Development of a loop-mediated isothermal amplification (LAMP) assay for the detection of Anaplasma marginale

• Published in: Experimental & applied acarology 2019-01, Vol.77 (1), p.65-72
• Main Authors: Giglioti, Rodrigo, Bassetto, César Cristiano, Okino, Cintia Hiromi, de Oliveira, Henrique Nunes, de Sena Oliveira, Márcia Cristina
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Anaplasma - isolation & purification; Anaplasma centrale; Anaplasma marginale; Anaplasmosis; Anaplasmosis - diagnosis; Anaplasmosis - microbiology; Animal Ecology; Animal Genetics and Genomics; Animal Husbandry; Animal Systematics/Taxonomy/Biogeography; Animals; Assaying; Babesia bigemina; Babesia bovis; Bacterial Outer Membrane Proteins - isolation & purification; Biomedical and Life Sciences; Bovidae; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - microbiology; Cattle industry; Cross Reactions; Deoxyribonucleic acid; Diagnostic systems; Diagnostic Tests, Routine - veterinary; DNA; Entomology; Life Sciences; Livestock; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - veterinary; Parasitemia; Parasites; Primers; Proteins; Sensitivity; Sensitivity and Specificity
• ISSN: 0168-8162; 1572-9702
• DOI: 10.1007/s10493-018-0327-y

Parasitemia generated by Anaplasma marginale causes significant losses in the cattle industry. A major constraint to the effective control and management of anaplasmosis in livestock is the lack of a rapid and reliable diagnostic test to identify the parasite and allow for immediate therapy. In the present study, we developed a novel DNA-based assay for the detection of A. marginale in bovine blood samples, using loop-mediated isothermal amplification (LAMP). DNA from six cattle and hemoparasite samples ( Babesia bovis , Babesia bigemina , Anaplasma centrale and A. marginale ) were tested for specificity, sensitivity and cross-reactions. The developed LAMP procedures were also confirmed and compared with the qPCR method. The same gene sequence (major surface protein 1b, msp1b ) of A. marginale was used to design a set of primers for the LAMP and qPCR assays. The results showed that LAMP is specific, as no positive signal was observed for the other tested hemoparasites. However, the sensitivity of the qPCR assay was ten times higher than LAMP. Our findings indicate that this LAMP method has a good sensitivity and high specificity for the detection of A. marginale and may have a potential application in the detection and differentiation of bovine anaplasmosis.