[Detection of urine DD3/PSA mRNA ratio by duplex TaqMan RT-PCR assay and evolution of its primary application].
OBJECTIVE
To establish a duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio, and to evaluate its effect in diagnosis of prostate cancer (PCa).


METHODS
Urine samples were obtained from 34 patients with PCa and 44 patients with benign prostate hypertrophy (BPH) by prostate massage. A duplex TaqMan RT-PCR assay was developed: PCR primers were designed to amplify the fragments between the exon1 and exon2 in the PSA mRNA and between the exon1 and exon3 in the DD3 mRNA, and the PCR TaqMan-MGB probes were labeled with HEX and FAM respectively in 5' for PSA mRNA. LNCaP cells were used as template. DD3/PSA mRNA ratio was measured. Receiver operating characteristic curve (ROC) was drawn so as to evaluate its diagnostic efficacy.


RESULTS
Sequencing showed that the PCR products were specific for PSA mRNA and DD3 mRNA. The minimum detection level was approximately 0.6 cells/reaction for PSA mRNA and was 60 cells/reaction for DD3 mRNA in the LNCaP cell cDNA. The intra- and inter-assay coefficients of variation of DD3/PSA mRNA were 3.8%-4.7% and 4.1%-4.9% respectively. Urine DD3/PSA mRNA ratio in PCa group was significantly higher than the BPH group (P < 0.01). When the cutoff value was defined as 0.254, the area under curve (AUC) of DD3/PSA mRNA ratio was 0.746 (95% CI: 0.630-0.862), and the sensitivity and specificity were 64.7% and 77.3% respectively. The urine DD3/PSA mRNA ratio positive rate was not correlated with clinical and pathological parameters.


CONCLUSIONS
A duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio has been established with an excellent clinical performance and specificity for PCa, saving time and reducing costs. It may be a promising method in the early diagnosis of PCa.
Chen
Feng-Ping
FP
Center for Cynical Laboratory Diagnosis, First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China.
Chen
Li-Li
LL
Shen
Mo
M
Chen
Wei
W
Tao
Zhi-Hua
ZH
Wu
Xiu-Ling
XL
Hu
Yuan-Ping
YP
Li
Cheng-di
CD
Chen
Zhan-Guo
ZG
Chen
Xiao-Dong
XD
D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D017430:Prostate-Specific Antigen / Q000235:genetics*; D011470:Prostatic Hyperplasia / Q000235:genetics / Q000473:pathology / Q000652:urine; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology* / Q000652:urine; D012333:RNA, Messenger / Q000235:genetics / Q000652:urine*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000951:Antigens, Neoplasm; D012333:RNA, Messenger; C479341:prostate cancer antigen 3, human; D017430:Prostate-Specific Antigen