Accurate nucleic acid quantification in a single sample tube without the need for calibration

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Traditional nucleic acid quantification (NAQ) is essential for clinical diagnostics, forensics, and food analysis.
• Existing NAQ methods are laborious, time-consuming, expensive, and prone to errors from calibration curves and tube-to-tube variations.
• Current methods struggle with inhibitor detection and require sample dilution, compromising accuracy and efficiency.

Purpose of the Study:

• To develop a novel, accurate, and convenient nucleic acid quantification method.
• To eliminate the need for calibration curves and reduce errors in NAQ.
• To create a method robust against inhibitors and suitable for rapid, on-site applications.

Main Methods:

• Introduced a single-tube quantitative PCR (stqPCR) technique.
• Utilized an internal quantitative standard DNA (IQS-DNA) co-amplified with the target DNA.
• Quantification based on the difference in quantification cycle values (ΔCq) between IQS-DNA and target DNA.

Main Results:

• Achieved high accuracy in NAQ for plasmid, serum standard, and clinical samples (R² > 0.97).
• Demonstrated robust performance in the presence of inhibitors, unlike commercial kits requiring significant dilution.
• Integration with microfluidics enabled super-fast NAQ (<30 min) and sample-to-answer testing (<40 min).

Conclusions:

• The stqPCR method provides accurate and reliable nucleic acid quantification without calibration curves.
• This technique overcomes limitations of traditional methods, including inhibitor interference and pipetting errors.
• stqPCR holds significant potential for advancing laboratory and on-site NAQ applications.