title: Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences.
abstract: We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/μl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.
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 Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China.
authors.forename: Aihong
authors.initials: A
authors.lastname: Liu
authors.forename: Guiquan
authors.initials: G
authors.lastname: Guan
authors.forename: Pengfei
authors.initials: P
authors.lastname: Du
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Liu
authors.forename: Huitian
authors.initials: H
authors.lastname: Gou
authors.forename: Junlong
authors.initials: J
authors.lastname: Liu
authors.forename: Jifei
authors.initials: J
authors.lastname: Yang
authors.forename: Youquan
authors.initials: Y
authors.lastname: Li
authors.forename: Milin
authors.initials: M
authors.lastname: Ma
authors.forename: Qinli
authors.initials: Q
authors.lastname: Niu
authors.forename: Qiaoyun
authors.initials: Q
authors.lastname: Ren
authors.forename: Qi
authors.initials: Q
authors.lastname: Bai
authors.forename: Hong
authors.initials: H
authors.lastname: Yin
authors.forename: Jianxun
authors.initials: J
authors.lastname: Luo
mesh_terms: D000818:Animals; D002417:Cattle; D002681:China; D017931:DNA Primers / Q000737:chemistry; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000592:standards; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016796:Theileria annulata / Q000235:genetics / Q000302:isolation & purification*; D013801:Theileriasis / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D012337:RNA, Ribosomal, 18S