A NEW BK PCR ASSAY WITH IMPROVED DETECTION OF ALL RECENTLY DESCRIBED VARIANT GENOTYPES

Introduction:  Our recent study comparing the performance of 7 different primer/probe sets for the detection of BK virus demonstrated that many existing PCR assays were unable to accurately measure samples of the recently described genotypes III and IV groups.  In addition, our current clinical assay based on the commonly used Pep-1/Pep-2 primer set was relatively insensitive and also amplified closely-related JC virus sequences.  Therefore, we sought to re-design our assay to include a 2 primer-probe set multiplex assay capable of detecting all variant genotypes, to be very sensitive, and to lack cross-amplification with JC virus.    

Methods:  All available VP1 and T region sequences were extracted from Genbank, aligned, and then the consensus sequence used in Primer Express (ABI) to create a variety of possible primer-probe combinations.  Two sets with complete sequence matches across all genotype-specific mutations and with a maximum number of differences from JC sequences were selected for further study.  With use, both sets less efficiently detected genotype III samples, so a second probe was added to the large T region set, and the 3’ primer of the VP1 region set moved slightly.  Amplification conditions were then maximized with both an in-house prepared master mix and a commercial master mix (ABI Taqman Fast).   The primer sets were tested with JC virus samples and purified DNA from genotype III and IV samples.  In addition, the large T region III probe(s) were tagged with Cy5 to enable specific detection of either the VP1 or T region amplicons separately, or to detect the genotype III samples in a multiplex format.  Finally, the new assay was run in parallel with the old clinical assay with 230 sequential serum and urine samples submitted for BK testing.   Assays were performed on ABI 7900, 7700, 7500, and StepOne instruments. 

Results:  Both new primer/probe sets had better amplification efficiency that our previous assay with standard material (BK Dunlop reference strain plasmid – from ATCC).  They worked well in a multiplex format, with either one or both of the two large T region probes tagged with Cy5.  They also were easily adapted to different master mix formulations, working equally well with either an in-house mix or the ABI fast mix.  Neither primer set amplifed JC virus samples.  In the parallel testing, similar results were obtained for 202 of the 230 clinical samples.  Of the 28 discrepant samples, 2 were positive with both old and new assays, but > 1 log higher with the new assay.  Nine additional samples with BK virus quantities of >200 copies/ml were negative with the old assay and positive with the new one.  A total of 14 samples with quantities of virus near the assay sensitivity or <200 copies/ml, were positive with the new assay but negative with the old, while only 3 were positive with the old assay and negative with the new assay. 

Conclusions:

1.      This new multiplex assay has very good amplification efficiency and improved sensitivity. 

2.      The primer/probe sets are robust.  Because of the multiplex design, the detection of BK should be insensitive to random point mutations in the virus. 

3.      The assay works well in both standard and fast-block master mix formats in multiple PCR instruments.  

4.      The primer/probe sets amplify all known genotypes.  Because of the genotype III specific probe, it may be used in a multicolor format to specifically detect most genotype III samples.