Optimization and performance evaluation of double-stranded probe in real-time PCR.

Area of Science:

• Molecular Biology
• Nucleic Acid Detection
• Biotechnology

Background:

• TaqMan probe-based real-time PCR (qPCR/RT-qPCR) offers high sensitivity and specificity.
• However, TaqMan probes exhibit higher background signals, potentially compromising detection accuracy.

Purpose of the Study:

• To design and optimize double-stranded probes (DSPs) for highly sensitive nucleic acid detection using qPCR/RT-qPCR.
• To evaluate the performance of novel DSPs compared to existing probe types.

Main Methods:

• Developed double-stranded probes (DSPs) for detecting hepatitis B virus (HBV) DNA and SARS-CoV-2 RNA.
• Systematically optimized DSP parameters including strand length, hybridization free energy (ΔG), and sticky end length.

Main Results:

• Optimized DSPs with similar strand lengths, ΔG of approximately -30 kcal/mol, and 4-6 nt sticky ends showed significantly improved analytical performance.
• DSPs demonstrated superior fluorescence signal intensity and sensitivity compared to other probe types.

Conclusions:

• Double-stranded probes (DSPs) offer enhanced sensitivity for molecular diagnostics.
• DSPs improve the detection rate of low-concentration samples in qPCR/RT-qPCR assays.