title: Rapid and sensitive detection of Babesia bovis and Babesia bigemina by loop-mediated isothermal amplification combined with a lateral flow dipstick.
abstract: Babesia spp. are apicomplexan protozoan parasites of the red blood cells of mammals and are transmitted by ticks. Bovine babesiosis mainly caused by Babesia bovis and Babesia bigemina occurs worldwide, which is a great threat to animal health. Microscopy examination is a gold standard for the diagnosis of babesiosis. However, its sensitivity is too low. This study was conducted to establish a simple, efficient and fast LAMP-LFP method used for early diagnosis of animal babesiosis. LAMP was developed with a set of four primers targeting and amplifying six distinct regions of cytochrome b gene of Babesia spp. under isothermal conditions. Afterwards, a chromatographic lateral-flow dipstick (LFD) was used to detect LAMP products that were labeled with FITC at the 5' end, avoiding gel electrophoresis. The LAMP-LFD method was very specific, yielding no positive results with DNA templates of Theileria sergenti, Thenileria ovis, Theileria equi and Toxoplasma gondii. The LAMP-LFP was highly sensitive and could detect 0.85 fg B. bigemina DNA and 0.14 fg B. bovis DNA, 100-fold higher than a conventional PCR assay. This method could be adapted for quick and accurate diagnosis of bovine babesiosis in the fields in case the whole blood could be directly used, especially for identifying carrier animals with very low parasitaemia.
authors.affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Yimin
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Li
authors.affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Suhua
authors.initials: S
authors.lastname: Wang
authors.affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xueqiu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China. Electronic address: afdu@zju.edu.cn.
authors.forename: Aifang
authors.initials: A
authors.lastname: Du
mesh_terms: D000818:Animals; D001403:Babesia / Q000235:genetics; D016793:Babesia bovis / Q000235:genetics; D001404:Babesiosis / Q000175:diagnosis* / Q000469:parasitology; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000469:parasitology; D045303:Cytochromes b / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000662:veterinary*; D021141:Nucleic Acid Amplification Techniques; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D045303:Cytochromes b
keywords: A lateral flow dipstick; Cytochrome b; Loop-mediated isothermal amplification