title: A Loop-Mediated Isothermal Amplification Assay Targeting 16S rRNA Gene for Rapid Detection of Anaplasma phagocytophilum Infection in Sheep and Goats.
abstract: Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions under which no cross-reaction was observed with other closely related tick-borne parasites ( Anaplasma bovis , Anaplasma ovis , Theileria luwenshuni, Babesia motasi, and Schistosoma japonicum ) were established. The assay exhibited much higher sensitivity compared with conventional polymerase chain reaction (PCR) (1 copy vs. 1,000 copies). To evaluate the applicability of the LAMP assay, 94 field samples of sheep blood were analyzed for A. phagocytophilum infection by using LAMP, nested PCR, and conventional PCR assays at the same time. A positive LAMP result was obtained from 53 (56.4%) of the 94 samples, whereas only 12 (12.8%) and 3 (3.2%) tested positive by nested and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep/goats.
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Jinhong
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Xiaoxing
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Cui
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Yaqun
authors.initials: Y
authors.lastname: Yan
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Rongjun
authors.initials: R
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Fuchun
authors.initials: F
authors.lastname: Jian
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Longxian
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
authors.forename: Changshen
authors.initials: C
authors.lastname: Ning
mesh_terms: D041081:Anaplasma phagocytophilum / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D016873:Ehrlichiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D015511:Goat Diseases / Q000175:diagnosis / Q000469:parasitology*; D006041:Goats; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000469:parasitology*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S