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A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses
- Source :
- Veterinary parasitology. 258
- Publication Year :
- 2018
-
Abstract
- The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10?min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50?fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75?samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.
- Subjects :
- 0301 basic medicine
Recombinase polymerase amplification(RPA)
030231 tropical medicine
ved/biology.organism_classification_rank.species
Loop-mediated isothermal amplification
Neospora caninum
Recombinase Polymerase Amplification
complex mixtures
Sensitivity and Specificity
Chromatography, Affinity
Isothermal amplification
Hammondia hammondi
Recombinases
03 medical and health sciences
chemistry.chemical_compound
Feces
0302 clinical medicine
parasitic diseases
Molecular diagnostics
Animals
DNA Primers
Lateral flow(LF)strips
General Veterinary
biology
ved/biology
Coccidiosis
fungi
Neospora
Temperature
Toxoplasma gondii
General Medicine
030108 mycology & parasitology
DNA, Protozoan
biology.organism_classification
Molecular biology
enzymes and coenzymes (carbohydrates)
chemistry
Molecular Diagnostic Techniques
Aborted Fetus
Sarcocystis
Parasitology
Cattle
Nested polymerase chain reaction
Nucleic Acid Amplification Techniques
DNA
Subjects
Details
- ISSN :
- 18732550 and 03044017
- Volume :
- 258
- Database :
- OpenAIRE
- Journal :
- Veterinary parasitology
- Accession number :
- edsair.doi.dedup.....3bb59a09e313ce684c8de054b5b4b56d