Single-tube real-time multiple allele-specific PCR for genotyping chicken Mx gene G2032A SNP.

1. A non-synonymous, single nucleotide polymorphism (G to A) at position 2032 (Ser 631 Asn) of the chicken Mx gene has been demonstrated to be related to resistance to antiviral activity. This study developed a new real-time PCR-based allelic discrimination assay for the rapid genotyping of the chicken Mx gene G2032A SNP. The distribution of the Mx gene G2032A SNP genotypes and the allele frequencies of A and G alleles among different chicken breed populations were screened with the use of this method. 2. We combined previously described allele-specific PCR and SYBR Green I-based real-time PCR melting curve analysis with a novel primer design strategy. A pair of outer nested primers was designed to amplify a fragment containing the SNP site, and two 3'-specific, allele-specific primers were combined with the outer primers to amplify SNP-specific fragments. Genotypes were identified based on the characteristic melting temperature of the SNP-specific fragments. 3. Genotyping assignments were successfully performed on samples from 8 chicken breeds, which were analysed by agarose gel electrophoresis of the PCR products and compared with results obtained from the direct sequencing of the outer primer amplicon. Five native breeds from Southern China carried a relatively higher frequency of the resistant A allele than the three commercial chicken lines. 4. This single-tube real-time multiplex allele-specific PCR assay is rapid, reliable, sensitive and easy to perform. It is appropriate for high-throughput sample analysis in large population-based Mx SNP genotyping studies.