The forward primer was 5?-GTTCATGGCTCGTCATGAGAC-3? and located between 145 and 165 bp of dfr ORF. The reverse primer was 5?-AATTCGACATCACTCCAGTTC-3? and located between 535 and 557 bp of the ORF. A PCR product of 400 bp was purified and cloned to pGEM-T (Promega).