Reevaluating and optimizing polyomavirus BK and JC real-time PCR assays to detect rare sequence polymorphisms

Area of Science:

• Molecular diagnostics
• Virology
• Clinical microbiology

Background:

• Polyomavirus diagnostics rely on PCR-based molecular assays.
• Regular updates of target sequences are crucial for optimal assay performance.
• In-house BK polyomavirus (BKV) and JC polyomavirus (JCV) real-time PCR assays require review.

Purpose of the Study:

• To review and update in-house BKV and JCV real-time PCR assays.
• To assess the impact of genetic variations on BKV assay performance.
• To ensure robust detection of polyomaviruses in clinical samples.

Main Methods:

• Database analysis of BKV and JCV sequences.
• Comparison of original and updated BKV primer versions.
• Analytical sensitivity and specificity testing using cloned viral DNA and clinical samples.
• Probit analysis for sensitivity determination.

Main Results:

• BKV target region showed variations affecting assay performance; JCV target region remained stable.
• Updated BKV primers accommodated >95% of genetic variants.
• Analytical sensitivity: BKV assay 4.15 copies/reaction, JCV assay 3.37 copies/reaction.
• No cross-reactivity observed; updated and original BKV assays showed similar quantitative results in clinical samples.

Conclusions:

• The updated BKV and existing JCV real-time PCR assays are robust for clinical diagnostics.
• These assays can detect rare polyomavirus variants encountered in routine practice.
• Regular sequence updates are essential for maintaining the performance of molecular diagnostic assays.