Multiplex qPCR is a popular way of enhancing assays by combining multiple reactions in a single well. This approach can be extremely tempting, offering the potential for more efficient, cost-effective and reliable molecular diagnostics. However, multiplex qPCR comes with additional challenges and effort in setting up and optimising. If you’re trying to decide if it’s worth the investment, this blog post may help you to better understand the trade-offs.
If you’ve decided to take a multiplex approach, or are having difficulties with your assays, see our eBook on how to optimise and validate a multiplex qPCR assay.
Advantages of multiplexing
There are many plus points for a multiplex qPCR assay compared to carrying out each reaction in individual wells.
Perhaps the most obvious of these is reducing the amount of sample required. This can be important if the supply of the sample is limited, for example with clinical specimens that are difficult to collect.
Combining assays per well also reduces reagents costs. While there still needs to be sufficient dNTPs for all assays to reach completion, master mix components like polymerase can be used more efficiently by multiplexing.
Multiplexing also provides faster results by allowing many more assays to complete for each thermal cycler run. This is particularly important when setting up a high-throughput testing system.
Multiplex qPCR assays can also be more reliable. With all targets sharing the same well, there is less risk from pipetting errors. Also, by having the control assay in the same well as the target assay, developers can have more confidence in the results.
Challenges of multiplexing
So far, so good. But these benefits come at a cost. Multiplex assays are inherently more complicated than singleplex, and that requires more work in the early stages to take advantage of the improved speed, cost and reliability.
Combining multiple primers and probes in one well raises the risk of interactions between these oligos, which could affect the assay performance. The sequences need to be carefully designed to avoid unintentional sequence homology leading to primer dimers.
Each probe must also be carefully paired with a dye that doesn’t overlap with the emission spectra of the others. Any crosstalk between the signals can lead to false positives or negatives if they are misinterpreted.
Then when it comes to optimising the assay conditions, it’s best to optimise each assay individually to understand the level of performance expected. Then, when reviewing the results from a multiplex reaction, it is much clearer to recognise if the performance of a particular assay has fallen too much and identify the source of the issue.
This can involve multiple iterations of tweaking the conditions to ensure that all assays perform as expected. For more information on optimising and validating a multiplex assay, read our eBook on the subject.
If a target is present in significantly higher amounts than others, you may need to lower the primer concentration for that target. Otherwise, the reagents may be quickly exhausted, reducing the performance of the other assays.
If just running a simple duplex reaction, typically a target paired with a control, the design and optimisation may be quite straightforward. However, the complexity rises significantly with higher-level multiplexes that potentially include five or six assays in one.
Multiplexing put into practice
Multiplex assays are common among molecular diagnostic tests for infectious diseases. Such tests might aim to identify which pathogen is the cause of the symptoms from a panel of diseases. Examples of these include tests for urinary tract infections (UTIs) or sexually transmitted infections (STIs).1 Multiplex assays have also been used to combine tests for flu and Covid to help distinguish between the diseases during flu season.2
Researchers have also used a triplex RT-qPCR to quantify the expression of cancer-causing genes in two human papillomaviruses (HPV) normalised on GAPDH expression levels.3 This could provide extra information to help in the follow-up of patients at higher risk of cervical cancer, for instance.
Multiplex tests are also popular to identify which genetic variants are present in a pathogen to inform the best response. These assays can reveal if a bacterial infection is resistant to certain antibiotics, allowing for more tailored and effective treatment.1 This can offer clinicians greater confidence in their prescribing decisions.4
Companies like SpeeDx are developing high-multiplex assays to identify resistance markers for STIs and UTIs – common diseases where antibiotics are heavily used. “In the past, we have operated by administering blanket antibiotics, the same antibiotic at the same dose, to everyone who comes in with urethritis,” said Professor Catriona Bradshaw, Genital Microbiota and Mycoplasma Group Lead at Monash University. “We need to develop this approach for all infectious diseases where resistance is an issue. It exists for certain infections like tuberculosis or HIV, but it’s just not there for many common infections.”
Multiplex qPCR was also used to detect specific strains of the bacteria Listeria monocytogenes in food.5 This could help to trace the source of the most clinically important strains that cause severe gastroenteritis.
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To find out how best to approach developing a multiplex qPCR assay, download our eBook guide dedicated to optimisation and validation. 
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References
- Davenport M et al. (2017) New and developing diagnostic technologies for urinary tract infections. Nat Rev Urol. 14(5):296-310. doi: 1038/nrurol.2017.20
- Abbasi et al. (2023) Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses. BMC Microbiol23, 335. doi: 1186/s12866-023-03048-9
- Bordigoni A et al.(2021) Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 Sci Rep 11, 4039. doi: 10.1038/s41598-021-83489-2
- Pandolfo AM et al.(2021) Intensivists’ beliefs about rapid multiplex molecular diagnostic testing and its potential role in improving prescribing decisions and antimicrobial stewardship: a qualitative study. Antimicrob Resist Infect Control 10, 95. doi: 1186/s13756-021-00961-4
- Cheng J et al. (2024) A novel multiplex PCR based method for the detection of Listeria monocytogenes clonal complex 8. International Journal of Food Microbiology 409:110475 doi: 1016/j.ijfoodmicro.2023.110475
