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

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.

Figure 1

Alignment of the nucleotide sequence corresponding to the VP2(VP3) C-terminus region. Nucleotide alignment of reference sequences for Simian Agent 12 (NC_007611.1; nucleotides 1347-1589), Simian virus 40 (NC_001669.1; nucleotides 1378-1620), JCV (NC_001699.1; nucleotides 1342-1560), and BKV (NC_001538.1; nucleotides 1437-1679) is shown. The positions of nucleotide polymorphisms among 487 JCV isolates relative to the reference sequence are shown below the JCV reference sequence. The guanine at position 150 (bold) is replaced by cytosine in six out of 487 JCV isolates. The locations of polymorphisms among 271 BKV isolates are shown below the BKV reference sequence and the number of BKV isolates with that particular set of polymorphism(s) is given in brackets. The positions of the primers (underlined) and probe (boxed) are indicated on the BKV reference sequence. There are no observed polymorphisms at the Polyoma_4.2(f) forward primer in either BKV or JCV sequences available at this time. There are no known polymorphisms at the relative position of the BKV_MGB probe (boxed) among either the BKV isolates or JCV isolates. There are three nucleotide differences between JCV and BKV at the probe binding site. There are three nucleotide differences between SV40 and BKV at the BKV_5.1(r) reverse primer binding site.

Figure 2

Amino Acid Sequence Alignment of the VP2 and VP3 C-terminus from representative viruses of the Polyomaviridae Family. Alignment of the amino acid sequence of the VP2/VP3 C-terminus was constructed using ClustalW2 from reference sequences of 17 different members of the Polyomaviridae family. BKV, JCV, SV40 and SA12 sequences were compared and residues that are conserved between at least three out of these four closely related members of the Polyomaviridae family are indicated in bold. The alpha-helix region of the BKV VP2/VP3 gene is shown in red. The location of the primers (underlined) and the probe (boxed) are indicated. The region in the BKV VP2/VP3 gene having two reading frames due to the overlap with the N-terminus of the VP1 gene is indicated in blue. The NLS and DNA binding region are located downstream of the alpha helix sequence. Residues are numbered according to the reference sequence for the BKV VP2 gene (YP_717937.1).

Figure 3

BKV Real-Time Quantitative PCR. The BKV standard was serially diluted prior to being assayed by real-time quantitative PCR. A. Raw amplification curves from precision study with primers BKV_5.1(r) and BKV_4.2(f). Input BKV DNA was 10 μl at concentrations of 10⁶ copies/μl, 10⁵ copies/μl, 10⁴ copies/μl, 10³ copies/μl, 10² copies/μl, 10 copies/μl and <10 copies/μl. B. Standard curve. Results of the regression were: slope -3.484, y-intercept 39.05 and efficiency 1.936. Similar results were obtained with other primer combinations.