Abstract:

BACKGROUND: For the BK virus (BKV) and BK virus-associated nephropathy， there lack of standardized laboratory diagnostic procedures and non-invasive testing methods.
OBJECTIVE: To establish a real-time fluorescent quantitative PCR method for determining the BKV level of urine and peripheral in renal transplant recipient and to evaluate its clinical application.
METHODS: According to the BK virus genome, BKV-F, BKV-R and Taqman fluorescent probe BKV-P were designed by us. 5' extremity of Taqman fluorescent probe BKV-P was labeled with fluorophores. Except 5' extremity, other sites were marked quenching group; the results of PCR reaction were obtained after detecting the samples.
RESULTS AND CONCLUSION: The positive PCR products were preformed with gene sequencing, the result confirmed by BLAST was the BK virus gene sequence; 56 samples were detected with this method, 20 cases of BKV in serum samples and 20 cases of BKV in urine samples were positive and had a good S-type amplification curve. Dynamic range tests showed that there was a good correlation among the 103-1010 copies/mL standard curves. Five urine samples from healthy individuals, five blood samples and six blood samples of common pathogens in clinical were negative, there was no S-type amplification curve. 
The real-time fluorescent quantitative PCR assay established in this study was qualitative and quantitative, and has the ability of sensitivity and specificity, low-cost and less false-positive. The general determining results can be obtained in 30 minutes and suitable for large-scale clinical application.