Development of fluorescent quantitation PCR method for detection of urinary CK20

Objective:To develop a novel real-time fluorescence PCR method using Taqman probe for convenient,fast,sensitive and specific detection of urinary CK20 and use in early diagnosis and follow-up of transitional cell carcinoma of the bladder(TCCB).Methods:CK20 gene from cultured T24 cells was amplified by conventional RT-PCR,the standard quantitative plasmid was constructed by T-Aclone method.Taqman probe and primers was designed according to the sequence of CK20 cloned gene,the real-time PCR method for determination of CK20 mRNA was established and evaluated.Results:The developed real-time PCR method showed high sensitivity(10 2copies /μl) and good specificity;the linear range was 102 ~109 copies /μl,the coefficient variation(CV) was 0.89 % in intra-assay and 2.45% in day to day.Conclusion:A novel real-time fluorescence quantitative PCR method for detection of CK20 was established.It provided a basis for developing new diagnostic kits using the Taqman probe for clinical application.