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A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses

Tian, A.L.; Elsheikha, Hany M.; Zhou, Dong-Hui; Wu, Yao-Dong; Chen, Mu-Xin; Wang, Meng; Chen, Dan; Zhang, Xi-Chen; Zhu, Xing-Quan

Authors

A.L. Tian

Dong-Hui Zhou

Yao-Dong Wu

Mu-Xin Chen

Meng Wang

Dan Chen

Xi-Chen Zhang

Xing-Quan Zhu



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.

Journal Article Type Article
Publication Date Jul 15, 2018
Journal Veterinary Parasitology
Print ISSN 0304-4017
Electronic ISSN 0304-4017
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 258
APA6 Citation Tian, A., Elsheikha, H. M., Zhou, D., Wu, Y., Chen, M., Wang, M., …Zhu, X. (2018). A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses. Veterinary Parasitology, 258, https://doi.org/10.1016/j.vetpar.2018.06.004
DOI https://doi.org/10.1016/j.vetpar.2018.06.004
Keywords Neospora caninum; Molecular diagnostics; Isothermal amplification; Recombinase polymerase amplification(RPA); Lateral flow(LF)strips
Publisher URL https://www.sciencedirect.com/science/article/pii/S0304401718302164
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc-nd/4.0

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LF-RPA diagnostics for Neosporosis.pdf (3.6 Mb)
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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc-nd/4.0


LF-RPA diagnostics for Neosporosis.pdf (3.7 Mb)
PDF

Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc-nd/4.0





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