Enhancing Sensitivity and Specificity in qPCR Assay Development

qPCR assay development
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Polymerase chain reaction (PCR) is a reliable technique for generating multiple copies of a DNA segment. First developed in 1983, PCR can generate millions of DNA copies. Today, it has become one of the most commonly used tools in biotechnology and molecular biology laboratories. Besides coupling it with other technologies, such as the Meso Scale Discovery assay, have advanced biological and clinical research to the next stage of transformation.

Today, there are multiple types of PCR assays, including qPCR, multiplex PCR, fast cycling PCR, ddPCR assay, and much more. ddPCR gene expression analysis is increasingly being employed in genomic studies. Besides, the ddPCR service has now become an integral component of analytical testing. The current article focuses on qPCR assay development to enhance its sensitivity and specificity. Besides, it provides a technical guide for qPCR CROs. 

qPCR assay development

qPCR or quantitative PCR is a DNA amplification technique that can identify, characterize, and quantify non-DNA sequences in a study sample. Hence, a thoroughly optimized and validated qPCR assay is crucial for generating reliable research. 

Assay optimization and validation are vital even for predesigned and commercially available assays. Assay optimization ensures the method is sensitive enough for the target of interest. Researchers can modify multiple parameters to attain optimal assay performance. A thoroughly designed qPCR assay will perform well under different experimental conditions. However, most assay parameters depend on selected reagents, primer concentrations, instruments, and temperature. Hence, irrespective of the target RNA or DNA, the following steps can help researchers ensure successful qualification:

  • Primer design validation
  • Primer concentration optimisation
  • Primer annealing temperature optimization
  • Probe concentration optimization
  • Optimizing reaction components
  • Validating assay performance

Validating primer design is critical when using primers from commercial supplies or previous publications. Some primary considerations for validating primer designing include:

  • Homologous primers for the desired target sequence
  • Correct reverse complement primer
  • Appropriate splice variants
  • Avoiding SNPs

Usually, a final concentration of 500 nM for primers and 250 nM for probes yields satisfactory results for probe-based qPCR. However, for SYBR green I-based detection, a lower concentration can minimize nonspecific amplification. Besides, a standard curve analysis can help verify optimal conditions for qPCR assay. 

qPCR assays generally use a two or three-step temperature cycling program. Two-step temperature cycles are used with dual-labeled probes, while a three-step cycle is suitable for complex target sequences. 

A final probe concentration of 250 nM is used mostly in qPCR assays. However, researchers may reduce costs by using lower concentrations when maximum sensitivity is not needed. Scientists must optimize probe conditions by testing it in several concentrations along with the lowest concentration of target nucleic assay and optimized concentrations of primers. 

Some qPCR assays are sensitive to reaction and buffer conditions. Scientists can overcome these challenges by modifying reaction buffers through magnesium chloride optimization, adding PCR enhancers, or adjusting instrument ramp rates. These modifications can help optimize multiplex qPCR reactions.


qPCR assays are increasingly becoming critical in biological and clinical research. However, adequate efforts in qPCR assay development and validation will remain important in enhancing its sensitivity and specificity and expanding its applications in different pharmaceutical domains.