Validation of a nested PCR protocol in time real in a tube and genetic sequencing for detection and characterization of species and genotypes of Cryptosporidium in birds fecal samples

Author(s)

Bruna Nicoleti Santana

• Document type: Doctoral Thesis
• Press: Araçatuba. 2021-09-21.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária. Araçatuba
• Defense date: 2021-08-27
• Advisor: Marcelo Vasconcelos Meireles

Abstract

Protozoa of the genus Cryptosporidium infect the gastrointestinal tract and occasionally the respiratory, biliary, and urinary tracts, causing clinical and subclinical disease in birds. Species-specific diagnosis of cryptosporidiosis is commonly performed by conventional nested PCR followed by genetic sequencing. The objective of this study was to validate a one-tube real-time nested PCR protocol followed by melting curve analysis and genetic sequencing to detect and characterize Cryptosporidium species and genotypes in birds. The reaction was standardized using the SsoFast® EvaGreen Supermix (Bio-Rad) and the CFX96 real-time PCR system (Bio-Rad). A total of 443 avian genomic DNA samples were analyzed by conventional nested PCR and one-tube nested real-time PCR. After validation one-tube real-time nested PCR was used to determine the prevalence of Cryptosporidium spp. in 64 commercial broiler flocks in the western region of the state of Santa Catarina. By conventional nested PCR, 36/443 (8.1%) samples were positive for Cryptosporidium spp. In contrast, one-tube nested real-time PCR showed 90/443 (20.3%) positive samples for Cryptosporidium spp. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. Analytical specificity evaluation did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability calculation showed the same result in 27 out of 30 samples (90%). Regarding the reproducibility of nPCR-TR-1T, the same result was observed in 24 of the 30 samples examined (80%). It was possible to perform sequencing on all 90 samples amplified by one-tube real-time nested PCR. The following species were identified: C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium genotype I in birds. Genetic sequencing of conventional nested PCR amplicons was possible in 10/36 (27.8%) of positive samples, with identification of the same species identified by one-tube real-time nested PCR. Cryptosporidium spp. prevalence in broiler flocks was 0.2% (1/64), with identification of C. baileyi in one sample. The results of this study demonstrated that one-tube nested real-time PCR, in comparison with conventional nested PCR, presents faster results, less possibility of contamination with amplicons, and lower consumption of reagents, since it is performed in only one step and the results are obtained in real time, without the need for agarose gel electrophoresis. (AU)

• FAPESP's process: 18/03791-2 - Validation of a real-time PCR assay associated with genetic sequencing for detection and characterization of Cryptosporidium species and genotypes in faecal bird samples
• Grantee: Bruna Nicoleti Santana
• Support Opportunities: Scholarships in Brazil - Doctorate