Rapid detection of Staphylococcus pseudintermedius and Staphylococcus coagulans by combining fast VPCR with nucleic acid lateral flow immunoassay (NALFIA).

Abstract

In dogs, pyoderma is primarily caused by coagulase-positive Staphylococcus pseudintermedius (SP), S. coagulans (SC) and S. aureus (SA). Standard culture and biochemical methods for identifying these organisms are laborious, and potential misidentification is a possibility. Molecular methods, like PCR, are a viable alternative for early and accurate diagnosis of these organisms. In this study, we collected 83 swab samples from pyoderma cases of dogs and compared the sensitivity and specificity of the fast VPCR in conventional agarose gel electrophoresis with Nucleic Acid Lateral Flow Immunoassay (NALFIA) in detecting SP and SC. Bacterial DNA was extracted by a simple and quick NaOH lysis method. Primers targeting the rodA gene of SC and the spsK gene of SP were used in PCR, and labelled primers of the same genes were used in NALFIA. The limit of detection of SP and SC in VPCR with gel electrophoresis was 10 CFU/ml and 10 CFU/ml for NALFIA. Though VPCR with gel electrophoresis was found to be more sensitive than NALFIA, it required specialized equipment like agarose gel electrophoretic apparatus with power pack and gel documentation system and carcinogenic ethidium bromide dye for detection of the bands in agarose gel. The VPCR, combined with NALFIA, offers sensitive and reliable detection of these bacteria in 45 min, even to the naked eye. The simplicity, portability, and cost-effectiveness of the NALFIA make them particularly suitable for resource-limited settings and point-of-care testing.