Related Experiment Video
Updated: May 3, 2026
Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry
Published on: July 13, 2019
High-resolution melting analysis for mutation scanning in the non-coding control region of JC polyomavirus from
Kazuo Nakamichi1, Shigeru Tajima, Chang-Kweng Lim
1Department of Virology 1, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan, nakamich@nih.go.jp.
Abstract:
JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. JCV isolates from PML patients have hypervariable mutations in the noncoding control region (NCCR) of the viral genome. Although nucleotide sequencing analysis of NCCR mutation is useful for the confirmation of PML diagnosis and basic studies examining JCV variants, it is often labor-intensive, time-consuming, and expensive. This study was conducted to evaluate the feasibility of a high-resolution melting (HRM) analysis technique for the rapid and low-cost scanning of NCCR mutations. The real-time PCR-HRM assay was developed with a pair of primers targeting the NCCR, and mutational patterns of NCCRs were compared using sequence-confirmed JCV DNA clones and CSF DNAs from PML patients. The NCCR patterns of DNA clones of the archetype JCV and PML-type variants could be differentiated by PCR-HRM. The mutational patterns of the rearranged NCCR clones were similar to those of JCV variants in the original CSF specimens as judged by nested PCR-HRM using pre-amplified targets. In addition, nested PCR-HRM could distinguish NCCR mutations in the JCV DNAs from each specimen at the patient level. These results indicate that the HRM-based assay affords a valuable technique for PML diagnosis and a versatile tool for the rapid scanning of NCCR mutations.
Insights
High-resolution melting (HRM) analysis offers a rapid, low-cost method for detecting JC polyomavirus (JCV) noncoding control region (NCCR) mutations. This technique aids in progressive multifocal leukoencephalopathy (PML) diagnosis by efficiently scanning viral variants.
Area of Science:
- Virology
- Molecular Diagnostics
- Neuroscience
Background:
- JC polyomavirus (JCV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease.
- PML-associated JCV isolates exhibit hypervariable mutations in the noncoding control region (NCCR).
- Current NCCR mutation analysis via nucleotide sequencing is labor-intensive, time-consuming, and costly.
Purpose of the Study:
- To evaluate the feasibility of high-resolution melting (HRM) analysis for rapid and cost-effective scanning of JCV NCCR mutations.
- To establish a real-time PCR-HRM assay for differentiating JCV variants.
Main Methods:
- Development of a real-time PCR-HRM assay targeting the JCV NCCR.
- Comparison of NCCR mutational patterns using sequence-confirmed JCV DNA clones and cerebrospinal fluid (CSF) DNA from PML patients.
- Utilized nested PCR-HRM with pre-amplified targets for analyzing rearranged NCCR clones and CSF specimens.
Main Results:
- PCR-HRM successfully differentiated archetype JCV and PML-type variant NCCR patterns.
- Mutational patterns in rearranged NCCR clones mirrored those found in original CSF specimens.
- Nested PCR-HRM distinguished NCCR mutations in JCV DNA from individual patient specimens.
Conclusions:
- HRM-based assay is a valuable technique for PML diagnosis.
- The developed assay provides a versatile tool for rapid scanning of JCV NCCR mutations.
- This method enhances the efficiency of identifying and characterizing JCV variants in PML patients.
