Identification of Salmonella,Campylobacter jejuni and Enterohemorrhagic E.coli by Denaturing High-performance Liquid Chromatography

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1 BIOTECHNOLOGY BULLETIN PCR multiplex PCR mpcr denaturing high-performance liquid chromatography DHPLC O157 H7 fimy gyra O157 H7 rfbe 3 O157 H7 PCR bp PCR O157 H7 1.5 CFU/ ml 15 CFU/ ml O157 H7 15 CFU/ ml O157 H7 PCR -DHPLC O157 H7 O157 H7 Identification of Salmonella,Campylobacter jejuni and Enterohemorrhagic E.coli by Denaturing High-performance Liquid Chromatography Xu Junyi Cao Jijuan Zheng Qiuyue Liu Shuyan Xu Yang Liaoning Entry-Exit Inspection and Quarantine Bureau of China Dalian AbstractA new molecular technique for the analysis of Salmonella Campylobacter jejuni and enterohemorrhagic E.coli based on the separation of PCR -amplified target fragments by denaturing high-performance liquid chromatography DHPLCwas reported. A multiplex PCR mpcrassay was developed by using 3 sets of primers that specifically amplify segments of the fimy gyra and rfbe genes the PCR Products of which were and 499 bp respectively. Analysis of 22 strains demonstrated that this PCR system was specific. The detection limit of the mpcr was 1.5 CFU/ ml for Salmonella 15 CFU/ ml for Campylobacter jejuni and 15 CFU/ ml for enterohemorrhagic E.coli 0157:H7. 10 of Campylobacter jejuni and 1 of O157 H7 were detected in 226 chicken meat samples. These results indicated that the multiplex PCR -DHPLC assay can be used for specific and sensitive detection of salmonella Campylobacter jejun and enterohemorrhagic E.coli O157 H7. Key wordssalmonella Campylobacter jejun Enterohemorrhagic E.coli O157 H7 liquid chromatography Denaturing high-performance 50%,,,, : : (2006BAK02A13) : (1979-),,, ; skyxjy@yahoo.com

2 128 Biotechnology Bulletin [1] [4],,,,, (HUS) (TTP),, PCR,, (DHPLC) [2] 90%, DHPLC,, [3] Reiter s,, [5],, PCR, DHPLC,, 3 1, DNA, ; ATCC, (CMCC)(1) 42, (5% O 2 10% CO 2 85% N 2 )24 h; (5% O 2 10% CO 2 85% N 2 ), (TSA) 37, 24 h 1 Campylobacter jejuni ATCC DNA Campylobacter jejuni genomic DNA ATCC 33560D-5 1 Campylobacter coli ATCC DNA Campylobacter coli genomic DNA ATCC BAA-1061D 1 Campylobacter fetus ATCC DNA Campylobacter lari genomic DNA ATCC BAA-1060D 1 Escherichia coli ATCC Enteropathogenic E.coli ATCC Enterotoxigenic E.coli ATCC Enterohemorrhagic E.coli ATCC ! Enteroinvasive E.coli ATCC "#$% Salmonella enteritidis ATCC & (#$% Salmonella. typhimurium ATCC )*+#$% Salmonella. cholerae ATCC ,-. (#$% Salmonella. enterica paratyphi A ATCC /012 Proteus vulgaris CMCC "456% Yersinia enterocolitica ATCC Vibrio parahaemolyticus ATCC *+8 Vibrio cholerae CMCC :;<=>% Listeria innocua ATCC ?@ABCD<=>% Liateria monocytogenes ATCC EFGHIJ Staphylococcus aureus ATCC

3 : DNA PE (PerkinElmer, ); (TakaRa MiniBEST Bacterial Genomic DNA Extraction kit),taq PCR Centrifuge 5804(Eppendorf, ) ( ) ; (Campylo- 1.2 NAV (Transgenomic, ); bacter Agar Base) BD ; GenBank, Primer Premier 5.0 3, MART-II fimy gyra (Mart Microbiology B.V., ); PCR rfbe (2) 53 bp Salmonella Campylobacter jejuni Enterohemorrhagic E.coli fimy gyra rfbe ( ) GTGAAATTATCGCCACGTTCGGGCAA TCATCGCACCGTCAAAGGAACC CAAATAAAGTTAGAGGTAGAATGT CCATAAGCACTAGCTAGCTGAT ATTGCGCTGAAGCCTTTG CGAGTACATTGGCATCGTG h 41.8% A,58.2% B;6.8 min, % A,61.8% B;9.9 min,, 5 ml 8.5%, 0.5 ( CFU/ml), min;: ( :150W Xenon, CFU/ml CFU/ml CFU/ml :15 nm, :15.3 nm, 1.4 DNA 2 1 DNA, DNA 1.5 PCR 1.6 DHPLC DNA 1.5 PCR(multiplex PCR,mPCR)1.8 PCR :10 PCR μl, (10 μmol/l) 1 μl,dntp (10 mmol/ L)4 μl,taq DNA (5 U/μl)0.25 μl, DNA 2 μl, ddh 2 O 25 μl : CFU/ml, min;94 60 s,56 60 s,72 60 s,35 ;72 7 min ;4 DNA, PCR 1.6 DHPLC :PS-DVB & C18 DNASep (4.6 mm 50 mm, 3 μm); 50 : min,55% A,45% B;0.5 min, 50.2% A,49.8% B;3.6 min, 36.3% A,63.7% B;13 min, 35% A,65% B :0.9 ml/ : 350 nm 2 s); :PCR 5 μl 1.7 PCR mpcr-dhplc ATCC ATCC ATCC 35150, 1.9 PCR-DHPLC 226,

4 130 Biotechnology Bulletin , 2 3 PCRDHPLC 1 22 DNA mpc- 2.2 R-DHPLC, PCR 1.3 DHPLC 1 PCR ATCC ATCC (159 bp) ATCC 35150, (284 bp) CFU/ml, 10 (499 bp) DNA, PCR CFU/ml, mpcr-dhplc DNA, PCR-DHPLC 1.5 CFU/ml, 15 CFU/ml, 15 CFU/ml(3) 3 PCR-DHPLC PCR-DHPLC 1. ATCC33291, ATCC CFU/ml 13076, ATCC 35150;A. ;B. ;C Á ;2. ATCC 1.510Â ;3. ATCC 25922; Ã ATCC 43887;5. ATCC 35401; Ä ATCC 43893;7. CMCC 49027; Å ATCC9610;9. ATCC 1.510Æ ;10. ATCC PCR-DHPLC (min ) (A ) (min ) (B ) (min ) (C ) 2.3 PCR-DHPLC 226, PCR-DHPLC 7 mpcr-dhplc GB A. ;B. ;C. 2 3 PCR DHPLC (%) mpcr-dhplc GB

5 : , 1,, 1% 7,4 Franciosa [6],1 A B E F PCR-DHPLC, DHPLC, Hurtle [7] DHPLC, rrna, (VIDAS) (API), (Yersinia pestis) (Bacillus anthracis) (4), 2 [8] 3 DHPLC,, DHPLC, PCR-DHPLC,, PCR- PCR (VIDAS), API,, PCR-,,,, ; mpcr- PCR,, DHPLC, ;VIDAS,,, ;API, ~88. ; 2 Nataro JP Kaper JB. Clin Microbiol ~201.,,, 3 Allos BM. Clin Infect Dis ~1206. ; 4 Mckay AM. Lett Appl Microbiol ~135., 5 Colosimo A Guida V Flex E et al. Biotechniques , 706~708., 6 Franciosa G Pourshaban M De Luca A et al. Appl Environ Microbiol ~ Hurtle W Bode E Kaplan RS et al. J Clin Microbiol ~4766. (DHPLC) ~53.

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