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German Group Develops Low-cost Method for Long-term, Ambient Storage of PCR Reagents


NEW YORK (GenomeWeb) — In an effort to improve the long-term storage of multiplex nucleic acid assays, a German academic group has developed a protocol for air drying PCR primers and probes and shown that the reagents remained stable and effective for a year at ambient laboratory conditions.

According to the researchers, the technique could be a low-cost alternative to existing procedures such as freeze-drying, especially for manufacturing test cartridges or chips pre-loaded with reagents for multiplex real-time PCR testing.

As such, the researchers are now looking to extend their technique to include other elements of a real-time PCR reaction, such as polymerases, as well as to reagents used in alternative nucleic acid testing methods such as loop-mediated isothermal amplification.

Markus Rombach, a researcher at the University of Freiburg's Institute for Micromachining and Information Technology of the Hahn-Schickard-Gesellschaft (HSG-IMIT), and lead author on a Biotechniques paper describing the method, told PCR Insider this week that he is part of a group developing microfluidic and lab-on-a-chip platforms for miniaturizing existing nucleic acid assays, hence his team's interest in developing the storage method.

Methods such as freeze-drying (lyophilization) have proven effective for preserving complete real-time PCR assays, with vendors such as Biofortuna building their businesses around the technique. However, lyophilization is relatively expensive, Rombach said, and requires the use of multiple heavy-duty machines.

"The reason we don't want to [use] lyophilization … is that we want to be able to spot or print those primers or probes … on our chip," Rombach said. "That will be the best solution for us to fabricate these microfluidic or lab-on-a-chip platforms. Primers and probes are always in such small amounts for one reaction that you wouldn't be able to just put some lyophilized primers and probes in your end cavities on a chip."

Meantime, other academic groups have demonstrated ways to stabilize and store different primers with a single dual-labeled hydrolysis probe treated with three different additives — trehalose, gelatin, and polyethylene glycol — achieving stability for up to three months.

However, Rombach noted that clinical regulations promulgated by various global standards organizations stipulate that a minimum shelf life of 12 months is required for in vitro diagnostics. Further, with the increasing use of multiplex qPCR assays in, for example, infectious disease testing or cancer biomarker panels, Rombach and colleagues wanted to test methods for long-term ambient storage of multiplex primers and fluorophores.

"We want to implement … multi-parameter analysis on a single sample," Rombach said. "We're kind of always in the position that we extract some DNA, then mix it with a master mix and split it into several amplification wells where we have the specific primers and probe sets. That's kind of a geometric multiplexing, as we call it — splitting the sample into different aliquots, and then distributing them to the final cavity where the amplification will run or take place. Then you can [assay for] different targets with different primer sets, and even use different fluorophore colors."

To develop their method, the HSG-IMIT researchers investigated the stability of four differently labeled hydrolysis probes with typical labels and emissions wavelengths used in nucleic acid diagnostics: 6-FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2.

To stabilize these reagents, they tested two different sugars, trehalose and xanthan, the former of which had been shown previously to be effective at stabilizing enzymes and nucleic acids and the latter of which is commonly used to extend shelf life in the food and cosmetics industry.

They mixed 0.2 µL of each fluorphore-quencher combination with 0.3 µL of forward and reverse primers, and added to those mixtures either 1 µL of trehalose for a 56mM final concentration or 1 µL of xanthan for a 2.78 mM final concentration. They spotted aliquots of these mixtures on cyclein olefin polymer strips — an emerging material for lab-on-chip applications — then air dried the reagents at ambient laboratory conditions, sealed them in aluminum bags filled with nitrogen and a drying agent, and aged the samples at ambient conditions for one year.

After aging the reagents, they then tested the functionality of each of the primer and fluorophore-quencher combinations by conducting real-time PCR on a standard double-stranded DNA target.

They found that the 6-FAM fluorophore-quencher combination was extremely stable even without additives, yielding satisfactory real-time PCR results. Meantime, real-time PCR using trehalose-treated prestored probes labeled with the remaining three fluorophore-quencher combinations corresponded to results obtained using untreated, un-aged primers and probes. However, the xanthan-treated reagents all resulted in reduced fluorescence signal during real-time PCR, generally yielding unsatisfactory real-time PCR data.

"This is already solving some problems we've had in the past," Rombach said. "Now [we want to] integrate the rest of the PCR amplification reagents, because with lyophilized reagents you always face problems that they attract the water molecules in the surrounding air if you're not working in a nitrogen atmosphere."

The end goal, he added, is to develop a chip with pre-stored reagents to screen for multiple nucleic acid targets using a single clinical sample such as blood, urine, or nasopharyngeal swabs.

In fact, Rombach is involved in another project at HSG-IMIT called RESPOC, an EU-funded effort to develop point-of-care molecular diagnostics to detect bacterial and viral respiratory pathogens in less than one hour.

"We're just trying to implement a nucleic acid testing assay for Bordetella pertussis and Streptococcus pneumonia as the targets," Rombach said. "Maybe we'll use this technique we just published for pre-storing the specific primers."

Furthermore, the RESPOC project is moving toward using isothermal amplification methods such as LAMP because of their compatibility with point-of-care testing. As such, "we now need to check if this technique also applies to LAMP primers," Rombach said. "Until now we have just tried it for PCR primers and probes."