Because multiplex PCR assays are run simultaneously on the same sample, they offer higher throughput and less processing, which improves overall cost effectiveness.
Multiplex PCR assays:
On the surface, then, multiplexing seems like a logical choice. Running multiple analyses in one sample must be more efficient than repeated singleplex assays, saving labs time and money. But the answer is more complex.
Multiplex PCR can present optimisation challenges. First, not all combinations of tests can be run together without compromising performance and accuracy. Each primer pair and probe in the test is designed to amplify and detect a unique target; the more primers in a single reaction, the more likely you are to see off-target amplifications in the results. Having multiple assays in one well can also lead to competing PCR reactions, which could mean false negative results.
That’s why assay optimisation is first required to ensure the accuracy of a multiplex test. Labs also compare multiplex conditions to each individual reaction to verify performance. Performance verification includes the evaluation and optimisation of primers and probes, additives, and cycling conditions. This helps to identify the right combination of conditions and ingredients that meet the same levels of efficiency as a corresponding singleplex reaction. This process, however, can be time consuming and costly.
How can labs improve their outcomes and their operational efficiency? The answer lies in an optimised multiplex master mix.
A multiplex master mix is a convenient, ready-to-use solution that includes pre-optimised concentrations of buffers, DNA polymerase, dNTPs, and additives that can support amplification and identification of multiple nucleic acid targets.
Ideally, since the reactions are often set up at room temperature, the master mix should have thermostable enzymes.
A multiplex master mix has been repeatedly tested and optimised by the supplier to ensure that multiplex reactions are of comparable specificity and sensitivity to corresponding singleplex tests. The mix should seamlessly enable the detection of targets from a wide range of sample materials.
Using a multiplex master mix can reduce lab costs by:
Multiplex assays can streamline workflow and simplify handling for improved efficiency and cost control. Using optimised master mixes in multiplex testing can deliver accuracy, sensitivity, and specificity that is comparable to singleplex testing, minimising time-consuming assay optimisation. This can help labs improve operational efficiency, reduce overall costs, and improve their economic outlooks.