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

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. © 2017 The Author(s).

• Fecha de publicación: 2017
• Institución: Universidad del Rosario
• Repositorio: Repositorio EdocUR - U. Rosario
• Idioma: eng
• OAI Identifier: oai:repository.urosario.edu.co:10336/22749
• Acceso en línea: https://doi.org/10.1186/s13071-017-2549-y; https://repository.urosario.edu.co/handle/10336/22749
• Palabra clave: Dna; Parasite antigen; Primer dna; Protozoal dna; Article; Controlled study; Cryptosporidium parvum; Dna determination; Dna sequence; Gene; Gene amplification; Hammondia hammondi; Limit of detection; Loop mediated isothermal amplification; Nc 5 gene; Neospora caninum; Neosporosis; Nonhuman; Polymerase chain reaction; Protozoon; Sarcocystis; Sarcocystis cruzi; Sarcocystis hominis; Sequence alignment; Toxoplasma gondii; Animal; Bovine; Cattle disease; Coccidiosis; Dog; Dog disease; Feces; Female; Genetics; Isolation and purification; Neospora; Nucleic acid amplification; Parasitology; Pregnancy; Procedures; Sensitivity and specificity; Temperature; Veterinary; Animals; Cattle; Cattle diseases; Coccidiosis; Dna primers; Dog diseases; Dogs; Feces; Female; Limit of detection; Neospora; Nucleic acid amplification techniques; Polymerase chain reaction; Pregnancy; Sensitivity and specificity; Temperature; Loop-mediated isothermal amplification; Nc-5 gene; Neospora caninum; Neosporosis; Semi-nested pcr; protozoan; protozoan; Antigens; Dna
• License: Abierto (Texto Completo)