Validation and application real-time PCR assay targeting ssra gene for detection of bartonella species from bodent blood

Abstract

Bartonella genus is one of the emergency pathogens infected in various mammalian hosts, including some species cause infectious disease in human. Numerous rodent species and human share a common habitat, especially in the South of Vietnam. Rodents can easily transmit the vector containing Bartonella to human community exposed with them. A real-time PCR targeting on ssrA gene developed for detecting Bartonella in ruminant blood is an effective tool for epidemiological studies all over the world. This study bases on verifying the specificity, sensitivity of the new assay and applying to detect Bartonella spp. in Zoonotic and Vector Borne Disease Surveillance in PI HCMC. The specificity of the assay determined by performing real-time PCR on four different microorganisms generated an excellent result with no amplification of all microorganisms. The sensitivity evaluated by carrying out the test on DNA dilutions reaching 1fg per reaction was an ideal level for most of PCR assays. The PCR efficiency was estimated through the slope of standard curve and provided a reasonable value in a range 90-105%. Precision was performed real-time PCR on triplicates for each dilutions and also considered by calculating coefficients of variation (CV). The precision is good enough to trust the procedure. Using this method, we detected over 68% samples infected Bartonella in total, three times as sensitive as culture (21.05%). Keywords: Bartonella, validation, real-time PCR, ssrA gene, Vietnam.