Design, optimization, and application of a conventional PCR assay with an internal control for detection of ‘CandidatusMycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Abstract

'Candidatus Mycoplasma turicensis' (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and 'Candidatus M. haemominutum' (CMhm) but not for CMtc. Although real-time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false-negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Species-specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10-fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica-based protocol and tested using the PCR assay. Primer concentration, annealing temperature, and MgCl(2) concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.