Proceedings, The 15th Congress of FAVA 27-30 October FAVA - OIE Joint Symposium on Emerging Diseases Bangkok, Thailand

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Detections of Clostridium perfringens and Alpha Toxin in Broiler Intestinal Content by Real-time Polymerase Chain Reaction with Taqman Probe

S. Athirote1, S. Boonfu1, P. Suksaknuansak1, R. Chatsantikul1, N. Sitthiode1, S. Raksasuk 2 , W. Lewkongstaporn2, B. Enkvetchakul1* 1Cargill Meats (Thailand) Limited 2, Biogenomed Co.,Ltd. *Corresponding author Keyword : Clostridium perfringens, Real-time polymerase chain reaction Introduction C. perfringens is an anaerobic bacteria. Commonly encountered in infections both human and animal. It produces toxins, α-toxin, which causes the cell membrane damage and disrupt normal cellular function. The necrotic enteritis (NE) is a major sym- ptom usually found in the animal which cause starve and death (1). Diagnosis of NE requires isolation of the pathogen but this conventional methods are generally time consumption. The real-time PCR is the rapid method which can be used to detect the DNA of C. perfringens and the α-toxin production strain by using the taqman probe which specific for 16s rDNA and α -toxin gene (2). Materials and Methods Bacterial strain:, bacterial cultures used for sensitivity, and specificity testing were obtained from American Type Culture Collection (ATCC). Samples and enrichment procedures:, the collected intestinal contents from broilers various ages were inoculated to Cooked meat medium and incubated at 420C for 24 h. Sample were then cultured on blood agar and incubated microaerophilically at 370C 24 h. and then investigated by Gram stain. DNA template preparations: all pure cultures, fecal samples and enriched samples were extracted by filter membrane spin column technique of High pure PCR template preparation kit/Roche and then were quantified by spectrophotometer and the 10 fold serial dilution to the 10-9 dilution was used to perform sensitivity testing. Real-time PCR assay conditions: the amplification reactions were carried out in total of 20 µl. All reaction mixtures contained DNA template, primer, Taqman probe specific for 16s rDNA and α -toxin gene (table. 1) were added to amplify DNA.

Table 1 Sequences used to test of C. perfringens and α -toxin (2)

Sequences C.perfringens Forward primer 5’TgA AAg ATg gCA TCA TCA TTC AAC Reverse primer 5’ggT ACC gTC ATT ATC TTC CCC AAA Taqman probe 5’ (6 Fam) CCg ACC TgA gAg ggT gAT Cgg CC(Tamra) α -toxin Forward primer 5’ TTC TAT CTT ggA gAg gCT Atg ACA TAT TTT Reverse primer 5’ TTT CAA ACT TAA CAT gTC CTg CgC Taqman probe 5’ (6 Fam) TAg ATA CTC CAT ATC ATC CTg CTA

ATG TTA CTg CCg TTg A (Tamra) Results and Discussion The results depicted in table 2 show that the real-time PCR tests for C. perfringens and α-toxin were the specific and were shown to be negative when tested with other bacterial samples. The sensitivity results shown in figure 1 and 2, the last sensitive dilution of C. perfringens was 10-7 (1.17 ρg DNA) / ml and α-toxin was 10-6 (11.7 ρgDNA/ml). Result s indicated that detection of C. perfringens DNA by real-time PCR was sensitive and specific. The times used in detection C. perfringens by real- time was faster than the conventional method (4 h vs 72 h). Table 2 Specificity test of C.perfringen and α-toxin

Organism C.perfringens detection

Alpha toxin

Clostridium perfringens ATCC 13124 + +

Mycoplasma gallisepticum (TS11) - -

Salmonella enteritidis - -

Salmonella typhimurium - -

Campylobacter jejuni - -

Listeria monocytogenes - -

E.coli ATCC 25922 - -

Pseudomonas aeruginosa ATCC 13048 - -

Staphylococcus aureus ATCC 65381 - -

Bacillus cereus ATCC 11778 - -

Streptococcus pyogen ATCC 19615 - -

27-30 October Proceedings, The 15th Congress of FAVA Bangkok, Thailand FAVA - OIE Joint Symposium on Emerging Diseases                         

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Fig. 1 Sensitivity results of C. perfringens detection

Fig. 2 Sensitivity results of α -toxin detection Acknowledgements Special thanks to Dr. Thiti Praditpechara and Mr. Prasit Meakwattana for the financial support. References 1 Gurjar et al., 2008. Mol. Cell. Probe 22: 90-95. 2 Skanseng et al., 2006. Mol. Cell. Probe 20: 269-

279.

10 -6= 11.7 ρg/ ml

10 -7 or 1.17 ρg/ml