PCR Campylobacter jejuni C. coli

Transcription

1 Jpn. J. Food Microbiol., 28(3), , 2011 PCR Campylobacter jejuni C. coli Evaluation of Selective Enrichment Media for Detection of Campylobacter jejuni and C. coli by Real-Time PCR Yoshimi D6I:, Yoshio AH6>, Ichiro FJGJ@6L6, Katsuhiro A>@6L6, Tomoe IH=>=6G6 and Toshiro KJGD@> (Kanagawa Prefectural Institute of Public Health, Shimomachiya, Chigasaki-shi, Kanagawa ; Corresponding author) A study on evaluating Preston broth and Bolton broth for reliable detection of Campylobacter jejuni and C. coli from food samples was performed. Both broths were spiked with combination of C. jejuni and C. coli strains, and the growth of strains was determined by quantitative real-time PCR assay. The number of C. jejuni in Preston broth rose to CFU/ml, while the number of C. coli ranged from less than 10 to 10 6 CFU/ml. The number of C. jejuni and C. coli spiked in Bolton broth increased to CFU / ml. The result indicated that the Bolton broth excellent ability for growth of broth C. jejuni and C. coli. Trial for detection of C. jejuni and C. coli from retailed meat was carried out with culture method using Preston and Bolton enrichment broths and a quantitative realtime PCR assay. The result of trial showed that the Bolton broth has higher performance for detecting C. coli than Preston broth has, and Preston broth is suitable to detect C. jejuni. These results suggested that use of both Preston and Bolton enrichment broths for detecting C. jejuni and C. coli is essential, and a quantitative real-time PCR assay is useful for rapid detection of Campylobacter. Key words: PCR Campylobacter jejuni, Campylobacter coli, Preston broth, Bolton broth, real-time Campylobacter jejuni/coli ) ) 10) Campylobacter jejuni/coli

2 187 Preston 2, 8) Preston 12) (ISO) Campylobacter jejuni/ coli 1995 Preston 2006 Bolton 5, 6) Bolton 12) 4) Preston Bolton C. jejuni C. coli 1) C. jejuni C. coli C. jejuni C. coli Campylobacter jejuni/coli 1. Preston Bolton C. jejuni C. coli PCR 2. PCR C. jejuni C. coli 1. C. jejuni C. coli 1) C. jejuni 3 1 ATCC 33560, 2 EK , EK C. coli 3 1 ATCC 43478, EK , EK Blood Agar Base No. 2 (Oxoid) BHI broth (Difco) 10 ml 37, CFU/ml CFU/ml 2) Bolton Bolton Broth (Oxoid) Modified Bolton Broth Selective Supplement (Oxoid) Preston Nutrient Broth No. 2. (Oxoid) Campylobacter Growth Supplement (Oxoid) Preston Campylobacter Selective Supplement (Oxoid) 5 3) CFU/ml 0.1 ml 10 ml CFU/ml A: C. jejuni ATCC C. coli ATCC B: C. jejuni EK C. coli EK C: C. jejuni EK C. coli EK ) 1 5 Blood Agar Base No. 2 (Oxoid) ) 5) PCR DNA 1ml 16,000 rpm 3 PrepMan Ultra Sample Preparation Reagent (ABI) 200 ml ,000 rpm 3 50 ml 2 4 DNA PCR LightCycler TM (Roche Diagnostics) SYBR Green I C. jejuni C. coli C. jejuni Light Cycler Probe Design Software 2.0 hipo Forward; 5 -CCT ATG CTT ACA ACT GCT GA-3, Reverse; 5 -CGA TGA TGG CTT CTT CGG-3 C. coli 3) ceue CCceuE-F (5 -ACGCGCA- CAAGGCATACTT- 3 ) CCceuE-R (5 -CCAGTATT- CAGGATCAAGATAAATGATTT-3 ) DNA 2 ml, (20 pmol/ ml) 0.2ml, SYBR Green Realtime PCR Master Mix- Plus- (TOYOBO) 10 ml, Plus solution 2 ml, 5.6 ml 20 ml PCR (65 95 )

3 188 Vol. 28 No Fig. 1. Generation of the standard curve for the real-time PCR (A) real-time PCR analysis of serial 10-fold dilutions of C. jejuni (ATCC 33560) DNA. (A) Threshold cycles (CT) values were plotted against the calculated CFU ( /ml). (B) Real-time PCR analysis of serial 10-fold dilutions of C. coli (ATCC 43478) DNA. Threshold cycles (CT) values were plotted against the calculated CFU ( /ml) C. jejuni ATCC C. coli ATCC Nutrient Broth No. 2 (Oxoid) DNA DNA Blood Agar Base No. 2 (Oxoid) DNA C. jejuni C. coli PCR Fig. 1 (A) (B) DNA Threshold Cycle (Ct) r C. jejuni C. coli 10 3 CFU/ml C. jejuni 82.3 C. coli PCR 1) ) 100 g 25 g ml 1 5 3) (MPN) 3 5 Preston Bolton , ml CCDA Selective Supplement (Oxoid) Campylobacter Blood-Free Selective Agar Base (Oxoid) m-ccda Preston Campylobacter Selective Supplement (Oxoid) 5 Campylobacter Agar Base (Oxoid) Preston C. jejuni C. coli 100 g MPN 10 C. jejuni C. coli ( 15/100g) 4) PCR 10 MPN3 Preston Bolton DNA PCR C. jejuni C. coli 5) Preston Bolton PCR c 2 1. Preston Bolton C. jejuni C. coli Preston Bolton C. jejuni C. coli 24 C. jejuni C. coli PCR Table 1 C. jejuni C. coli A C Preston C.

4 189 Table 1. Comparison of the quantitative value and the colony counts on isolating media from the enrichment culture (Preston and Bolton) when C. jejuni and C. coli were simultaneously inoculated Preston broth Bolton broth Combination of isolates Detection by real- time PCR (CFU/ml) Number of colonies confirmed Detection by real- time PCR (CFU/ml) Number of colonies confirmed C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli A C. jejuni: ATCC CFU C. coli : ATCC CFU B C. jejuni: EK CFU C. coli : EK CFU C C. jejuni: EK CFU C. coli : EK CFU a) Five colonies from each blood agar plate were analyzed. Table 2. Domestic meat a) Imported meat Samples Detection of C. jejuni and C. coli in retail chiken meat by enrichment cultures (Preston and Bolton broth) (MPN/100 g) C. jejuni C. coli Preston Bolton Preston Bolton a) Distributed under refrigeration jejuni C. coli A 2 B 1 Bolton C. coli C. jejuni A 2 PCR Preston C. jejuni A C CFU/ml C. coli A B CFU/ ml, C Bolton C. jejuni Preston A C CFU/ml C. coli 10 7 CFU/ml 2. C. jejuni C. coli C. jejuni C. coli Preston Bolton (MPN) 3 Table 2 10 C. jejuni 4 Preston Bolton 4 3 Preston 3 Bolton 1 C. coli 2 Preston Bolton 2 2 Preston 1 Bolton , 5, 7, 9 C. jejuni C. coli C. jejuni Preston 3 (6, 7, 9) 2 (6, 9) Bolton C. coli Bolton 2 (7, 9) 1 (7) Preston 3. Preston Bolton Campylobacter jejuni/coli MPN 10 Preston Bolton 30 PCR m-ccda Preston 2 Table PCR C. jejuni Preston 18 12, 6 (60.0 ), Bolton 15 12, 3 (50.0 ) C. coli Preston 9 6, 3 (30.0 ), Bolton 10 6, 4 (33.3 ) C. jejuni C. coli Preston Bolton m-ccda, Preston C. jejuni Preston m-ccda 12 (40.0 ), Preston 15 (50.0 ),

5 Table 3. Comparison of the PCR assay and culture for the detection of C. jejuni and C. coli in enrichiment cultures Detection of C. jejuni Detection of C. coli Samples Sample No. (No. of test) No. positive ( ) in real-time PCR No. positive ( ) on subculture to selective agar No. positive in real-time PCR No. positive ( ) on subculture to selective agar m-ccda Preston m-ccda Preston Preston Bolton Preston Bolton Preston Bolton Preston Bolton Preston Bolton Preston Bolton Domestic meat a) 1 (3) (3) (3) (3) (3) Sub total (80.0) 12 (80.0) 9 (60.0) 2 (13.3) 10 (66.6) 6 (40.0) 6 (40.0) 6 (40.0) 3 (20.0) 2 (13.3) 2 (13.3) 6 (40.0) Imported frozen meat 6 (3) (3) (3) (3) (3) Sub total 15 6 (40.0) 3 (20.0) 3 (20.0) 1 (6.0) 5 (33.3) 1 (6.0) 3 (20.0) 4 (26.6) 0 (0.0) 4 (26.6) 2 (13.3) 4 (26.6) Total (60.0) 15 (50.0) 12 (40.0) 3 (10.0) 15 (50.0) 7 (23.0) 9 (30.0) 10 (33.3) 3 (10.0) 6 (20.0) 4 (13.3) 10 (33.3) a) Distributed under refrigeration 190 Vol. 24 No

6 191 Bolton m-ccda 3 (10.0 ), Preston 7 (23.0 ), C. coli Preston m-ccda 3 (10.0 ), Preston 4 (13.3 ), Bolton m-ccda 6 (20.0 ), Preston 10 (33.3 ) PCR C. jejuni C. coli 4 3, 5, 7, 9 Campylobacter jejuni/coli Preston Bolton C. jejuni C. coli Preston C. coli 1) C. jejuni C. coli Table 1 PCR Preston C. jejuni C. coli C. jejuni EK07-250, Bolton (C. coli) Table 2 C. jejuni Preston C. coli Bolton 2 Preston C. coli C. coli 3 Campylobacter jejuni/coli 3) PCR Table 3 Preston Bolton Bolton 3) Campylobacter jejuni/coli Preston 9, Bolton 7 Bolton (p 0.05). Table 3 C. jejuni ISO Bolton m-ccda (10 ) Preston Preston (50 ) (p 0.05) ISO Bolton m-ccda Preston Bolton PCR C. jejuni C. coli m-ccda Campylobacter jejuni/coli Bolton m-ccda b- b- (ESBL) 13) ESBL 2) Bolton m-ccda Campylobacter jejuni/coli 13) Bolton Campylobacter jejuni/coli Preston Campylobacter jejuni/coli Bolton Preston PCR PCR C. jejuni C. coli PCR Table 1 Table 3 PCR Preston Bolton PCR PCR 3, 9 Table 2 MPN C. jejuni C. coli 2 C. coli C. jejuni

7 192 Vol. 28 No C. jejuni C. coli Preston Bolton PCR Preston Bolton Campylobacter jejuni C. coli PCR PCR Preston C. jejuni CFU/ml C. coli C. jejuni Bolton 10 6 CFU/ml Preston Bolton C. jejuni C. coli, Preston Bolton C. jejuni C. coli MPN Preston C. jejuni, Bolton C. coli PCR PCR 1) 39, 4 7(2009). 2) 55, (2009). 3) PCR p , (2006). 4) 26, (2009). 5) International Organization for Standardization, ISO Microbiology of food and animal feeding stu#s. Horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method. ISO, Geneva, Switzerland (1995). 6) International Organization for Standardization, ISO : Microbiology of food and animal feeding stu#s-horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method. ISO, Geneva, Switzerland (2006). 7) 3, (2009). 9) p , (2004). 9) p , (2004). 10) Campylobacter jejuni Campylobacter coli p. 64, (2006). 11) 8, 9 16 (2006). 12) 3, (2009). 13) Jasson, V., Sampers, I., Botteldoorn, N., López-Gálvez, F., Baert, L., Denayer, S., Rajkovic, A., Habib, I., De Zutter, L., Debevere, J., Uyttendaele, M.: Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth. Int. J. Food Microbiol., 135, (2009).