Overcoming the challenge of primer-dimers in multiplexed PCR assays

Although PCR is often viewed as a gold standard for molecular diagnostic tests, the problem of non-specific primer interactions can seriously limit its speed and sensitivity. LGC Biosearch Technologies has supported developing an innovative approach to primer design that overcomes this significant hurdle and creates new opportunities for faster, less expensive and higher quality molecular diagnostic tests.

Designing a better primer

Assay design requires striking a tricky balance between sensitivity and specificity. In PCR-based assays, primer design exemplifies this challenge – primers need to be long enough to bind tightly to the target but not so long that they anneal to the wrong sequence.covid-sample

The growth in demand for multiplexed tests increases the complexity of primer design, as targeting multiple sequences further raises the risk of the different primers amplifying each other. These primer-dimers can be a major problem for PCR test developers as they weaken the signal and can even cause false negatives and positives.

“These errors dramatically reduce the accuracy of molecular diagnostic PCR tests and can lead to false results and inaccurate diagnoses in healthcare, which can be detrimental to patient health,” says Dr Kenneth Bramwell, VP of In Vitro Diagnostics at Co-Diagnostics, Inc.

Co-Diagnostics has developed an approach to increase the sensitivity and specificity of PCR through its Co-Primers® technology. These Co-Primers have two target recognition sequences linked together – a short primer and a longer capture sequence.

The primer sequence is short enough that it would not ordinarily amplify the template. However, the capture sequence is designed to tightly bind to a region near the primer’s complementary target. By anchoring the Co-Primers in place, the capture sequence brings the primer sequence close to the target, enabling sufficient annealing for amplification.

This design means that the short primer does not anneal to any sequence unless there is a corresponding region to which the capture sequence can bind, vastly reducing primer-dimer formation.


Improved multiplexed diagnostic assays

Reducing primer-dimers leads to improved multiplexed assays, a key factor for developing more comprehensive and easier-to-use multi-target diagnostics. “With the Co-Primers technology, we can carry out multiplexes within a singular molecular diagnostic test to simultaneously identify multiple diseases that would otherwise have to be tested for individually. This enables us to utilise singular samples and significantly increase our throughput, saving a significant amount of time and money. Plus, a reduction in primer-dimers and shorter primer development time further cut the overhead costs of PCR testing, helping make infectious disease diagnostics more affordable across the globe,” says Bramwell.

Co-Primers are already being used in India and elsewhere for multiplexed tests like a combined dengue and chikungunya test, and for a combined Influenza A/B and COVID-19 test, among others.

Bramwell also says that this technology could benefit applications beyond human healthcare, such as the food and agricultural industries: “Co-Primers can improve our ability to detect beneficial single-nucleotide polymorphisms (SNPs) in crops, such as adaptations that can increase production rates or increase resistance to diseases. Similar to detecting pathogens in humans, Co-Primers can also detect harmful bacteria, such as E. coli, in food.”


Delivering on an industrial scale Nexar blog

Brent Satterfield, co-founder of Co-Diagnostics, developed the Co-Primers technology in 2013.1 LGC Biosearch Technologies synthesised these innovative molecules for the original study, with the two key sequences connected by a polyethylene glycol linker that prevents the polymerase from extending through the capture sequence. This also included manufacturing prototype Co-Primers in which the 5ʹ ends of the primer and capture sequence were linked together, using reverse amidites to switch the strand polarity.

“LGC Biosearch Technologies was an early partner that was willing to accept this challenge and provide the expertise necessary to develop and master the chemical synthesis and the purification of Co-Primers on an industrial scale. They have been critical in helping bring this innovative new technology to fruition. We really couldn’t have done it without them,” says Bramwell.

In addition to reducing the propagation of primer-dimers, Co-Primers can also increase the signal through greater probe efficiency. The capture sequence can include a dye and quencher, such as BHQ Probes, to function as a hydrolysis probe. As the probe must bind to the template for amplification to occur, it is more likely to be hydrolysed by the polymerase and release the fluorophore. This led to 2.5 times the fluorescent signal of normal hybridisation probes.1

Scaling up from developing an initial concept to delivering on an industrial level requires partnering with a supplier who can adapt and customise according to your needs. LGC Biosearch Technologies has a long history of supporting scientific innovators to build a safer world.


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  1. Satterfield BC (2014) Cooperative Primers. The Journal of Molecular Diagnostics 16(2):163 – 173 DOI:https://doi.org/10.1016/j.jmoldx.2013.10.004

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