq values, which represent the number of cycles needed to reach a set threshold fluorescence signal level.
Schneider said that Anapa hopes to make SNA-modified oligos commercially available later this year.