Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis

Abstract

BackgroundDogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum.MethodsLAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.ResultsSpecificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).ConclusionsThis is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2330-2) contains supplementary material, which is available to authorized users.

Rapid and Sensitive Detection of Salmonella in Chickens Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

“…All of the invA sequences were aligned with DNAMAN 4.0 [22], and the conserved regions were used for analysis across the Salmonella species. We designed LAMP primers using PrimerExplorer v3 (http://primerexplorer.jp; [27]), based on the invA genes in the previously published S. enterica genome (GenBank Accession No: NC003197.1). We designed two outer primers (F3 and B3), two inner primers (FIP and BIP), a loop primer (LP), and a probe (HP).…”