Detection of Babesia gibsoni in dogs by combining recombinase polymerase amplification (RPA) with lateral flow (LF) dipstick
Babesia gibsoni is a protozoan parasite responsible for the majority of reported cases of canine babesiosis in China. Currently, microscopic examination of the Giemsa-stained thin blood smears is the main diagnosis method in clinic. Here, we report the recombinase polymerase amplification-lateral flow (LF-RPA) dipstick detection method for targeting B. gibsoni cytochrome c oxidase subunit I (cox I) gene. The reaction takes only 20–30 min under isothermal temperatures between 30 and 45 °C. Specificity was evaluated using DNA from related apicomplexan parasites and their host, while the sensitivity was calculated based on the DNA from the experimental B. gibsoni-infected dogs. Results indicated that the LF-RPA method is 20 times more sensitive than the conventional PCR based on 18S rRNA and has no cross reaction with any other test DNAs. The applicability of the LF-RPA method was further evaluated using 15 samples collected from clinic. Thirteen of the 15 samples (86.67%) were detected as positive by LF-RPA, while 10 of them (66.67%) were found positive by conventional PCR. Overall, the novel LF-RPA assay is effective for the detection of B. gobsini and has considerable advantages over the conventional PCR in sensitivity, specificity, simplicity in operation, less time consumption, and visual detection. The LF-RPA method may facilitate the surveillance and early detection of B. gibsoni infection in dogs.
KeywordsBabesia gibsoni Recombinase polymerase amplification Lateral flow Diagnosis Babesiosis
The authors would like to thank the members of our lab.
This work was supported by the National Key Research and Development program of China (2017YFD0501201), the National Key Basic Research Program (973 program) of China (Grant No. 2015CB150300), and the Natural Science Foundation of Hubei Province (2017CFA020).
Compliance with ethical standards
This study was approved by the Scientific Ethic Committee of Huazhong Agricultural University. All pet dogs were handled in accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China (Permit number: HZAUCA-2016-007). All blood samples were collected under the owner’s informed consent.
Conflict of interest
The authors declare that they have no conflict of interest.
- Aboge GO, Jia H, Terkawi MA, Goo Y, Kuriki K, Nishikawa Y, Igarashi I, Suzuki H, Xuan X (2007b) A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay. Vet Parasitol 149(1–2):85–94. https://doi.org/10.1016/j.vetpar.2007.06.025 CrossRefPubMedGoogle Scholar
- Birkenheuer AJ, Levy MG, Breitschwerdt EB (2003) Development and evaluation of a Seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. J Clin Microbiol 41(9):4172–4177. https://doi.org/10.1128/jcm.41.9.4172-4177.2003 CrossRefPubMedPubMedCentralGoogle Scholar
- Boyle DS, McNerney R, Teng Low H, Leader BT, Pérez-Osorio AC, Meyer JC, O'Sullivan DM, Brooks DG, Piepenburg O, Forrest MS (2014) Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification. PLoS One 9(8):e103091. https://doi.org/10.1371/journal.pone.0103091 CrossRefPubMedPubMedCentralGoogle Scholar
- Eichenberger RM, Štefanić S, Naucke TJ, Šarkūnas M, Zamokas G, Grimm F, Deplazes P (2017) An ELISA for the early diagnosis of acute canine babesiosis detecting circulating antigen of large Babesia spp. Vet Parasitol 243:162–168. https://doi.org/10.1016/j.vetpar.2017.06.030 CrossRefPubMedGoogle Scholar
- Ikadai H, Tanaka H, Shibahara N, Matsuu A, Uechi M, Itoh N, Oshiro S, Kudo N, Igarashi I, Oyamada T (2004) Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol 42(6):2465–2469. https://doi.org/10.1128/jcm.42.6.2465-2469.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- Jaroenram W, Owens L (2014) Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome. J Virol Methods 208:144–151. https://doi.org/10.1016/j.jviromet.2014.08.006 CrossRefPubMedGoogle Scholar
- Jia H, Aboge GO, Terkawi MA, Goo YK, Nishikawa Y, Kuriki K, Lee KK, Jang HK, Kim S, Fujisaki K, Xuan X (2009) Genetic diversity of two selected antigen loci in Babesia gibsoni Asian genotype obtained from Japan and Jeju island of South Korea. Vet Parasitol 162(1–2):142–146. https://doi.org/10.1016/j.vetpar.2009.02.025 CrossRefPubMedGoogle Scholar
- Masatani T, Hayashi K, Andoh M, Tateno M, Endo Y, Asada M, Kusakisako K, Tanaka T, Gokuden M, Hozumi N, Nakadohzono F, Matsuo T (2017) Detection and molecular characterization of Babesia, Theileria, and Hepatozoon species in hard ticks collected from Kagoshima, the southern region in Japan. Ticks Tick-Borne Dis 8(4):581–587. https://doi.org/10.1016/j.ttbdis.2017.03.007 CrossRefPubMedGoogle Scholar
- Matsuu A, Ono S, Ikadai H, Uchide T, Imamura S, Onuma M, Okano S, Higuchi S (2005) Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA. J Vet Diagn Investig 17(6):569–573. https://doi.org/10.1177/104063870501700608 CrossRefGoogle Scholar
- Narantsatsral S, Goo YK, Battsetseg B, Myagmarsuren P, Terkawi MA, Soma T, Luo Y, Li Y, Cao S, Yu L, Kamyingkird K, Aboge GO, Nishikawa Y, Xuan X (2011) Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay. Exp Parasitol 129(2):196–202. https://doi.org/10.1016/j.exppara.2011.07.011 CrossRefPubMedGoogle Scholar
- Soliman H, Kumar G, El-Matbouli M (2018) Recombinase polymerase amplification assay combined with a lateral flow dipstick for rapid detection of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonids. Parasit Vectors 11(1):234. https://doi.org/10.1186/s13071-018-2825-5 CrossRefPubMedPubMedCentralGoogle Scholar