A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

• Main Authors: Andrea Estefanía Ramos, Marina Muñoz, Jesús Alfredo Cortés-Vecino, Paola Barato, Manuel Alfonso Patarroyo
• Format: Article
• Language: English
• Published: BMC 2017-11-01
• Series: Parasites & Vectors
• Subjects: Neospora caninum; Neosporosis; Loop-mediated isothermal amplification; Semi-nested PCR; Nc-5 gene
• Online Access: http://link.springer.com/article/10.1186/s13071-017-2549-y

Abstract Background Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Results Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. Conclusion The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.