Template-ready PCR method for detection of human telomerase reverse transcriptase mRNA in sputum

Area of Science:

• Molecular Biology
• Oncology
• Biochemistry

Background:

• Human telomerase reverse transcriptase (hTERT) mRNA is a known lung cancer biomarker in tissue.
• Conventional methods struggle to detect hTERT mRNA in sputum samples.
• Need for sensitive, non-invasive diagnostic tools for lung cancer.

Purpose of the Study:

• To develop a novel PCR protocol for detecting sputum hTERT mRNA.
• To enhance sensitivity and specificity for lung cancer diagnosis using sputum biomarkers.
• To validate a new non-invasive molecular diagnostic approach for lung cancer.

Main Methods:

• Development of Template-Ready PCR (TRPCR) protocol utilizing probes as amplification templates.
• Removal of free probes and dsDNA via aspiration and restriction digestion.
• Detection of hTERT mRNA by amplifying probes within heterocomplexes with target RNA.

Main Results:

• TRPCR demonstrated higher sensitivity compared to reverse transcription-quantitative PCR (RT-qPCR).
• Receiver Operating Characteristic (ROC) curve analysis showed high sensitivity and specificity for lung cancer detection (Ct cutoff 33).
• In a large cohort, TRPCR correctly identified 84.2% of lung cancer patients and 96.0% of non-malignant cases.

Conclusions:

• TRPCR is a highly sensitive and specific method for detecting sputum hTERT mRNA.
• This assay offers a promising non-invasive molecular diagnostic tool for lung cancer.
• The TRPCR assay significantly advances early lung cancer detection capabilities.