Screening of Multiple Drug Resistant Genes of Eimeria tenella Infesting Chicken in China

Abstract

Total RNA of Eimeria tenella drug-resistant strains from Qinhuangdao, Tanshan and Shijiazhuang were extracted with Trizol. DDRT-PCR was established by 3 anchored primers and 20 arbitrary primers. The products of PCR were analyzed on the denaturing polyacrylamide gels by silver-straining. Twenty-six differential bands were excised from the gels and reamplified with the same sets of primers. The products were purified, and 12 differential bands (20～50ng) were recuperated. Twelve plasmid cloning identification differential bands were identified by the dot-blot hybridization. The results showed that 3 bands were positive. And then the positive cloning was sequenced and compared to homology. The results showed that through comparison of the nucleotide acid sequence, the similarity was 99% among the sequence S116 from mRNA of Shijiazhuang multiple-resistant strain with the sequence 882bp length in the first chromosome of E. tenella in Genbank and Sanger, which was an unknown protein. The sequence of T311 and Q19 from mRNA of Tangshan and Qinhuangdao multiple-resistant strains were unknown sequences. These novel cDNA fragments associated with drug resistance might be involved in the process of the drug resistances of E. tenella.

Keywords

• Chicken
• Denaturing polyacrylamide gel
• E. tenella
• Gene cloning
• mRNA differential display
• PCR