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a) DNA sample from C. perfringens reference strains type A (ATCC 13124, CECT 486), type B (ATCC 3626), type D (CN3978) and type E (ATCC 27324) were used to validated the different toxin genes.
b) A panel of 27 related microorganisms including other clostridium strains (C. difficile CECT531, C. sporogenes CECT485) and several bacteria, viruses and parasites resulted negative.
c) A total of 35 field strains of C. perfringens (porcine=21, bovine=7, ovine=6 and caprine=1) were evaluated. The 89% (29/35) of these strains were clasified as type A and 11% (4/35) as type D.
d) Presence of toxin genes were additionally evaluated in 28 C. perfringens positive samples of small intestine from porcine (n=15), bovine (n=6), ovine (n=5) and caprine (n=2). Toxin alpha was detected in all the samples, toxin beta in 14% of samples, toxin epsilon in 25%, toxin iota in 11%s, enterotoxin in 14% and toxin Beta-2 in 78% of samples.
• We studied the reportable range for this qPCR assay using a purified specific synthetic oligonucleotide (CLS1 positive control).
• This qPCR kit can identify C. perfringens Alpha toxin from clinical samples and field strains.
• Maximum quantification limit of C. perfringens Alpha toxin ≥1010 copies/rxn.
• Minimum quantification limit of C. perfringens Alpha toxin = 50 copies /rxn.