Three multiplex species-specific PCR-based assays were developed and used to identify blood meal source of engorged females of Palaearctic midge species of veterinary interest.
This assay combines PCR amplification of the small subunit (ssu) ribosomal RNA gene (491 500 bp fragments) using genus specific primers, followed by a multiplex species-specific ligation detection reaction (LDR).
Samples positive for Brucella were subjected to multiplex species-specific Brucella amplification.
Positive B4/B5 samples were subjected to multiplex species-specific Brucella PCR amplification.
Therefore, considering the importance of differential diagnosis, particularly for epidemiologic studies or eradication programs [ 13], the aim of this study was to develop and validate a multiplex species-specific PCR assay for simultaneous detection of B. ovis, A. seminis, and H. somni.
We isolated genomic DNA from ACCNS and CAC2 cells and amplified it by PCR using multiplex 12 species-specific primer sets designed to amplify a specifically sized product only in the presence of target species [33].
PCR reactions were performed using multiplex 12 species-specific primer sets that target the 5' variable region of the COI gene [33].
These loci were designed into a single diagnostic multiplex suitable for species identification and population genetics studies.
Conventional multiplex PCR for species identification was performed with primers specific for amplifying the genes porA (N. meningitidis), lytA (S. pneumoniae), and bexA (H. influenzae) for all samples, and capsule genogrouping was carried out for porA PCR-positive samples (7, 8 ).
In the present study, we demonstrated that multiplex PCR assay with species specific primers does not amplify DNA from other bacteria species that can potentially cause epididymitis in rams, including S. aureus, M. haemolytica, C. pseudotuberculosis, and T. pyogenes as well as O. anthropi, a species phylogenetically related to B. ovis (Table 4).
Additionally, the species-specific multiplex assay was demonstrated to be specific for the target species and did not exhibit cross reactions with other organisms that may also cause infectious ovine epididymitis.
