Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR

Abstract

Erysipelothrix rhusiopathiae is a gram-positive rod capable of causing erysipelas in swine. To establish a method for specifically detecting E. rhusiopathiae for practical applications, such as for the inspection of slaughterhouses, the feasibility of using primers derived from the DNA sequence coding for 16S rRNA in a PCR-specific detection system was investigated. Oligonucleotide primers were designed to amplify a 407-bp DNA fragment by PCR. The amplification was specific to the Erysipelothrix DNA but not to that of other bacterial genera tested. This PCR-based method efficiently and specifically detected the Erysipelothrix DNA sequence in joint and spleen samples from mice within 6 h, and application of the 407-bp DNA segment from samples containing very low numbers of bacteria (<20 bacteria per spleen from mice) was possible. Although this PCR amplification is specific for the Erysipelothrix genus, which contains at least two species, E. rhusiopathiae and E. tonsillarum, it can be concluded that all Erysipelothrix strains detected by this PCR system in diseased pigs are E. rhusiopathiae because only E. rhusiopathiae is virulent for pigs. These results show that this PCR amplification system using the DNA sequence coding for 16S rRNA is very rapid and reliable and avoids cumbersome and lengthy cultivation steps, demonstrating that this system could be used for practical applications.

Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional PCR assays

“…isolates. Conventional PCR assays (Makino et al 1994;Shimoji et al 1998;Takeshi et al 1999;Yamazaki 2006) Table 3 Intra-assay and inter-assay variation in cycle threshold (C T ) values for the spa multiplex real-time PCR assay…”

Erysipelothrix rhusiopathiae bloodstream infection – A 22‐year experience at Mayo Clinic, Minnesota

“…Such technology has been used successfully in veterinary studies but is still under investigation in humans (Romney et al, 2001). A PCR amplification system using the DNA sequence coding for 16S rRNA was demonstrated to be rapid and reliable in identifying Erysipelothrix species in joint and spleen samples from mice (Makino et al, 1994).…”

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

“…The main disadvantage of this PCR is the difficulty in identification of the four species as PCR products differ in size by as little as three base pairs. Moreover, conventional PCR methods (Makino et al 1994;Okatani et al 2000;Shimoji et al 1998;Takeshi et al 1999;Yamazaki 2006) are qualitative and need postPCR processing of the samples. The multiplex real-time PCR assay developed in the present study allows automated product detection with improved specificity and sensitivity.…”

“…and about 6-8 days to determine its serotype (Hassanein et al 2000(Hassanein et al , 2001. Recently, several conventional PCR assays (Makino et al 1994;Shimoji et al 1998;Takeshi et al 1999;Yamazaki 2006) have been proposed that can replace the traditional time-consuming methods of routine culture detection. Each method has its own advantages and disadvantages.…”