Single tube multiplex PCR detection of 27 cystic fibrosis mutations and 4 polymorphisms using neonatal blood samples collected on Guthrie cards

In response to recommendations for cystic fibrosis (CF) carrier screening of the American College of Medical Genetics/American College of Obstetrics and Gynecology (ACMG/ACOG), we evaluated a commercial kit for mutation panel testing (Roche CF Gold Linear Array Panel). This kit tests for 25 CF mutations and 4 polymorphisms; it comprises an analyte specific reagent for single tube multiplex polymerase chain reaction (PCR) amplification and subsequent allele specific oligonucleotide (ASO) hybridization. Neonatal whole blood samples collected on Guthrie card filter paper served as the DNA source. Following a wash step to remove whole blood PCR inhibitors, multiplex PCR amplification could be performed either on DNA that was heat extracted from Guthrie cards or directly on the filter paper matrix itself. In 13 CF patient samples, the CF mutation results obtained with this kit agreed completely with those obtained from a reference laboratory that performs an 87 CF mutation panel. The kit was reliable, despite the small sample size (3 mm diameter punch of the Guthrie card), the presence of PCR inhibitors in whole blood, and protracted storage of blood samples (up to 9 yr at room temperature). The kit was convenient, cost competitive, and practical for use in a small CF screening laboratory.