Development of multiplex TaqMan probe PCR Assays for typing of up to 6 genetic markers per reaction

Background

TaqMan probes and fluorescent endpoint detection for allelic discrimination is commonly used for genotyping of Single Nucleotide Variations (SNVs). Assays are usually desigend for typing one di-allelic SNV per reaction using two fluorescent dyes. Recent systems for quantiative PCR enable the detection of up to 6 colors. We aimed to develop multiplex assays for up to 3 di-allelic SNVs in one reaction and used Human Platelet Antigens (HPA) and Human Neutrophil Antigens (HNA) as model.

Methods

Primers and probes were designed for the SNVs coding for HPA-1a/b, -5a/b, -15a/b and HNA-3a/b, -4a/b, -5a/b according to reference sequences. Probes were labeled with FAM (HPA-1a, HNA-3a), HEX (HPA-1b, HNA-3b), TAMRA (HPA-5a, HNA-4a), TexasRed (HPA-5b, HNA-4b), Cy5 (HPA-15a, HNA-5a) or Cy5.5 (HPA-15b, HNA-5b). Multiplex assays for HPA and for HNA were used to genotype DNA samples from blood donors and the results were compared with reference data in the donor files. PCR amplification was performed in standard cyclers and fluorescence was measured after PCR by using the QuantStudio 5 system (Thermo Fisher Scientific).

Results

Re-typing of HPA-1, -5, -15 and HNA-3, -4, -5 in 475 blood donor samples revealed 100 % concordance with typing data in the donor files previously obtained by using monoplex TaqMan assays.

Conclusion

We were able to develop multiplex assays for time and cost efficient genotyping of 3 SNVs per reaction. The assays are used in the ongoing blood donor typing of HPA and HNA at our institute. Additional multiplex assays will be developed and also tested in digital PCR for the use in noninvasive prenatal diagnosis.

Offenlegung Interessenkonflikt:

No conflicts of interest