A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses
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...
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| Format: | Article |
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Elsevier
2018
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| Online Access: | https://eprints.nottingham.ac.uk/52373/ |
| _version_ | 1848798710649061376 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Tian, A.L. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:24:06Z |
| format | Article |
| id | nottingham-52373 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:24:06Z |
| publishDate | 2018 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-523732020-05-04T19:46:24Z https://eprints.nottingham.ac.uk/52373/ 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 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. Elsevier 2018-07-15 Article PeerReviewed Tian, A.L., Elsheikha, Hany M., Zhou, Dong-Hui, Wu, Yao-Dong, Chen, Mu-Xin, Wang, Meng, Chen, Dan, Zhang, Xi-Chen and Zhu, Xing-Quan (2018) A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses. Veterinary Parasitology, 258 . pp. 24-29. ISSN 0304-4017 Neospora caninum; Molecular diagnostics; Isothermal amplification; Recombinase polymerase amplification(RPA); Lateral flow(LF)strips https://www.sciencedirect.com/science/article/pii/S0304401718302164 doi:10.1016/j.vetpar.2018.06.004 doi:10.1016/j.vetpar.2018.06.004 |
| spellingShingle | Neospora caninum; Molecular diagnostics; Isothermal amplification; Recombinase polymerase amplification(RPA); Lateral flow(LF)strips 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 A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title | A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title_full | A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title_fullStr | A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title_full_unstemmed | A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title_short | A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses |
| title_sort | novel recombinase polymerase amplification (rpa) assay for the rapid isothermal detection of neospora caninum in aborted bovine fetuses |
| topic | Neospora caninum; Molecular diagnostics; Isothermal amplification; Recombinase polymerase amplification(RPA); Lateral flow(LF)strips |
| url | https://eprints.nottingham.ac.uk/52373/ https://eprints.nottingham.ac.uk/52373/ https://eprints.nottingham.ac.uk/52373/ |