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Newborn Bloodspot Screening Test Using Multiplexed Real-Time PCR to... - PubMed - NCBI

Newborn Bloodspot Screening Test Using Multiplexed Real-Time PCR to... - PubMed - NCBI

 2014 Dec 11. pii: clinchem.2014.231019. [Epub ahead of print]

Newborn Bloodspot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency.



Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of the motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn bloodspot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well.


An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members.


DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range.


SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.
© 2014 The American Association for Clinical Chemistry.

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