Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

Abstract

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis Avaliação da sensibilidade da PCR em uma etapa com base no gene p28 de Ehrlichia canis e sua aplicação no diagnóstico da erliquiose canina.

Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

“…A matter of concern is the progression of CME to the chronic form, which is characterised by the presence of physical and hematological signs similar to those seen during the acute phase. CME is a multisystemic disease with clinical signs overlapping other infections, theerfore its diagnosis can be improved by using specific diagnostic tests combined with physical and hematological evaluation (de et al 2011, Harrus and Waner 2011, Nakaghi et al 2010. Blood smears for the detection of morulae, serology, cell culture, and PCR are common tests used.…”

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

“…This approach will permit veterinarians to prescribe an appropriate treatment method for, and evaluate pathogen clearance in, infected animals (Harrus and Waner, 2011). Although nested PCR assays are the most sensitive technique for the detection of Ehrlichia sp in infected blood samples (Stich et al, 2002;Nakaghi et al, 2010), their application in clinical settings is limited by the need for a high-precision thermal cycler and the excessive number of steps, causing possible cross-contamination (Nakaghi et al, 2010). In contrast, LAMP is performed in a water bath and the results, which can be observed with the naked eye without the need for a specialized gel electrophoresis unit, are obtained within 1 h (Mori and Notomi, 2009).…”

“…In this scenario, molecular techniques are often combined with clinical and hematological evaluation strategies to improve the diagnosis of CME. PCR, nested PCR, and real-time PCR assays, techniques based on the amplification of different genes with variable sensitivity, have been previously used to detect E. canis in dogs (McBride et al, 1996;Stich et al, 2002;Doyle et al, 2005;Kledmanee et al, 2009;Nakaghi et al, 2010;Peleg et al, 2010;Cardozo et al, 2011). The 16S rRNA nested PCR assay is the most common assay used in the detection of E. canis; however, a nested PCR assay for the detection of the E. canis p30 gene has been recently shown to be more sensitive (Stich et al, 2002).…”

Recombinant gp19 as a potential antigen for detecting anti-Ehrlichia canis antibodies in dog sera

“…CME can also be confirmed via imported diagnostic kits including dot-ELISA (Immunocomb@) or ELISA (SNAP@ 3DX); immunoblot assays; molecular biology techniques such as polymerase chain reaction (PCR), which can be nonspecific depending on the sample used; and cell culture, which is a method that is used less frequently (NAKAGHI et al, 2010).…”

“…Sensitive and specific molecular techniques were evaluated for the detection of antigens, but they are not yet available for use in routine diagnostics due to their high costs (CADMAN et al, 1994;NAKAGHI et al, 2010;WANER et al, 1998).…”