Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

PLoS One. 2020 Mar 16;15(3):e0230445. doi: 10.1371/journal.pone.0230445. eCollection 2020.

Abstract

Single-nucleotide polymorphisms (SNPs) is associated with efficacy of specific drugs. Although there are several methods for SNP genotyping in clinical settings, alternative methods with lower cost, higher throughout and less complexity are still needed. In this study, we modified Kompetitive Allele Specific PCR to enable multiplex SNP genotyping by introducing additional fluorescent cassettes that specifically help to differentiate more amplification signals in a single reaction. This new format of assay achieved a limit of detection down to 310 copies/ reactions for simultaneous detection of 2 SNPs with only standard end-point PCR workflow for synthetic controls, and genotyped 117 clinical samples with results that were in 100% agreement with hospital reports. This study presented a simplified, cost-effective high-throughput SNP genotyping alternative for pharmacogenetic variants, and enabled easier access to pharmaceutical guidance when needed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Genotype
  • Genotyping Techniques / methods*
  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Pharmacogenetics / methods*
  • Polymorphism, Single Nucleotide / genetics*

Grants and funding

The experiment was supported from Special fund support project for clinical medicine development of Beijing Municipal Hospital Administration(ZYLX201805) and Major new drug creation national major science and technology special project "construction of clinical research demonstration platform for cardiovascular disease drugs"(2017ZX09304017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.