title: [Establishment of a real-time PCR assay for simultaneously detecting human BKV and CMV DNA and its application in renal transplantation recipients].
abstract: To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.
authors.affiliation: (The First Hospital of Wenzhou Medical College, Wenzhou 325000, China. chhahaa@163.com
authors.forename: Chun-Wu
authors.initials: CW
authors.lastname: Zhang
authors.forename: Xiao-Qian
authors.initials: XQ
authors.lastname: Chen
authors.forename: Yong-Heng
authors.initials: YH
authors.lastname: Bai
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Pan
authors.forename: Si-Lu
authors.initials: SL
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Cai
authors.forename: Peng
authors.initials: P
authors.lastname: Xia
authors.forename: Cun-Zao
authors.initials: CZ
authors.lastname: Wu
authors.forename: Bi-Cheng
authors.initials: BC
authors.lastname: Chen
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D017124:Conserved Sequence; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D004279:DNA, Viral / Q000097:blood; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood; D016030:Kidney Transplantation / Q000009:adverse effects; D008297:Male; D008875:Middle Aged; D011120:Polyomavirus / Q000235:genetics / Q000302:isolation & purification*; D027601:Polyomavirus Infections / Q000175:diagnosis* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D016559:Tacrolimus / Q000097:blood; D013997:Time Factors; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000821:virology; D019562:Viral Load; D055815:Young Adult
chemical_list: D004279:DNA, Viral; D007166:Immunosuppressive Agents; D016559:Tacrolimus