1、KSKSKSKS SKSKSKS KSKSKS SKSKS KSKS SKS KS 2006 12 21 http:/www.kats.go.krKS H 1208 KS H 1208： 2006 (2011 )H 1208： 2006 : ( ) ( ) BK ( ) : (http:/www.standard.go.kr) ： ： 2006 12 21 ： 2011 12 29 2011-0654 ： ： ( 02-509-7273) (http:/www.kats.go.kr). 10 5 , . ICS 07.100 KS H 1208： 2006(2011 ) Detection m
2、ethods for antimicrobial resistance and virulence genes in lactic acid-producing bacteria 1. . , . 2. (1) (2) . (1) esp, gelE, agg, efaAfs, efaAfm, cylA . (2) vanA, vanB . 3. . . 3.1 KS M 0126 ( ) KS M 10100 (PCR) KS M 10200 3.2 CLSI(Clinical and Laboratory Standards Institues) MM1 A2 Molecular Diag
3、nostic Methods for Genetic Diseases： Approved Guideline(2006) MM3 A2 Molecular Diagnostic Methods for Genetic Diseases： Approved Guideline(2006) 4. . KS M 10200, KS M 0126 . 4.1 (probiotic) . 4.2 (culture medium) . . 4.3 (blood agar) TSA(Trypticase Soy Agar)(3) 5 % . (3) (trypsin) , BBL Trypticase s
4、oy agar H 1208： 2006 2 . 4.4 (Polymerase Chain Reaction：PCR) (Deoxyribonucleic Acid： DNA) DNA DNA . PCR (denaturation), (annealing), (extension) , DNA . PCR DNA , , dNTP, Taq (Taq polymerase) , PCR . 4.5 PCR (denaturation) PCR , 94 1 . DNA DNA DNA DNA . 4.6 PCR (annealing) DNA . DNA . PCR . DNA , .
5、Tm = (4G C) (2A T) , . 4.7 PCR (extension) DNA 72 2 . Taq . 4.8 (primer) DNA DNA . PCR 1730 (mer) . 4.9 (mer) (single strand) DNA . 4.10 (base pair：bp) (double strand) DNA . 5. PCR PCR KS M 10100 (PCR) . 6. PCR 6.1 PCR . (Glycopeptide) (vancomycin) (teicoplanin) . vanA vanB . esp , , , gelE , agg ,
6、efaAfm efaAfs , (cytoly sin) cylA . 6.2 6.2.1 (Enterococcosel) . (esculine) H 1208： 2006 3 . 6.2.2 (cell lysis buffer) 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 0.5 % (sodium dodecyl sulfate), 100 g/mL (protease K), 10 U (mutanolysin) . 6.2.3 CTAB(Cetyltrimetyl Ammonium Bromide) 0.7 M NaCl, 10 % CT
7、AB . 6.2.4 TE(Tris-EDTA) 10 mM Tris, 1 mM EDTA, pH 8.0 . 6.2.5 1 . 1 DNA 5-GCT ATT CAG CTG TAC TC-3 vanA 5-CAG CGG CCA TCA TAC GG-3 783 bp 5-CAT CGC CGT CCC CGA ATT TCA AA-3 vanB 5-GAT GCG GAA GAT ACC GTG GCT-3 297 bp 5-TTG CTA ATG CTA GTC CAC GAC C-3 esp 5-GCG TCA ACA CTT GCA TTG CCG AA-3 933 bp 5-
8、ACC CCG TAT CAT TGG TTT-3 gelE 5-ACG CAT TGC TTT TCC ATC-3 419 bp 5-AAG AAA AAG AAG TAG ACC AAC-3 agg 5-AAA CGG CAA GAC AAG TAA ATA-3 1 553 bp 5-GAC AGA CCC TCA CGA ATA-3 efaAfs 5-AGT TCA TCA TGC TGT AGT A-3 705 bp 5-AAC AGA TCC GCA TGA ATA-3 efaAfm 5-CAT TTC ATC ATC TGA TAG TA-3 735 bp 5-TGG ATG AT
9、A GTG ATA GGA AGT-3 cylA 5-TCT ACA GTA AAT CTT TCG TCA-3 517 bp 6.2.6 3(dNTPs) DNA PCR 3 3 (dATP), 3 (dCTP), 3 (dGTP), 3 (dTTP) . . 6.2.7 DNA (thermostable DNA polymerase) PCR DNA Taq (Taq polymerase) . DNA dNTPs (phosphodiester) DNA 5 3 . 6.2.8 PCR (PCR buffer) PCR DNA , pH , 10 X PCR . . 6.2.9 (ag
10、arose gel) DNA 1.0 % (agarose) . 1.0 % 0.5 X TBE . 6.2.10 0.5 X Tris-Borate EDTA (0.5 X TBE) 44.5 mM Tris, 44.5 mM Boric acid, 1 mM EDTA H 1208： 2006 4 . 6.3 37 48 . . 6.4 PCR 6.4.1 DNA DNA CTAB . 600 L 37 2 . 5 M NaCl 100 L CTAB 80 L 65 10 , 5 . DNA ： (24： 1) DNA . 30 DNA , 70 %(v/v) TE DNA PCR . D
11、NA 260 nm 280 nm (A260/A280) 1.8 DNA PCR DNA . 1.8 , . 6.4.2 PCR 14.3 L, 10 X PCR 2.5 L, dNTP 2.0 L, 1 0.5 L, Taq 1 U PCR PCR DNA 5 L . 6.4.3 PCR PCR PCR 2 , , 30 . 2 PCR vanA 95 52 72 vanB 95 52 72 esp 95 63 72 gelE 95 52 72 agg 94 52 72 efaAfs 94 52 72 efaAfm 94 52 72 cylA 94 57 72 6.4.4 PCR PCR D
12、NA . VanA VanB . 6.4.5 DNA 0.5 X TBE 1.0 % . (well) (gel loading dye) (bromophenol blue) PCR 5 L 23 cm 100 V 25 . 600 ng/mL (ethidium bromide) 10 . UV DNA . 100 bp (marker) 3 L DNA . H 1208： 2006 5 () () ( ) 8 , . 8 (Culture Collection of Antimicrobial Resistant Microbes： CCARM) 6 (CCARM 5007, CCARM
13、 5014, CCARM 5015, CCARM 5016, CCARM 5021, CCARM 5022) (Korean Collection for Type Cultures： KCTC) 1 (KCTC 5292), (American Type Culture Collection： ATCC) 1 (ATCC 29212) . A.1 vanA, vanB VanA CCARM 5007 CCARM 5021 783 bp DNA vanA . VanB CCARM 5014, 5015, 5016 CCARM 5022 297 bp DNA vanB . 5007 En.fae
14、cium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 En.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 M 100 bp DNA ladder A.1 vanA, vanB 500 bp 1.0 kb 1.5 kb5007 5014 5015 5016 5021 5022 5292 2
15、9212 M H 1208： 2006 6 A.2 esp CCARM 5007, 5014, 5015, 5016 KCTC 5292 933 bp DNA esp . 5007 En.faecium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 En.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC
16、29212 M 1 kb DNA ladder A.2 esp 1.0 kb3.0 kb 5007 5014 5015 5016 5021 5022 5292 29212 M H 1208： 2006 7 A.3 gelE CCARM 5021, CCARM 5022, KCTC 5292, ATCC 29212 419 bp DNA gelE . M 1 kb DNA ladder 5007 En.faecium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 501
17、6 5021 En.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 A.3 gelE M 5007 5014 5015 5016 5021 5022 5292 29212 1.0 kb 3.0 kb H 1208： 2006 8 A.4 agg CCARM 5021, 5022 1 553 bp DNA agg . M 100 bp DNA ladder 5007 En.faecium CCARM 5007 5014 En.faeciu
18、m CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 En.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 A.4 agg 500 bp M 5007 5014 5015 5016 5021 5022 5292 29212 1.0 kb 1.5 kb H 1208： 2006 9 A.5 efaAfm CCARM 5007, 5014, 5015,
19、 5016 735 bp DNA efaAfm . 5007 En.faecium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 En.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 M 1 kb DNA ladder A.5 efaAfm 1.0 kb 3.0 kb5007 5014 50
20、15 5016 5021 5022 5292 29212 M H 1208： 2006 10 A.6 efaAfs CCARM 5021, CCARM 5022, KCTC 5292, ATCC 29212 705 bp DNA efaAfs . M 100 bp DNA ladder 5007 En.faecium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 En.faecalis CCARM 5021 5022 En.faecalis CCA
21、RM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 A.6 efaAfs 500 bp 1.5 kb M 5007 5014 5015 5016 5021 5022 5292 29212 H 1208： 2006 11 A.7 cylA ATCC 29212 517 bp DNA cylA . 5007 En.faecium CCARM 5007 5014 En.faecium CCARM 5014 5015 En.faecium CCARM 5015 5016 En.faecium CCARM 5016 5021 E
22、n.faecalis CCARM 5021 5022 En.faecalis CCARM 5022 5292 En.faecalis KCTC 5292 29212 En.faecalis ATCC 29212 M 100 bp DNA ladder A.7 cylA 500 bp 1.0 kb 5007 5014 5015 5016 5021 5022 5292 29212 M 153787 1 92 3(13) (02)26240114 (02)2624 01489 http:/ KSKSKSSKSKS KSKS SKS KSKS SKSKS KSKSKSKorean Agency for Technology and Standards http:/www.kats.go.kr KS H 1208： 2006 Detection methods for antimicrobial resistance and virulence genes inlactic acid-producing bacteriaICS 07.100
