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

Human telomerase reverse transcriptase (hTERT) mRNA in tissue is a biomarker of lung cancer, but hTERT mRNA in sputum had not been successfully detected with conventional reverse transcription PCR methods. Here, we developed a novel PCR protocol: Template-Ready PCR (TRPCR), to detect sputum hTERT mRNA, in which probes serve as templates of amplification. While free probes and dsDNA were removed in template preparation through aspiration and restriction digestion, probes that formed into heterocomplex with target RNA remained intact for PCR amplification. By fishing out the heterocomplex and amplifying the probes, TRPCR achieved sensitivity higher than reverse transcription-quantitative PCR (RT-qPCR). ROC curve of sputum hTERT mRNA by TRPCR assay showed the discrimination in high sensitivity and specificity between patients with lung cancer and lung cancer-free donors at the PCR Ct cutoff of 33. We further validated this approach through TRPCR assay of sputum from 858 lung cancer patients and 480 non-malignant pulmonary disease patients. 722 (84.2%) cases from 858 with lung cancer patients were detected as positive, whereas 461 (96.0%) cases from 480 non-malignant pulmonary disease patients were detected as negative, suggesting that TRPCR assay of sputum hTERT mRNA can serve as a non-invasive molecular diagnosis of lung cancer.