Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil

Abstract

Objectives: The objective of this study was to develop a quantitative 5' nuclease real-time polymerase chain reaction (PCR) assay to diagnose infections caused by Bartonella species.

Methods: Between January and April 2013 whole blood samples were collected by convenience from 151 cats (86 domiciled and 65 stray cats). The feline blood samples were subjected to a novel quantitative 5' nuclease real-time PCR (qPCR) for Bartonella species targeting the nictonamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) and conventional PCR assays targeting intergenic transcribed spacer, ribC, gltA, pap31 and rpoB, followed by sequencing and basic local alignment search tool analysis.

Results: The qPCR assay detected as few as 10 copies of plasmid per reaction. Forty-six (54.4% domiciled and 45.6% stray cats) of 151 sampled cats showed positive results in nuoG qPCR for Bartonella species. The absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 1.1 × 10(4) to 1.3 × 10(4). Eighteen (39.1%) of 46 positive samples in the qPCR were also positive in conventional PCR assays. The sequencing confirmed that Bartonella henselae and Bartonella clarridgeiae circulate in cats in midwestern Brazil.

Conclusions and relevance: The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species.

Figure 1

Optimization of the annealing temperature and concentrations of the primers and hydrolysis probe. The best annealing temperature and concentrations were detected through a range of gradients. Analysis of the results showed that 52.8°C and 1.2 µM were the best annealing temperature and concentration of primers and hydrolysis probe, respectively. RFU = relative fluorescence units

Figure 2

Specificity analysis of the nuoG real-time polymerase chain reaction assay. Amplification curve was observed for only Bartonella species. Positive controls were DNA. No amplification was verified for Ehrlichia species, Anaplasma species, Neorickettsia species, Orientia species, Rickettsia species, Trypanosoma species or Plasmodium species. RFU = relative fluorescence units