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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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IDSP-IA: Immunotriggered Double Stem-Loop Probe-Mediated Isothermal Amplification for Attomolar Protein

Jiangyan Zhang¹, Yinuo Wang¹, Qing Li¹

¹Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Materials Science, Hebei University, Baoding 071002, P. R. China.

Analytical Chemistry

|October 6, 2025

Summary

A new assay enables ultrasensitive protein detection for disease management. This immunotriggered double stem-loop probe-mediated isothermal amplification (IDSP-IA) assay offers high specificity and a wide dynamic range for biomarker quantification.

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Area of Science:

Biochemistry
Molecular Biology
Assay Development

Background:

Accurate quantification of low-abundance proteins is crucial for disease management.
Existing protein detection methods face challenges in sensitivity and specificity.

Purpose of the Study:

To develop a novel assay for ultrasensitive and specific protein detection.
To establish a modular and universally adaptable platform for protein quantification.

Main Methods:

Developed an immunotriggered double stem-loop probe-mediated isothermal amplification (IDSP-IA) assay.
Utilized a monoclonal capture antibody (mAb1) for target protein capture and enrichment.
Employed an aptamer-oligonucleotide switch (Apt-OS) for signal transduction to nucleic acid triggers, initiating isothermal amplification.

Main Results:

Achieved a limit of detection (LOD) of 1.3 fg/mL for alpha-fetoprotein (AFP) and 1.4 fg/mL for interleukin-6 (IL-6).
Demonstrated a wide dynamic range (5 fg/mL to 50 pg/mL) with no cross-reactivity from other serum proteins.
Clinical validation showed excellent correlation with established electrochemiluminescence and chemiluminescence immunoassays.

Conclusions:

The IDSP-IA assay provides a robust, flexible, and universally adaptable tool for attomolar protein biomarker quantification.
This assay offers high specificity, ultrasensitivity, and a modular design suitable for diagnostic and biomedical applications.