| Objective: Construct quantitative plasmid standard pMD18-T-CK20 and develop a novel real-time PCR method using Taqman probe for convenient, fast, sensitive and specific detecting CK20 in urine and using in early diagnosis and follow-up of transitional cell carcinoma of bladder (TCCB).Methods: 1. CK20 gene from cultured T24 cells origined bladder cancer was amplified by conventional RT-PCR, the standard quantitative plasmid pMD18-T-CK20 was constructed by T-A clone method and analyzed by restriction enzyme digestion, PCR identification and DNA sequencing. 2. Taqman probe and primers were designed according to the sequence of CK20 cloned gene, the real-time PCR method for determination of CK20 mRNA was established, the reaction condition and system were optimized and evaluated (linear range, sensitivity, specificity , reproducibility ) . 3. The CK20 mRNA level was screened by real-time PCR in 95 urine samples including 60 TCCB patients, 20 non-cancer disease patients and 15 health volunteers, and analyzed according to the following parameters: urinary cytology, tumor stage and grade.Results:1. Standard preparation: By restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMD18-T-CK20 was successful, and corresponded with request of quantitative standard.2. Development and evaluated of quantitative PCR method: we established a new quantitative detection method and the reaction condition and system were optimized.①The better reaction condition was as follows: Each cycle was of 5 min each at 95°C, 30 sec at 95°C, and 70 sec at 51°C, 40 cycles. The better reaction system was 25μl volumes containing 10×PCR buffer: 2.5μl; MgCl2 (25 mmol/l): 3.5μl; dNTPs (2.5 mmol/l): 2μl, Taq polymerase (5 U/μl): 0.35μl; forward primer (10 pmol/μl): 2.2μl; reverse primer (10 pmol/μl): 2.2μl; Taqman probe (10 pmol/μl): 2.2μl; cDNA: 2μl; ddH2O: 8.05μl.②Technology evaluated : The developed real-time PCR method showed high sensitivity(102 copies/μl) and good specificity , the linear range was102 ~109 copies/μl, the coefficient variation (CV) was 1.59 % in intra-assay and 2.34% in day to day.3. Clinical application:①For 60 TCCB patients urinary cytology was positive in 28 (46.7%), control group had no false-positive results (specificity 100%); CK20 expression was positive in real-time PCR of 51 cases (85%) of TCCB, but control group was positive in 2 cases (specificity 94.3%) with a cutoff value of crossover point (CT) = 30. Two methods have significant variation in sensitivity (p < 0.01).②CK20 mRNA values in TCCB group (mean 27712.95 copies/ul) were significantly higher than in benign disease group (mean 74.72 copies/ul) and control group (mean 8.61 copies/ul) (p < 0.01, p < 0.01, respectively).③CK20 mRNA values increased gradually with higher tumor grade(Mean G1: 21680.5, G2: 30985.82, G3: 36333.89 copies/ul ) and stage( Mean Tis/Ta: 20672.16, T1-2: 29213.19, T3-4: 35585.63 copies/ul ): G1 differs significantly from G2 (p = 0.016); Tis/Ta differs significantly from T1-2 (p =0.022).Conclusion: CK20 real-time PCR is an operation simple, sensitive, specific and outcome data method to detect free bladder cancer cells in urine, and could be recommended for being wide application in the early clinical diagnostics of TCCB and monitoring of recurrence, and conduce to identify stage and grade of TCCB.
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