Rapid detection of Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays

Yuelin Liu; Jialin Xiang; Yaxin Gao; Jinfeng Wang; Libing Liu; Ruiwen Li; Jianchang Wang

doi:10.1016/j.heliyon.2023.e20794

Rapid detection of Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays

Heliyon. 2023 Oct 7;9(10):e20794. doi: 10.1016/j.heliyon.2023.e20794. eCollection 2023 Oct.

Authors

Yuelin Liu¹, Jialin Xiang², Yaxin Gao¹, Jinfeng Wang², Libing Liu², Ruiwen Li¹, Jianchang Wang²

Affiliations

¹ College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
² Food Microbiology and Animal Quarantine Laboratory, Technology Center of Shijiazhuang Customs District, Shijiazhuang, China.

Abstract

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.'s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Keywords: 18S rRNA gene; Cryptosporidium spp.; Isothermal amplification; LFS RPA; Point-of-care detection; Real-time RPA.