The results obtained using deoxyribonucleic acid (DNA) amplification-based tests must be accurate and reproducible. One such test that simultaneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in this report. An investigation was conducted into how changes of primer, DNA template and Taq DNA polymerase concentrations and of polymerase chain reaction annealing temperatures affect the test. A total of 383 DNA samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate results are obtained even if the test is performed under suboptimal conditions. Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remaining 1.6% of samples were not mistyped, rather they were not scored owing to failure to detect control DNA sequences. These were also archival DNA preparations rather than freshly prepared samples from venous blood. Careful primer design and optimization of reaction conditions are important in the development of multiplex deoxyribonucleic acid amplification-based diagnostic tests. Providing the recommended protocols are followed, the test described here is simple to carry out, gives accurate results and works well if performed within defined operational windows for each reaction variable.