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

Andrea Estefanía Ramos,Marina Muñoz,Paola Barato,Jesús Alfredo Cortés-Vecino,Manuel Alfonso Patarroyo

doi:10.1186/s13071-017-2549-y

Abstract

BackgroundNeospora 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.ResultsSix 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.ConclusionThe 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.

Highlights

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
Eighty-three Nc-5 gene sequences from different isolates were recovered from databases and aligned to check how conserved the primers designed for the loop-mediated isothermal amplification (LAMP) method were; 38 sequences were excluded from further analysis as they did not have the required length for bioinformatics analysis
A further group of 9 sequences was excluded as lacking clinical relevance, since the host from which they came did not play a relevant role in N. caninum’s biological cycle and isolates infecting these species lacked epidemiological interest, leaving 36 sequences to be analyzed

Summary

Introduction

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. Regarding molecular diagnosis of infection, polymerase chain reaction (PCR)-based tests have the advantage of being able to amplify small amounts of parasite DNA in different types of biological samples and are directed towards amplifying different genes, the N. caninum Nc-5 repeat sequence and internal transcribed spacer (ITS1) being most frequently used [14]. Different PCR formats have been developed recently for increasing sensitivity in detecting N. caninum DNA, i.e. nested, semi-nested, and real-time PCR [9]. PCR diagnosis implies the use of sophisticated equipment such as a thermocycler for amplifying nucleic acids, as well as requiring personnel trained in such area and additional time for detecting the amplified products [15]

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Results

Discussion

Conclusion