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Minerva Pediatrica 2016 Dec 22

Copyright © 2016 EDIZIONI MINERVA MEDICA

lingua: Inglese

A novel TaqMAMA assay for allelic discrimination of immunoproteasome subunit PSMB8 in pediatric patients

Massimiliano BERGALLO 1, Paola MONTANARI 1, Elisa LOIACONO 2, Ilaria GALLIANO 1

1 Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy; 2 Nephrology, Dialysis and Transplantation, University Hospital Città della Salute e della Scienza di Torino, Regina Margherita Hospital, Turin, Italy


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BACKGROUND: A more recent improvement in MAMA was made when the allele selectivity of MAMA primers was combined with the 5’ fluorogenic exonuclease (TaqMan) assay. This strategy referred to as TaqMAMA.
METHODS: A SNP rs9357155 at the PSMB8 locus (GenBank access number NM_148919.3.) on chromosome 6 showing a C/T transition in position 32842071, has been chosen as model in this study.
RESULTS: Tested assays have provided to be linear over two log10 of magnitude and have efficiencies close to the average of 80.3%. Average Interference Limit values of TaqMAMA have been considered around the 0.6%. Coefficient of variation (CV) for PSMB8c and –t was approximatively 1%.
CONCLUSIONS: Pre-validation procedure described in this paper can be applied, not only to the allele quantification, but also to many other Real Time PCR applications.


KEY WORDS: TaqMAMA - PSMB8 - SNPs - PCR real time - ASPCR

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