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Updated: Feb 6, 2026

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRT-PCR) for Precise Quantitation of Messenger RNA
Yuanyuan Sun1, Hui Tian1, Chenghui Liu1
1Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , P. R. China.
Abstract:
Precise detection of the low copy numbers of messenger RNA (mRNA) mutation in single cells is of great significance but still remains challenging. Herein, by integrating the outstanding features of a rationally designed peptide nucleic acid (PNA) clamp for highly selective discrimination of single-nucleotide variation, and droplet digital PCR for ultrasensitive and precise quantification, we have developed a robust one-step droplet digital reverse transcription PCR (ddRT-PCR) method which enables precise mRNA mutation detection in single cells with ultrahigh specificity to clearly discern as low as 0.01% mutated mRNA in a high background of wild-type mRNA. Because of its outstanding single-molecule level sensitivity and ultrahigh specificity, this ddRT-PCR method holds great promise for studying cellular heterogeneity at the single cell level, as well as for the precise quantification of mutant mRNAs in complex plasma or serum for liquid biopsy.
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