Open Access Methodology

Development of a real-time quantitative PCR assay for detection of a stable genomic region of BK virus

Kosuke K Iwaki1*, Suhail H Qazi1, Jean Garcia-Gomez1, Deanna Zeng1, Yasuhiro Matsuda1, Kazuko Matsuda1, Monica E Martinez1, Mieko Toyoda2, Arputharaj Kore3, Wesley T Stevens4, Miroslaw Smogorzewski5, Daisuke D Iwaki1, Yasir Qazi5 and Yuichi Iwaki1

Author Affiliations

1 Metic Transplantation Laboratory, USC, Keck School of Medicine, Los Angeles, CA, USA

2 Transplant Immunology Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA, USA

3 Division of Transplantation, Department of Surgery, Loma Linda University Medical Center, Loma Linda, CA, US

4 Department of Pathology, Loma Linda University Medical Center, Loma Linda, CA, US

5 Internal Medicine, USC, Keck School of Medicine, Los Angeles, CA, US

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Virology Journal 2010, 7:295  doi:10.1186/1743-422X-7-295

Published: 29 October 2010

Abstract

Background

BK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available.

Results

Employing a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%).

Conclusion

Identifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.