Rapid point-of-care detection of BK virus in urine by an HFman probe-based loop-mediated isothermal amplification assay and a finger-driven microfluidic chip.

BACKGROUND

BK virus (BKV)-associated nephropathy (BKVN) is one of the leading causes of renal dysfunction and graft loss in renal transplant recipients. Early monitoring of BKV in urine is crucial to minimize the deleterious effects caused by this virus on preservation of graft function.

METHODS

We report a simple, rapid, sensitive loop-mediated isothermal amplification (LAMP) assay using an HFman probe for detecting BKV in urine. To evaluate the performance of the assay, a comparison of the HFman probe-based LAMP (HF-LAMP) assay with two qPCR assays was performed using urine samples from 132 HIV-1 infected individuals. We further evaluated the performance of HF-LAMP directly using the urine samples from these HIV-1 infected individuals and 30 kidney transplant recipients without DNA extraction. Furthermore, we combined the HF-LAMP assay with a portable finger-driven microfluidic chip for point-of-care testing (POCT).

RESULTS

The assay has high specificity and sensitivity with a limit of detection (LOD) of 12 copies/reaction and can be completed within 30 min. When the DNA was extracted, the HF-LAMP assay showed an equivalent and potentially even higher sensitivity (93.5%) than the qPCR assays (74.2-87.1%) for 132 urine samples from HIV-1 infected individuals. The HF-LAMP assay can be applied in an extraction-free format and can be completed within 45 min using a simple heat block. Although some decreased performance was seen on urine samples from HIV-1 infected individuals, the sensitivity, specificity, and accuracy of the extraction-free BKV HF-LAMP assay were 95%, 100%, and 96.7% for 30 clinical urine samples from kidney transplant recipients, respectively.

CONCLUSION

The assay has high specificity and sensitivity. Combined with a portable finger-driven microfluidic chip for easy detection, this method shows great potential for POCT detection of BKV.