title: Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.
abstract: Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu, 730046, People's of Republic China, jliu1211@gmail.com.
authors.forename: Liu
authors.initials: L
authors.lastname: Junlong
authors.forename: Youquan
authors.initials: Y
authors.lastname: Li
authors.forename: Aihong
authors.initials: A
authors.lastname: Liu
authors.forename: Guiquan
authors.initials: G
authors.lastname: Guan
authors.forename: Junren
authors.initials: J
authors.lastname: Xie
authors.forename: Hong
authors.initials: H
authors.lastname: Yin
authors.forename: Jianxun
authors.initials: J
authors.lastname: Luo
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000469:parasitology*; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity; D016794:Theileria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016796:Theileria annulata / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013801:Theileriasis / Q000175:diagnosis / Q000469:parasitology*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan