Quantitation Of The Plasma HTERT DNA HCC Patients By Fluorescent Quantitative PCR And Its Significance

ObjectiveTo construct plasmid pMD18-T-hTERT as standard, the quantitative real-time polymerase chain reaction(FQ-PCR) using SYBR Green was developed to quantify the plasma human telomerase reverse transcriptase (hTERT) DNA of patients with hepatocellular carcinoma (HCC). The clinical significance of plasma hTERT DNA was analyzed.Methods1. The method of extracting plasma DNA was developed and confirmed by amplification of hTERT gene. As standard, the plasmid pMD18-T-hTERT was constructed by T-A clone method, the plasmid DNA was extracted and confirmed by PCR and DNA sequencing. Pimers were designed according to the sequence of hTERT gene. The FQ-PCR was established to quantify the plasma hTERT DNA and the methodologies including imprecision, specificity and linear range were evaluated.2. Plasma and the associated clinical characters of 60 HCC patients were collected, 21 HBV patients and 29 healthy persons were enrolled as control. Plasma DNA was extracted and quantified by the established method. Its diagnostic efficiency and relationship to clinical characters were analyzed. In addition,plasma hTERT DNA levels of 19 HCC patients after operation was compared with the preoperative levels.Results1. Extraction of plasma DNA and evaluation of developed FQ-PCR method:â‘ Established the method of extracting plasma DNA successfully.â‘¡The plasmid of pMD18-T-hTERT was constructed successfully and confirmed by PCR and DNA sequencing.â‘¢The optimum reaction system was 20.0μl volumes containing SYBR Premix Ex Taq (2×) : 10.0μl, forward primer (10μM) : 0.8μl,reverse primer (10μM) : 0.8μl, ROX reference dye II: 0.4μl, DNA: 2.0μl, ddH₂O: 6.0μl. The optimum condition of FQ-PCR was as follows:10 s at 95℃,5s at 95℃,and 34 s at 60℃,40 cycles. The developed FQ-PCR method showed small imprecision (the coefficient variation was 0.86% within runs and 1.44% between days), high specificity and good linear range(10³ï½ž10⁸ copies/μl).2. Relationship between plasma hTERT DNA of HCC patients and clinical characters:â‘ The levels of plasma hTERT DNA in the HCC patients {(4.18×10⁴±4.94×10⁴) copies/μl} were significantly higher than that of HBV patients {(1.44×10⁴±6.61×10³) copies/μl} and healthy controls {(1.21×10⁴±6.63×10³) copies/μl} (P<0.01).â‘¡ROC curve showed a sensitivity of 64% and specificity of 90% in the ability to detect HCC at a cut-off of 1.87×10⁴ copies/μl.â‘¢Plasma hTERT DNA levels were closely relevant to the tumor size, TNM stage, portal vein cancer embolus and serum AST level,but there was irrelevant to the lymph node metastases, peripheral blood leukocyte count, serum ALT and AFP levels (P>0.05). Of interest, the plasma hTERT DNA levels in HCC patients with TNM stageâ… -â…¡and those with AFP≤20 ng/ml were higher than that of HBV and healthy controls with statistical significance (P<0.05).â‘£Of 19 HCC patients, the plasma hTERT DNA level after operation was significantly lower than that before operation (P<0.05).ConclusionEstablished the method of extracting plasma DNA successfully. Taking plasmid pMD18-T-hTERT as standard, FQ-PCR method to quantify plasma hTERT DNA was developed successfully with good methodological perfomance. The levels of plasma hTERT DNA in HCC patients were significantly higher than that of HBV and healthy controls, and closely relevant to the tumor size, TNM stage, portal vein cancer thrombus and serum AST level. Quantification of plasma hTERT DNA by FQ-PCR may be a good and simple tool for early screening and monitoring of HCC with a potential clinical application.