Development of mismatch amplification mutation assay for the rapid differentiation of K vaccine strain from field isolates.

causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage but this stability test should still be evaluated as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 10 and 10 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.