Development of a Nucleic Acid Based Lateral Flow Immunoassay to Detect BK Virus in Renal Transplant Recipients

• Main Authors: YU, KUAN-YI, 余冠毅
• Other Authors: Liu, Cheng-Che
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/56421140324965919830

碩士 === 國防醫學院 === 生理學研究所 === 105 === BK virus, one of the human polyomaviruses, is a double-stranded DNA virus with circular genomes of around 5,000 base pairs. Many research reports in recent years has proved that BK virus-associated nephropathy is one of the main causes to decline the transplanted kidney survival. Most of BKV infections initially are asymptomatic and will laten within human body. The latened virus may be re-activated and replicated to infect the renal tubular cells casuing cell lysis when the human immune suppression or dysfunction. It results in not only loss of renal function but also the kidney allograft failure. Hence, it is very important to monitor BK virus quantitatively before or after renal transplantation. Current standard methods which applied to check BK virus in clinical patient's urine or blood are PCR or quantatitive real-time PCR using BKV-specific primers. And the pathological cytology of the Decoy cells in urine is also assisted for clinical diagnosis. But the methods mentioned above maybe highly false negative, time-consuming, labor-operation and expensive instruments required. Thus it is urgent to develop an accurate, rapid and low-cost assay for BKV detection. In this study, all of the sequences of BK, JC and SV40 viruses were firstly collected from NCBI databank and aligned to screen out the BKV-specific primers. The primers will be chemically modified onto the surface of gold nanoparticle (AuNP) as AuNP probes. And the bovine serum albumin (BSA) was applied to be the non-specific blocking on AuNP surface to develop a nucleic acid based lateral flow immunoassay. The streptavidin was used to capture the biotin-labeled complexes after the hybridization primarily. The red signal bands which produced by aggregated AuNP probes on the test-line (with anti-streptavidin antibody) and control-line (with anti-BSA antibody) of the nitrocellulose membranes were visible to the naked eye. In our results of the synthesized single stranded DNA template showed that the assay could be completed in 40 minutes after viral nucleic acid extraction and the visual limit of detection was 10 fmol. In the quantitative analysis results, this assay also showed a well linear correlation in the detection of target concentration between 10fmol ~ 2500 fmol. In summary, we successfully developed a prototype of nucleic acid based lateral flow device for BK virus detection. We will still optimze the device senisitivity in the following experiments and verify the clinical usefulness by detecing BK virus from patient’s urine.