Multiplex detection and SNP genotyping in a single fluorescence channel

PLoS One. 2012;7(1):e30340. doi: 10.1371/journal.pone.0030340. Epub 2012 Jan 17.


Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alphapapillomavirus / classification
  • Alphapapillomavirus / genetics
  • Aryl Hydrocarbon Hydroxylases / genetics
  • Cell Line, Tumor
  • Cytochrome P-450 CYP2C9
  • DNA / genetics
  • DNA Probes / chemistry
  • DNA Probes / genetics*
  • DNA, Viral / genetics
  • Fluorescence
  • Genotype
  • Genotyping Techniques / methods*
  • HeLa Cells
  • Human papillomavirus 16 / genetics
  • Human papillomavirus 18 / genetics
  • Human papillomavirus 31 / genetics
  • Humans
  • K562 Cells
  • Nucleic Acid Amplification Techniques / methods*
  • Nucleic Acid Conformation
  • Polymorphism, Single Nucleotide*
  • Reproducibility of Results
  • Species Specificity
  • Transition Temperature


  • DNA Probes
  • DNA, Viral
  • DNA
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases