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

文献类型： 外文期刊

第一作者： Tian, Ai-Ling

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

作者机构：

关键词： Neospora caninum; Molecular diagnostics; Isothermal amplification; Recombinase polymerase amplification (RPA); Lateral flow (LF) strips

期刊名称：VETERINARY PARASITOLOGY （ 影响因子：2.738； 五年影响因子：2.951 ）

ISSN： 0304-4017

年卷期： 2018 年 258 卷

页码：

收录情况： SCI

摘要： 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, Hanunondia 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.

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