The Veterinary Journal 173 (2007) 184–189
Cloning and nucleotide sequencing of the second internal transcribed spacer of ribosomal DNA for three species of Eimeria from chickens in Taiwan
Y.Y. Lien a, S.C. Sheu b, H.J. Liu a, S.C. Chen a, M.Y. Tsai a, S.C. Luo c, K.C. Wu c, S.S. Liu a, H.Y. Su c,*
a Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
b Department of Food Science, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
c Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
摘要
Coccidiosis of chickens caused by protozoan parasites of the genus Eimeria (Coccidia: Eimeriidae) is an enteric disease thatresults in great economic losses throughout the world, including Taiwan. Using polymerase chain reaction (PCR) with primers specific for the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), three species of Eimeria, E. tenella, E. maxima, and E. acervulina have been successfully characterised from chickens in Taiwan. The sizes of PCR products from various isolates representing these three species were between 370 and 580 base pairs (bp). After cloning and sequencing of the PCR products, high nucleotide sequence identity (96.8–100%) was observed within a species. In addition, ITS-2 nucleotide sequences for E. tenella had higher homology (98.5–99.3%) than E. maxima (81.6–96.5%) when compared with appropriate sequences deposited in GenBank. To our knowledge, this is the first report of a 412-bp ITS-2 sequence for E. acervulina from chickens. 2005 Elsevier Ltd. All rights reserved.
关键字: Coccidiosis; Eimeria; Ribosomal DNA; Nucleotide sequencing; Internal transcribed spacer
文中提及的产品
PCR products were purified using a QIAquick PCR Purification Kit (Qiagen Inc.) following the manufacturer's recommendations. Purified PCR products were cloned into 3015 bp of the pGEM-T Easy Vector (Promega). The ligated product was transformed into Escherichia coli INVaF competent cells (Invitrogen), and the transformed cells were allowed to recover in SOC medium (Invitrogen). These recovered cells were plated onto LB agar (MDBio) plates containing ampicillin, IPTG (MDBio) and X-Gal (MDBio) to facilitate blue/white selection of transformants. Fifteen white colonies were randomly selected from each agar plate and inoculated in a LB medium (MDBio) containing ampicillin, and the plasmid DNA was purified as previously described (Su et al., 2004) and then digested with EcoRI (Promega) (Sambrook et al.,1989) to verify whether the plasmid DNA contains the ITS-2 insert.