title: Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites.
abstract: A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers to construct a multiplex LAMP (mLAMP) method by combining these two sets totaling eight primers. The mLAMP method was distinguishable between B. bovis and B. bigemina, simultaneously, due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 10(3) and 10(5) times higher on the detection limits for B. bovis and B. bigemina, respectively, than those of the classical PCR methods. Of 40 blood samples collected from cattle living in Ghana, 12 and 27% were positively detected by the mLAMP for B. bovis and B. bigemina, respectively. Furthermore, 14 and 23% of 90 blood samples from cattle in Zambia showed mLAMP-positive reactions to B. bovis and B. bigemina, respectively. These findings indicate that this mLAMP method is a new convenient tool for simultaneous detection of the bovine Babesia parasites.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Iseki
authors.forename: Andy
authors.initials: A
authors.lastname: Alhassan
authors.forename: Naomi
authors.initials: N
authors.lastname: Ohta
authors.forename: Oriel M M
authors.initials: OM
authors.lastname: Thekisoe
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.forename: Andrew
authors.initials: A
authors.lastname: Nambota
authors.forename: Jun
authors.initials: J
authors.lastname: Yasuda
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
mesh_terms: D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D016793:Babesia bovis / Q000235:genetics / Q000302:isolation & purification; D001404:Babesiosis / Q000097:blood / Q000175:diagnosis / Q000469:parasitology* / Q000662:veterinary*; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000469:parasitology*; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000032:analysis / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards / Q000662:veterinary*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan