David E. Barton, Rosie O'Shea and Elizabeth Donohoe for The CRMGEN Consortium

Presenter - David E Barton, PhD

The use of appropriate Reference Materials (RMs) to validate test equipment or testing methods is an important part of any analytical testing system. Certified reference materials (CRMs) are RMs whose characteristics have been fully documented and validated. Currently, no CRMs are available for genetic testing. The CRMGEN project is a fourteen-centre collaboration funded by the European Commission's Measurement and Testing program (Contract G6RD-CT-2001-00581). We are developing reference measurement systems and producing CRMs for molecular genetic tests. Prototype RMs will be developed for a wide range of tests. These prototype RMs, developed in one of 4 genetics centres, will be validated in 7 other centres before extensive field trials. The knowledge gained in this process will be used to develop guidelines for the production of CRMs for any genetic test. Special emphasis will be given to the commutability of the candidate RMs, i.e. their ability to perform under a wide range of test protocols and conditions.

In Dublin, we have used PCR to produce RMs for the common mutations involved in hereditary haemochromatosis (H63D & C282Y). Working from a 3kb master template, we have produced RMs for the individual mutations and for both together. The RMs have been tested by the CRMGEN partners and are now undergoing field trials and stability studies. We have used a similar approach to develop a multiplex RM containing both mutant and normal alleles for four common cystic fibrosis mutations.
 
As the RM production process requires these PCR products to be brought back into pre-PCR areas, serial dilutions are carried out for each PCR product, in order both to minimise the risk of contamination in second-round PCR reactions and to negate the need for scale-up in the production phase of the project. Strategies for decontamination of PCR reaction tubes and racks were also investigated, to allow future safe handling of the final product. It was found that HCl was ineffective (up to 4M), but the use of a 1/10 dilution of sodium hypochlorite eliminated all contaminating PCR products.