Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples

Author(s)

Bruna Nicoleti Santana ^[1] ; Elis Domingos Ferrari ^[2] ; Alex Akira Nakamura ^[3] ; Giane Serafim da Silva ^[4] ; Marcelo Vasconcelos Meireles ^[5]

Affiliation

^[1] Universidade Estadual Paulista. Faculdade de Medicina Veterinária - Brasil

^[2] Universidade Estadual Paulista. Faculdade de Medicina Veterinária - Brasil

^[3] Universidade Estadual Paulista. Faculdade de Medicina Veterinária - Brasil

^[4] Instituto Biológico. Laboratório de Parasitologia Animal - Brasil

^[5] Universidade Estadual Paulista. Faculdade de Medicina Veterinária - Brasil

• Document type: Journal article
• Source: Revista Brasileira de Parasitologia Veterinária; v. 31, n. 1 2022-03-21.

Abstract

Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples. (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
• FAPESP's process: 18/06681-3 - Validation of real-time PCR followed by sequencing for detection and characterization of Cryptosporidium species and genotypes in fecal samples from birds and mammals
• Grantee: Marcelo Vasconcelos Meireles
• Support Opportunities: Regular Research Grants