| Popis: |
BK polyomavirus (BKV) is a common opportunistic pathogen in the community, infecting humans in their early life stages, often remaining in a latent state. BK virus-associated nephropathy occurs in 2 to 10% of kidney recipients, and up to 40 to 90% of these patients have permanent loss of graft function due to delay in diagnosis. In the present study, we have successfully optimized the component and thermal cycling for the long-PCR assay to amplify the BKV genome, including middle-concentration samples such as plasma (concentration about 105 copy/ml). We have successfully amplified the BKV genome in 41/50 samples, including 04 plasma and 39 urine samples. The optimized amplification protocol of full-length BKV genome includes the following steps: 1) Extraction of DNA-BKV from plasma and urine samples and quantification of BKV-DNA concentrations to ensure that about 105 copies/ml (plasma) and 107 copies/ml (urine); 2) Each full-length genome reaction in the volume of 50 μl containing 1X Phusion HF master mix (Thermo Scientific, USA); 0.5 μM of wBK_a(F+R) primers, 3-5 μl of genomic DNA. Cycling conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s; 58°C for 20 s; 72°C for 2 min, and 72ºC for 6 min and a 4oC hold; 3) All 5.1 kb purified PCR were verified and confirmed by restriction enzymes XbaI, EcoRI, and direct sequencing. |
