Thiết lập quy trình PCR-RFLP xác định kiểu gen vi rút BK phân lập từ bệnh nhân ghép thận

Abstract

The monitoring and treatment of patients after kidney transplant is important in maintaining transplant kidney function, including BK polyomavirus surveillance. BKV is a common opportunistic pathogen in kidney transplant patients and can cause BKV-associated nephropathy, which represents a major problem for these patients, causing graft dysfunction and graft loss. BKV is classified into four genotypes, I, II, III and IV. Here, thirty urine samples from kidney transplant patients were collected and quantified BKV-DNA loads by real-time PCR, viral loads were 105-1010 copies/ml. We successfully amplified and purified the specific VP1-BKV region with 580 bp in length using primers BK_(S+AS). Additionally, we also identified BKV genotypes by PCR-RFLP using the restriction enzymes RsaI and AluI. In total 20 out of 30 cases (66.7%) were genotype I and 10 cases (33.3%) were genotype IV in collected urine samples. Comparing the obtained PCR-RFLP results with direct sequencing analysis achieved complete concordance (30/30, 100%). We successfully developed a cost-effective and accurate BKV genotyping assay using PCR-RFLP, that contributes to the monitoring of patients after kidney transplantation.