Multiplex qPCR Analysis: Simultaneous Detection of Multiple Targets in a Single Reaction

Multiplex qPCR Analysis

qPCR analysis is a reliable technique to confirm the presence and quantify the target nucleic acid sequence in a biological sample. The generated PCR product can be monitored in real-time through the fluorescent signal measured and correlating to the amount of nucleic acid sequence in the reaction mixture. Besides, researchers detect the PCR product through an intercalating dye or a fluorophore-conjugated with the sequence. 

A multiplex qPCR assay can detect multiple sequences of interest in a single reaction mixture. Multiplex qPCR assays deliver high throughput analysis to assess several target sequences across hundreds of study samples. Hence, qPCR CROs offering PCR services and immunogenicity testing services are relying on qPCR assays for gene expression analysis. Moreover, qPCR assays have witnessed the development of advanced alternatives such as ddPCR assay and digital PCR services. ddPCR analysis divides the study sample into thousands of microdroplets, where each droplet consists of the target RNA or DNA. The current article discusses multiplex qPCR analysis for detecting multiple targets in a single reaction volume. 

Multiplex qPCR analysis

Depending on the assay design, researchers can use multiple primer probes or primer pairs with fluorophores to identify and detect target sequences through multiplex qPCR assays. Hence, thorough assay design and optimization are critical for generating the highest-quality data. Scientists can detect each amplified product independently by labeling target probes with unique fluorophores having non-overlapping emission and excitation spectra. 

When researchers have to assess multiple target sequences in a study sample, multiplex qPCR assays offer several technical and logistical benefits over singleplex assays. Multiplex qPCR assays save biological samples. This property is particularly critical when working with limited biological samples such as biopsy or tissue material. Besides, multiplex qPCR assays save time by minimizing reaction numbers required for multiple targets, eventually reducing costs by saving consumables and reagents. 

Besides saving costs and increasing efficiency, multiplex qPCR simplifies data analysis. qPCR assays compare targets in the same sample. Assessing target sequences in the same reaction negates the requirement of controlling well-to-well or plate-to-plate variability observed when running different qPCR reactions. 

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Multiplex qPCR assays have numerous applications across forensics, clinical diagnostics, and biomedical research. The multiplexing and high throughput ability of multiplex qPCR assays has made them the primary choice of technique in diagnosing infectious diseases. They are ideal for infectious disease research because they can simultaneously identify multiple pathogens and characterize them by evaluating genes predicting response to several antibiotics and medications. Additionally, multiplex qPCR can confirm the authenticity or detect contamination in food products. qPCR can also estimate DNA quality and quantity in forensic samples. 

The number of target sequences identified through multiplex qPCR assays depends on dye and channel compatibility. Hence, researchers should select dyes with non-overlapping emission and excitation spectra. Multiplex qPCR employs more probes and primers, which may produce non-specific products. Here, researchers can avoid primer dimer products to optimize qPCR assays.

In conclusion

Multiplex qPCR analysis is an ideal technique to simultaneously detect multiple targets in a single reaction volume. 

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