SF-qPCR: Strand Displacement-Based Fast Quantitative Polymerase Chain Reaction

Area of Science:

• Molecular Biology
• Biotechnology
• Clinical Diagnostics

Background:

• Nucleic acid testing (NAT) is crucial for identifying and quantifying DNA and RNA targets in life sciences and diagnostics.
• The polymerase chain reaction (PCR) assay, a common NAT method, involves a time-consuming amplification step.
• Optimizing PCR protocols is essential for faster and more efficient molecular diagnostics.

Purpose of the Study:

• To develop a simplified and faster quantitative PCR (qPCR) method for nucleic acid amplification.
• To reduce the amplification time of qPCR assays while maintaining sensitivity and accuracy.
• To adapt the qPCR protocol for rapid detection of clinically relevant DNA and RNA targets.

Main Methods:

• Modified a standard three-step qPCR protocol into a two-step process.
• Optimized temperatures by increasing annealing/extension temperatures and lowering denaturation temperature.
• Replaced Taq polymerase with SD polymerase, which possesses strand displacement activity for efficient amplification at lower denaturation temperatures.
• Validated the method, termed SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), using genomic DNA and SARS-CoV-2 RNA targets.

Main Results:

• The developed two-step SF-qPCR protocol successfully reduced amplification time to 25-40 minutes.
• The method demonstrated stable detection of DNA and RNA targets at concentrations as low as 10 copies.
• Amplification of the SARS-CoV-2 nucleocapsid (N) gene was achieved with high sensitivity.

Conclusions:

• The SF-qPCR method provides a sensitive and rapid approach for detecting DNA and RNA targets.
• This optimized protocol significantly shortens the time required for nucleic acid amplification in qPCR assays.
• SF-qPCR holds promise for improving the efficiency of clinical diagnostics and life science research.