球虫抗药性分子生物学检测技术的建立

Identification of the drug resistant strain by molecular biological technique

选择在敏感虫株中沉默、抗药虫株中表达的特异序列AGD5设计检测引物,采用RT-PCR方法,以总RNA反转录的cDNA为模板,RT-PCR方法能够鉴别马杜霉素抗药虫株和3株马杜霉素敏感虫株(2株对所有药物敏感的不同地理株,1株地克珠利抗药性虫株对马杜霉素敏感),此鉴定结果与鸡体试验的结果一致,说明敏感虫株和抗药虫株在该序列上的差异发生在转录水平。而以卵囊总DNA为模板的PCR方法不能鉴别敏感和马杜霉素抗药性虫株。

The sense and anti-sense primers were designed according the special sequence from the resistant strain by the result of the differential display PCR. The total RNA was purified with Trizol from merozoite of 1 maduramisin resistant E. tenella strain and 3 differntial maduramisin sensitive E. tenella strains. The DNA was purified with DNA purification kit from oocysts of the 4 differential strains. The drug resistance could be identified by RT-PCR. It was unanimous to the test results in chickens. But it could not be identified by PCR with DNA template.