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Validation of Real-Time PCR followed by sequencing for detection and Characterization of Cryptosporidium species and genotypes in fecal samples from birds and mammals

Grant number: 18/06681-3
Support type:Regular Research Grants
Duration: February 01, 2019 - January 31, 2021
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
Principal Investigator:Marcelo Vasconcelos Meireles
Grantee:Marcelo Vasconcelos Meireles
Home Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil


Protozoa of the genus Cryptosporidium belong to the phylum Apicomplexa, class Gregarinomorphea, present epicellular localization in the mucosal epithelia, particularly the gastrointestinal tract and, occasionally, also infect the respiratory, biliary and urinary tract, causing clinical and subclinical disease in vertebrates. Several Cryptosporidium species and genotypes are zoonotic. For molecular detection of Cryptosporidium spp. in fecal samples, in most situations it is necessary to perform the PCR in two steps (nested PCR), followed by genetic sequencing. This is the most used technique for this purpose and allows the characterization of all species and genotypes of Cryptosporidium. However, it is a technique that comprises two steps of amplification, which demands time, greater expense of reagents and, also, there is a greater possibility of contamination due to the use of amplicons from the first amplification to perform nested PCR. Real-time PCR is an alternative method to conventional PCR, and provides a faster diagnosis, reduction of possible contaminants in the laboratory, it is not necessary to perform electrophoresis to visualize the result, there is possibility of previewing preliminary results before the end of the reaction and it is possible to determine the degree of infection by the absolute quantification of the amplified DNA. Real-time PCR validation will be accomplished by the comparison of three real-time PCR protocols: 1) reaction using one step in one tube; 2) reaction using two steps (nested) in one tube; 3) reaction using two steps (nested) in two tubes. All the reactions will be performed using primers targeting the 18S rRNA gene and SsoFast Eva Green Supermix, following melting curve analyses. Real-time PCR validation will consist of the following steps: viability, standardization and assessment of analytical sensitivity and specificity, epidemiological sensitivity and specificity, repeatability and reproducibility using DNA from fecal samples (positive e negative for Cryptosporidium spp.) from several avian species. Absolute quantification of Cryptosporidium DNA will also be determined using DNA from fecal samples from domestic chicken, dogs, cattle and pigs seeded with Cryptosporidium parvum DNA. Therefore, the development of this project will allow the validation of a new method for detection of Cryptosporidium spp. in fecal samples of birds and mammals, thus providing an alternative to the currently available methods for this purpose, with high sensitivity, high specificity, with lower cost and less possibility of contamination. (AU)