Detection of clostridium perfringens toxin genes by multiplex PCR method in chicken meat parts and effects of different freezing temperatures on survival of clostridium perfringens

2. ABSTRACTThis study was carried out (i) to investigate the presence of C. perfringens in chicken meat parts (breast, wing, drumstick and leg) by culture methods (ii) to detect the cpa, cpb, etx, iA, cpe ve cpb2 toxin genes of isolates obtained from the chicken meat parts by multiplex PCR and (iii) to investigate the effects of different freezing temperatures on the survival of C. perfringens in chicken wings.A total of 200 samples, the fresh chicken breasts (n: 50), wings (n: 50), drumsticks (n:50) and legs (n: 50) were collected from various retail points including supermarkets, delicatessen stores and butchers in Elazig city. Results indicated that 47 of 50 wing samples (94 %), 40 of 50 leg samples (80 %), 34 of 50 drumstick samples (66 %) and 33 of 50 breast samples (66 %) were found contaminated with C. perfringens, and 558 out of 568 presumptive-positive isolates obtained from these samples were identified as C. perfringens based on the microscopic examination and biochemical tests. It was detected that 545 (97.6 %) of DNA products extracted from these 558 C. perfringens strains carried cpa toxin gene, 12 (2.1 %) of them carried both cpa and cpb2 toxin gene, one (0.1 %) of them carried both cpa and cpe toxin genes, according to the multiplex PCR results. In additon, it was determined that none of the isolates carried cpb, etx or iA toxin genes. As a conclusion, 545, 12 and 1 of 558 C. perfringens isolates were type A, cpb2 (+) type A and cpe (+) type A, respectively.Another purpose of the research was to investigate the survival ability of C. perfringens at different freezing temperatures. For this purpose, a total 24 chicken wings (c.a 80 g) were used for each repetition and the study was composed from 3 repetitions with a total of 72 wings. In the first group (frozen and stored at -12 ± 1 ?C), the numbers of the pathogen decreased significantly (P <0.05) compared to the second group (frozen and stored at -18 ± 1 ?C) and the third group (frozen at -40 ?C following stored at -18 ± 1 ?C), during 112 day storage. No significant difference between the 2nd and 3rd group was detected during the storage (P >0.05). Within the first group, significant differences between the days of the storage were found (P <0.05). At the end of the storage (112th day), C. perfringens level dropped below the detectable level (<1.0 log10 cfu/cm2) in 1st group, and decreased to 4.23 and 4.30 log10 cfu/cm2 in 2nd group and 3rd group, respectively.The results of the study indicate that the majority of the chicken meat parts are contaminated with C. perfringens. Consequently, the hygienic conditions from farm to fork should be ensured by implementing HACCP.In addition, it was determined that for a long time storage of the chicken wings at different freezing and storage temperatures have a significant effect on the survival of C. perfringens. It can be speculated that chicken meat frozen and stored at -18 ?C and below may still pose public health risk because of the treatment did not show the expected reduction on the survival of vegetative C. perfringens cells. However, freezing and storing the chicken meats at -12 ?C decreased the survival ability of vegetative C. perfringens and caused to drop below the detectable level of the pathogen at the end of the storage. It is concluded that the number of C. perfringens at -12 ?C decreased during the storage period and dropped below detetion limit by the 112th day. However, the potential negative effects on the shelf life of the product at this storage temperature should be taken into consideration.Keywords: Chicken meat parts, C. perfringens, toxin genes, multiplex PCR, frozen storage