|
Objective To construct pMD18-T-hTERT recombinant plasmid as a standard, the establishment of SYBR Green real-time fluorescence quantitative PCR (Real-time fluorescent quantitative PCR, FQ-PCR) method for detection of liver cancer (hepatocellular carcinoma, HCC) patients with plasma of human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) DNA levels, and analysis of its clinical significance. Method 1. Establish plasma DNA extraction method by conventional PCR amplification of the target gene hTERT confirm plasma DNA extraction. Human genomic DNA as a template, conventional PCR amplification of the hTERT gene, purified amplification products connected with pMD18-T vector to construct the recombinant plasmid as a standard for quantitative detection and amplified by PCR and sequencing. SYBR Green real-time PCR method to detect plasma of hTERT DNA designed the hTERT DNA primers, establish and methodological evaluation (including precision, specificity and linear range). Collected 60 patients with HCC preoperative plasma and the corresponding clinical data of 21 cases of hepatitis B (hepatitis B virus, HBV) patients and 29 healthy subjects, plasma control. Extracted plasma DNA FQ-PCR method using the established quantitative detection of plasma hTERT DNA level, hTERT DNA levels in plasma of HCC diagnosis efficiency, as well as the relationship between the clinical characteristics of patients with HCC. At the same time, the detection of 19 cases of postoperative plasma the hTERT DNA level, and compared with preoperative levels. Results of plasma DNA extraction and the establishment and evaluation of FQ-PCR method: ① successfully established plasma DNA extraction methods. ② successfully constructed the recombinant plasmid pMD18-T-hTERT, after PCR amplification and sequencing are consistent with the expected results. Optimal ③ FQ-PCR reaction system: SYBR Premix Ex Taq (2 ×) 10.0μl on downstream primer (10μM) 0.8μl, ROX reference dye II 0.4μl, template 2.0μl double distilled water (ddH < sub> 2 O) 6.0μl, total of 20.0μl. The optimal reaction conditions: 95 ° C denaturation for 10 s, 95 ° C denaturation 5 s, annealing at 60 ° C for 34 s, 40 cycles; This method is not smaller precision (the intra day coefficient of variation were 0.86 % and 1.44%), high specificity, linear range wide (10 3 the to 10 8 copies / μl). 2 plasma hTERT DNA and clinical features and laboratory parameters in patients with HCC: ① HCC patients plasma level of hTERT DNA {(4.18 × 10 4 ± 4.94 × 4 ) copies / μl} were significantly higher than HBV patients {(1.44 × 10 4 ± 6.61 × 10 3 ) copies / μl} and healthy controls {(1.21 × 10 4 ± 6.63 × 10 3 ) copies / μl}, the difference was statistically significant (P lt; 0.01). ② ROC curve, 1.87 × 10 4 copies / μl for the cutoff value, the sensitivity and specificity of the diagnosis of HCC were 64% and 90%. ③ HCC patients plasma hTERT DNA level and tumor diameter, TNM stage, portal vein tumor thrombus and is closely related to serum levels of AST (P lt; 0.05), with lymph node metastasis, peripheral blood leukocyte count, serum AFP and ALT levels were unrelated (P gt; 0.05 ); addition, TNM stage Ⅰ - Phase II and serum AFP ≤ 20 ng / ml in patients with HCC plasma hTERT DNA levels were significantly higher than HBV patients and healthy controls (P lt; 0.05). ④ detected 19 patients plasma the hTERT DNA level, significantly reduced compared with before surgery, and the difference was statistically significant (P lt; 0.05). Conclusion plasma DNA extraction methods and standard recombinant plasmid pMD18-T-hTERT successfully established FQ-PCR method to detect plasma hTERT DNA. HCC plasma hTERT DNA levels significantly higher than HBV patients and healthy controls, and tumor diameter, TNM stage, portal vein tumor thrombus and serum levels of AST are highly correlated. FQ-PCR detection of plasma the hTERT DNA level is expected to become an important tool of clinical HCC early screening and disease monitoring.
|