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Korean J. Food Sci. An. Vol. 33, No. 5, pp. 610~616(2013) DOI http://dx.doi.org/10.5851/kosfa.2013.33.5.610 ARTICLE 분유에오염된 Cronobacter sakazakii 검출을위한중합효소연쇄반응, 실시간중합효소연쇄반응, 등온검출법의비교 김영주 1 서승우 왕효우 서동주 이민화 손나리 이복희 최창순 * 중앙대학교식품공학부식품영양학과, 1 중앙대학교교육대학원영양교육전공 Comparison of Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, and Loop-Mediated Isothermal Amplification for the Detection of Cronobacter sakazakii in Milk Powder Young-Joo Kim 1, Sheungwoo Seo, Xiaoyu Wang, Dong Joo Seo, Min Hwa Lee, Na Ry Son, Bog-Hieu Lee, and Changsun Choi* School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University, Anseong 456-756, Korea 1 Department of Nutrition Education, Chung-Ang University, Anseong 456-756, Korea Abstract Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompa) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompa gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk. Key words: loop-mediated isothermal amplification (LAMP), Cronobacter sakazakii, ompa, specificity, sensitivity 서 *Corresponding author: Changsun Choi, Department of Food and Nutrition, Chung-Ang University, Anseong 456-756, Korea. Tel: 82-31-670-4589, Fax: 82-31-676-8741, E-mail: cchoi@cau.ac. kr 론 Cronobacter sakazakii는 Enterobacteriaceae과속하는그람음성간균이며, 포자를형성하지않는통성혐기성균이다 (Friedemann, 2007). 1980년 Enterobacter sakazakii라고명명되었으나, 2008년에 Cronobacter species로재분류되면서 C. sakazakii로학명이변경되었다 (Iversen et al., 2008). 현재알려진 Cronobacter spp. 는 C. sakazakii, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis이다 (Iversen et al., 2008). C. sakazakii는모든연령대에서질환을일으킬수있으나, 특히생후 1개월이하의신생아, 조산아, 저체중아등의영유아에게수막염, 패혈증, 신생아괴사성장염과같은증상을일으키는매우위험한급성기회감염균이다 (Arad et al., 2001; Farmer et al., 1980; Guillaume-Gentil, 2005). 현재까지집계된바에의하면, C. sakazakii의발생율은낮으나 C. sakazakii에감염된영유아의치사율은 33-80% 이상으로매우치명적이다 (Guillaume-Gentil, 2005). 1958년영국에서처음으로 C. sakazakii 감염에의해 2명의신생아가뇌수막염으로사망한사례를보고한이후 2003년까지발생한 76건의 C. sakazakii 식중독으로 19명이사망하였다 (Iversen and Forsythe, 2003). 610

Comparison of PCR and LAMP for the detection of C. sakazakii 611 C. sakazakii균의기존검출법은세균배양, 생화학적검사, 혈청학적분석이있지만이러한방법들은시간이많이소요되고정확하지않다는문제점이있다 (Derzelle et al., 2007). 미국 Food and Drug Administration(U.S. FDA) 는 C. sakazakii의검출법으로세균분리배양법, polymerase chain reaction(pcr), real-time PCR 방법을추천하였다. 시간이많이소요되며민감도가낮은세균분리배양법에비하여 PCR 은민감도와특이도가좋으나 DNA를확인하기위해전기영동과정과전문적인기술자가필요하기때문에보편적으로사용하기어려운단점이있다 (Orlandi and Lampel, 2000). Real-time PCR은 PCR보다민감도가높고 DNA 증폭정도를실시간으로확인이가능하고전기영동과정없이형광물질을이용하여신속하고정확하게확인할수있지만그과정이정교하고복잡하며고가의장비가필요하다는단점이있다 (Liu et al. 2006; Yamazaki et al., 2008). 최근에는등온조건하에서민감하고빠르게 DNA를증폭할수있는등온증폭법 (loop-mediated isothermal amplification, LAMP) 이개발되었다 (Notomi et al., 2000). LAMP 검출법은표적유전자를증폭한다는점에서는 PCR과유사하지만, 일정한온도에서온도변화없이특정유전자의증폭이가능하기때문에반응시간을단축할수있다. LAMP 는 DNA가닥의변성반응을위해등온조건하에서 Bst DNA polymerase와표적 DNA의특정염기서열을바탕으로특이적으로디자인된 4개혹은 6개의 primer와몇가지간단한장비만으로표적유전자를다량증폭할수있다 (Notomi et al., 2000). 이러한 LAMP기법은 Campylobacter jejuni, Campylobacter coli, Mycobacterium avium, Yersinia pseudotuberculosis, Mycoplasma pneumoniae, Porphyromonas gingivalis와같은병원성미생물의신속검출기술로활용되고있어식중독병원체의조기검출예방에활용할수있을것으로생각되고있다 (Enosawa et al., 2003; Saito et al., 2005; Yamazaki et al., 2008; Yoshida et al., 2005). 따라서본연구에서는 C. sakazakii를특이적으로증폭할수있는 LAMP 기술을개발하고, C. sakazakii가오염된조제분유에서 PCR, real-time PCR, LAMP 검출법의민감도와특이도를비교함으로써현장적용성을평가하였다. 재료및방법 공시균주 C. sakazakii의 LAMP 검출법개발을위하여 C. sakazakii ATCC 29004, C. sakazakii ATCC 29544 2개의표준균주를사용하였다. 또한, 본연구에서개발한 C. sakazakii LAMP 검출법의특이도검사를위해식품으로부터분리한 35개의 C. sakazakii 균주를중앙대학교식품영양학과식품미생물연구실에서제공받았으며, Arcobacter buzleri ATCC 49616, Bacillus cereus ATCC 13061, B. cereus ATCC 10876, Escherichia coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, Listeria monocytogenes ATCC 19114, L. monocytogenes ATCC 15313, Salmonella Typhimurium ATCC 43174, Salmonella Enteritidis ATCC 43076, Staphylococcus aureus ATCC 12600, S. aureus ATCC 10768, Vibrio parahaemolyticus ATCC 4664, V. parahaemolyticus ATCC 43996 을 American Type Culture Collection(ATCC; USA) 에서구매하여음성대조군으로사용하였다. C. sakazakii균주는 Brilliance TM Enterobacter Sakazakii Agar(Oxoid, UK) 에서 37 o C 조건으로 24시간 1차배양하여단일집락을선택하였으며 Tryptic Soy Broth(TSB; Difco, Becton Dickinson, USA) 에서 1차배양과동일한조건으로 2차배양하여 DNA 추출에사용하였다. A. buzleri는 Arcobacter Selective Broth (Oxoid) 에 1.2% Agar Technical(Oxoid) 을첨가하여사용하였으며 B. cereus는 Tryptic Soy Agar(Oxoid), E. coli O157: H7은 Sorbitol McConkey Agar(Oxoid), L. monocytogenes 는 Listeria Selective Agar Base(Oxoid), Salmonella spp. 는 Rappaport-Vassiliadis Soya Peptone Agar(Oxoid), S. aureus 는 Baird-Parker Agar(Oxoid), V. parahaemolyticus는 2% NaCl이함유된 TSA에각각 1차배양하여단일집락을선택하였으며, TSB에 2차배양한후 DNA 추출에사용하였다. DNA 추출 2차배양이끝난 37개의 C. sakazakii와 13개의음성대조군균주를각각 1.5 ml 튜브에옮겨담고 100 o C 끓는물에 10분간중탕한후 ice에서냉각시켰다. 8,000 rpm에 2분간원심분리하여상층액을새로운 1.5 ml 튜브에옮겨담았으며, 이것을각균주의 DNA로사용하였다. C. sakazakii의 LAMP 검출법개발 LAMP 검출법개발을위한표적유전자로는 C. sakazakii 의병원성과상관성이잘알려진 outer membrane protein A(ompA) gene(genbank accession number: GQ845410) 을표적유전자로선정하였다. LAMP 프라이머디자인에는 Explorer V4 software(fujitsu System Solution) 를이용하였으며, C. sakazakii ompa gene을증폭할수있는 LAMP 프라이머후보군을다수확보한후예비연구에서비특이반응이없는최적의프라이머를선정하여본연구에서사용하였다 (Table 2). LAMP 검출법을수행하기위해 1 reaction buffer (New England BioLabs, USA), 20 mm dntp, Bst polymerase 8 U, inner primer인 FIP와 BIP를각각 40 pmol, outer primer 인 F3와 B3을각각 20 pmol, template DNA 2 µl로반응혼합액을구성한후 diethyl pyrocarbonate (DEPC)-treated distilled water로최종용량을 25 µl로맞추었다. 이혼합액은 MJ MINI Personal Thermal Cycler(Bio-Rad, USA) 로 64 o C에서 60분, 80 o C에서 10분간반응시켰다. LAMP product

612 Korean J. Food Sci. An., Vol. 33, No. 5 (2013) Table 1. Comparison of PCR and LAMP detection in the reference bacterial stains used in the study No. Original ID LAMP PCR No. Original ID LAMP PCR 1 C. sakazakii 1 FDA + + 26 C. sakazakii FSM 292 FDA + + 2 C. sakazakii 2 FDA + + 27 C. sakazakii FSM 293 FDA + + 3 C. sakazakii 3 FDA + + 28 C. sakazakii FSM 294 FDA + + 4 C. sakazakii 5 FDA + + 29 C. sakazakii FSM 295 FDA + + 5 C. sakazakii 6 FDA + + 30 C. sakazakii FSM 297 FDA + + 6 C. sakazakii 2.40 FDA + + 31 C. sakazakii FSM 298 FDA + + 7 C. sakazakii 2.44 FDA + + 32 C. sakazakii FSM 299 FDA + + 8 C. sakazakii 2.46 FDA + + 33 C. sakazakii FSM 300 FDA + + 9 C. sakazakii 2.47 FDA + + 34 C. sakazakii FSM 302 FDA + + 10 C. sakazakii 2.80 FDA + + 35 C. sakazakii FSM 303 FDA + + 11 C. sakazakii 2.81 FDA + + 36 C. sakazakii 29004 ATCC + + 12 C. sakazakii 2.82 FDA + + 37 C. sakazakii 29544 ATCC + + 13 C. sakazakii 2.83 FDA + + 38 A. butzleri 49616 ATCC 14 C. sakazakii 2.84 FDA + + 39 B. cereus 10876 ATCC 15 C. sakazakii 279-1 FDA + + 40 B. cereus 13061 ATCC 16 C. sakazakii FSM 262 FDA + + 41 E. coli O157:H7 43889 ATCC 17 C. sakazakii FSM 265 FDA + + 42 E. coli O157:H7 43890 ATCC 18 C. sakazakii FSM 270 FDA + + 43 L. monocytogenes 15313 ATCC 19 C. sakazakii FSM 271 FDA + + 44 L. monocytogenes 19114 ATCC 20 C. sakazakii FSM 272 FDA + + 45 S. Enteritidis 13076 ATCC 21 C. sakazakii FSM 273 FDA + + 46 S. Typhimurium 43971 ATCC 22 C. sakazakii FSM 274 FDA + + 47 S. aureus 10768 KCCM 23 C. sakazakii FSM 275 FDA + + 48 S. aureus 12600 ATCC 24 C. sakazakii FSM 287 FDA + + 49 V. parahaemolyticus 43996 ATCC 25 C. sakazakii FSM 290 FDA + + 50 V. parahaemolyticus 4664 KACC Table 2. Oligonucleotide sequences of PCR, real-time PCR, and LAMP for the detection of C. sakazakii outer membrane protein A (ompa) gene Primer Oligonucleotide sequence Reference F3 5'-CCC GGT GAA GGA TTT AAC CG-3' LAMP B3 5'-CGT CGT TAG GGA TAA AGC CG-3' BIP 5'-CAG GCC GCA CCG AAA GAT AAC AAT CGT GGA ACT GGG ACC A-3' In this study FIP 5'-TGC AAT CGC GAT AGC CGT CTT TCA AGG AAA AGC GCA TGG C-3' Real-time PCR Forward Reverse Probe 5'-CCG GAA CAA GCT GAA AAT TGA-3' 5'-TCT TCG TGC TGC GAG TTT G-3' FAM-5'-ACT CTG ACA CAC CGC GCA TTC CTG-3'-TAMRA Liu et al., 2006 는 1% agarose gel을사용하여전기영동을실시하였으며, Launch DocIT LS (UVP Advanced BioImaging Software version. 5.5.5a, UK) 로분석하였다. LAMP 검출법의특이도검사 PCR에의하여얻어지는 DNA 산물과달리 LAMP 검출법은연속적인 DNA 증폭산물을생산하므로전기영동을실시하는경우사다리 (ladder-like) 와같은유형으로나타난다. 이러한 DNA 증폭산물의특이도검정을위해서증폭부위에존재하는특이염기서열을 GeneCutter (DNASTAR) 로확인하였다. C. sakazakii ompa 증폭산물의염기서열분석에서확인된부위를 HhaI과 NruI을사용하여 restriction fragment length polymorphism(rflp) 을수행하여특이도검정을하였다. RFLP는 1 reaction buffer, 10 U NruI 또는 HhaI, 2 µg의 Bovine serum albumin(bsa), 4 µl의 LAMP product DNA로반응혼합액을구성한후 DEPC-treated distilled water를이용하여최종반응용량을 20 µl로맞추었다. RFLP는 37 o C 조건에서 2시간반응시키고, 65 o C에서 25분간반응시켜효소를불활성화하였다. 결과는전기영동후 Launch DocIT LS로분석하였다. 양성대조군과음성대조군에대한 C. sakazakii LAMP 검출법의특이도를검증하기위하여표준균주를포함한 37 개의 C. sakazakii와 13개의병원성미생물에대하여 LAMP 및 PCR을실시하였다. LAMP 검출법은본연구에서개발한 C. sakazakii LAMP 검출법을사용하였으며 PCR은다음과같이수행하였다. PCR mixture는 1 reaction buffer (Bioneer, Daejeon, Korea), 20 mm dntp, Top polymerase 1 U, template DNA 2 µl와 forward, reverse primer를각각 20 pmol씩포함하였으며, LAMP 검출법개발을위하여제작된 outer primer인 F3와 B3를 PCR primer로써사용하였

Comparison of PCR and LAMP for the detection of C. sakazakii 613 Fig. 1. Restriction fragment length polymorphism (RFLP) of LAMP DNA products using HhaI and NruI restriction enzyme. M: 100-bp DNA ladder, Lane 1: Negative control, Lane 2: C. sakazakii LAMP product. Lane 3: Digestion of C. sakazakii LAMP product with HhaI restriction enzyme, Lane 4: Digestion of C. sakazakii LAMP product with NruI restriction enzyme. 다 (Table 2). PCR 반응은 94 o C에서 5분간 DNA를변성시킨후 94 o C에서 30초, 57 o C에서 30초, 72 o C에서 30초를 1 cycle로하여총 35 cycle로반응시킨후 72 o C에서 5분간더진행시켰다. PCR product는 1% agarose gel를이용하여전기영동을실시하고 Launch DocIT LS로분석하였다. 24시간배양하고, 균수를측정하여접종액으로준비하였다. 조제분유는 0.1% bacteriological peptone water(pw) 로 20% 분유액을만들었으며, 10 ml의분유액에 C. sakazakii ATCC 29004를 7 Log, 6 Log, 5 Log, 4 Log, 3 Log, 2 Log, 1 Log, 0 Log, -1 Log CFU/mL 수준으로각각인위오염시켰다. C. sakazakii가인위접종된샘플의 DNA는 Genomic DNA Extraction kit(bioneer) 를사용하여추출하였으며, LAMP, PCR, real-time PCR을각각실시하여 C. sakazakii 의검출민감도를비교분석하였다. LAMP 및 PCR법은 C. sakazakii의특이도검사와동일하게진행하였으며, real-time PCR은 Liu 등 (2006) 이선행연구에서보고한 TaqMan probe법을이용하여검출민감도를비교하였다. 1 premix Ex Taq(Takara Biotech, China), C. sakazakii 16S-23S internal transcribed spacer(its) 를표적유전자로증폭하는 forward와 reverse primer를각각 5 µm, probe 10 µm, template DNA 2 µl로반응혼합액을구성한후 DEPC-treated distilled water를이용하여반응용량을 25 µl로맞추었다. Thermal Cycler Dice Real Time System(Takara, Japan) 을이용하여 95 o C에서 10초, 55 o C에서 20초, 72 o C에서 15초간총 45 cycle로 PCR을수행하였다 (Liu et al., 2006). 결과및고찰 LAMP, PCR, real-time PCR의분유내 C. sakazakii 검출민감도비교실제조제분유나 powder형식품에서 C. sakazakii균의검출가능성을확인하기위해시판중인조제분유를구입하여검출민감도실험을실시하였다. 접종원은표준균주인 C. sakazakii ATCC 29004로 brain heart infusion broth에 LAMP 검출법의특이도 37개의 C. sakazakii균주와 13개의음성지표균인병원성미생물을사용하여 LAMP 검출법과 PCR법의특이도를검증한결과, LAMP 검출법및 PCR법모두 37개의 C. sakazakii균주에대해서는양성반응을보였으며음성지표균에대해서는모두음성반응을보였다 (Table 1 and Fig. 2). 본 Fig. 2. Specificity of loop-mediated isothermal amplification (LAMP) for the detection of C. sakazakii using reference strains. M: 100-bp DNA ladder, Lane 1: negative control, Lane 2: Cronobacter sakazakii ATCC 29004, Lane 3: Salmonella Enteritidis ATCC 13076, Lane 4: Salmonella Typhimurium ATCC 43971, Lane 5: Arcobacter butzleri ATCC 49616, Lane 6: Listeria monocytogenes ATCC 19114, Lane 7: Listeria monocytogenes ATCC 15313, Lane 8: Vibrio parahaemolyticus KCCM 4664, Lane 9: Vibrio parahaemolyticus ATCC 43996, Lane 10: Escherichia coli O157:H7 ATCC 43889, Lane 11: Escherichia coli O157:H7 ATCC 43890, Lane 12: Bacillus cereus ATCC 13061, Lane 13: Staphylococcus aureus ATCC 12600.

614 Korean J. Food Sci. An., Vol. 33, No. 5 (2013) 연구에서얻어진 LAMP product를 GC GC 염기서열을특이적으로절단하는제한효소 HhaI으로반응시킨결과, 2개의 cutting site가존재하여 66 bp, 90 bp, 24 bp 3개분절로관찰되었다. 또한 TCG CGA, AGC GCT 염기서열을특이적으로절단하는제한효소 NruI으로반응시킨결과, 한개의 cutting site가존재하여각각 164 bp, 79 bp 2개분절로관찰되었다. 이와같이 LAMP products에대한 RFLP 결과로 C. sakazakii LAMP 검출법에의해증폭된 DNA가 C. sakazakii-specific ompa임이확인되었다 (Fig. 1). 본연구에서표적유전자로선정한 C. sakazakii의 ompa 는유전자염기서열의변이가적고, 종특이적인특징이있어선행연구에서개발한 PCR 또는 real time PCR 개발에사용되었다 (Ye et al., 2009). ompa 유전자에의하여합성되는 outer membrane protein A는그람음성장내세균의세포외막에존재하는단백질로 N-말단에 β-barrel 도메인을가지며 C-말단은 α-helix구조로양친매성구조를가지고있다 (Alex and Georg, 1998; Singh et al., 2003). E. coli나 S. Typhimurium과같은그람음성장내세균과같이 C. sakazakii균의 outer membrane protein A도인간의소장상피세포에침입과병원성에관여한다고보고되어있다 (Mohan Nair and Venkitanarayanan, 2007; Singh et al., 2003). 또한 C. sakazakii의 outer membrane protein A는병원성을가진박테리오파지나박테리오신의수용체로작용하고, 대식세포의세포자살 (apoptosis) 을막아면역계로침투하는기능이알려져있다 (Alex and Georg, 1998; Singh et al., 2003). LAMP 검출법에의한분유내 C. sakazakii 검출민감도 LAMP 검출법에의한분유에접종된 C. sakazakii의검출한계는 1CFU/mL이었다 (Fig. 3). 반면, PCR법에의한검 출한계는 10 4 CFU/mL이었으며, real-time PCR법에의한검출한계는 10 2 CFU/mL이었다. 이를통해본연구에서개발한 C. sakazakii LAMP 검출법이 PCR에비해민감도가 10,000배, real-time PCR에비해 100배높은것을확인하였다. LAMP 검출법에사용되는 Bst DNA polymerase는 5' 3' exonuclease 부분이결핍되어있어유전자의여러구역에서 primer를동시에사용하더라도 inner primer가 outer primer 에방해받지않고증폭이가능하다. 따라서 LAMP 검출법은 4개혹은 6개의 primer로원하는유전자를신속하게검출할수있으며결과에있어서 PCR에비하여높은특이도를가진다 (Nagamine et al., 2002). 최근연구보고에따르면 LAMP 검출법은형광물질을이용하거나 pyrophosphate 이온에의한 magnesium pyrophosphate의백색침전물의혼탁도를이용하여시각적인판단을할수있기때문에전기영동과정이생략할수있다 (Hara-Kudo et al., 2005; Mori et al., 2001). 또한유전자증폭반응이 60-65 o C의등온조건하에수행되기때문에고가의장비가필요없이항온수조와같은장비만있으면분석이가능하다는장점이있다 (Hara-Kudo et al., 2007). Ye 등 (2009) 은 16S-23S rdna internal transcribed spacer (ITS), ompa gene, α-1,4-glucosidase gene(glua) 세가지유전자를검출할수있는 PCR로 243개의조제분유의 C. sakazakii 오염도를조사하였고, 그결과 ompa gene에대한 PCR이민감도와특이도가가장높은것으로보고하였다. Liu 등 (2009) 은 ITS를타겟으로디자인된 4개의 primer를사용하여 LAMP를수행하였으며분유에오염된 C. sakazakii 를 10 1 CFU/mL 수준까지검출하였다고보고하였다 (Liu et al., 2009). 본연구에서개발한 C. sakazakii LAMP 검출법 Fig. 3. Sensitivity of loop-mediated isothermal amplification (LAMP) for the detection of C. sakazakii in milk powder. M: 100-bp DNA ladder, Lane 1: positive control, Lane 2: negative control, Lane 3: 7 Log 10 CFU/mL C. sakazakii in milk powder, Lane 4: 6 Log 10 CFU/mL C. sakazakii in milk powder, Lane 5: 5 Log 10 CFU/mL C. sakazakii in milk powder, Lane 6: 4 Log 10 CFU/mL C. sakazakii in milk powder, Lane 7: 3 Log 10 CFU/mL C. sakazakii in milk powder, Lane 8: 2 Log 10 CFU/ ml C. sakazakii in milk powder, Lane 9: 1 Log 10 CFU/mL C. sakazakii in milk powder, Lane 10: 0 Log 10 CFU/mL C. sakazakii in milk powder, Lane 11: -1 Log 10 CFU/mL C. sakazakii in milk powder.

Comparison of PCR and LAMP for the detection of C. sakazakii 615 은 10 0 CFU/g까지검출되어 Liu 등의연구에비해더높은민감도를나타내었다. Hu 등 (2009) 은 C. sakazakii 16S- 23S rrna gene를증폭할수있는 LAMP 검출법을개발하고, 조제분유에인위적으로오염된 C. sakazakii를검출하였다. Hu 등 (2009) 은 PCR의검출한계가 10 3 CFU/mL인데반하여, LAMP법의검출한계는 1.1 CFU/mL로민감도가 1,000배높은것으로보고하였다. Hu 등 (2009) 의선행연구는본연구에서개발한 LAMP 검출법의검출한계인 10 0 CFU/g과동등한수준이었으나, 16S-23S rrna gene은 Cronobacter species와 Enterobacter species 간의상동성이높아특이도와민감도가떨어지는제한점이있었다. 이와대조적으로본연구는 C. sakazakii 특이도가높은 ompa gene을표적으로선정하여민감도와특이도가높은 LAMP 검출법을개발하였다는장점이있다. 본연구에서개발한 C. sakazakii LAMP 검출법은민감도와특이도가높을뿐만아니라분유에오염된병원체검출에도적용이가능하였다. 특히 1시간이내오염된병원체의유전자를증폭할수있는 LAMP 검출법은 C. sakazakii 를포함하여병원성미생물에의한식중독사고발생시신속하고쉽게원인체를규명할수있는현장적용이용이한효과적인도구로사용될것으로기대된다. 요약본연구에서는영유아에게치명적인감염을일으키는 C. sakazakii에대하여 LAMP 검출법을개발하였다. LAMP법에의한 C. sakazakii의검출율은 100% 였으며 13개의음성지표군에대해서는모두음성반응을보여특이도가매우높은것으로판단되었다. 또한, HhaI과 NruI 두개의제한효소를 LAMP product에반응시킨결과, 유전자의특정염기서열이절단되는것을확인하였으며, 이를통해 LAMP 검출법에의해증폭된 DNA가 C. sakazakii-specific ompa 임을확인하였다. 조제분유에오염된 C. sakazakii를 LAMP 법으로검출시검출한계는 10 0 CFU/mL이었으며이는기존의 PCR법이나 real-time PCR법에비해 100-10,000배높은수준으로민감도가매우높은것으로판단되었다. 이와같이높은특이도와민감도를가진 LAMP 검출법은 C. sakazakii와같은급성기회감염균이나병원성미생물에의한식중독발생시현장에서병원체를간편하고신속하게검출할수있는기술로기대된다. 감사의글본연구는농림수산식품부농림수산식품기술기획평가원수산실용화연구사업 (#112089-3) 지원에의하여이루어진것임. 참고문헌 1. Alex, P. and Georg, E. S. (1998) Structure of the outer membrane protein A transmembrane domain. Nat. Struct. Biol. 5, 1013-1017. 2. Arad, I., Baras, M., Gofin, R., Bar-Oz, B., and Peleg, O. (2001) Does parity affect the neonatal outcome of very low birth weight infants. Eur. J. Obstet. Gynecol. Reprod. Biol. 94, 283-288. 3. Derzelle, S., Dilasser, F., Maladen, V., Soudrie, N., Leclercq, A., Lombard, B., and Lafarge, V. (2007) Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories. J. Food Prot. 70, 1678-1684. 4. Enosawa, M., Kageyama, S., Sawai, K., Watanabe, K., Notomi, T., Onoe, S., Mori, Y., and Yokomizo, Y. (2003) Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis. J. Clin. Microbiol. 41, 4359-4365. 5. Farmer, J. J., Asbury, M. A., Hickman, F. W., and Brenner, D. J. (1980) Enterobacter sakazakii: A new species of Enterobacteriaceae isolated from clinical specimens. Int. J. Syst. Bacteriol. 30, 569-584. 6. Friedemann, M. (2007) Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int. J. Food Microbiol. 116, 1-10. 7. Guillaume-Gentil, O., Sonnard, V., Kandhai, M. C., Marugg, J. D., and Joosten, H. (2005) A simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples. J. Food Prot. 68, 64-69. 8. Hara-Kudo, Y., Nemoto, J., Ohtsuka, K., Segawa, Y., Takatori, K., Kojima, T., and Ikedo, M. (2007) Sensitive and rapid detection of vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J. Med. Microbiol. 56, 398-406. 9. Hara-Kudo, Y., Yoshino, M., Kojima, T., and Ikedo, M. (2005) Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol. Lett. 253, 155-161. 10. Hu, L., Zhang, W., Zhang, X., Yuan, Y., Zhang, Y., Zhang, H., Ma, X., and Su, X. (2009) Development of a loop-mediated isothermal amplification assay for rapid detection of Enterobacter sakazakii in powdered infant formula. Wei Sheng Wu Xue Bao. 49, 378-382. 11. Iversen, C. and Forsythe, S. J. (2003) Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula. Trends Food Sci. Tech. 14, 443-454. 12. Iversen, C., Mullane, N., McCardell, B., Tall, B. D., and Lehner, A. (2008) Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and

616 Korean J. Food Sci. An., Vol. 33, No. 5 (2013) Cronobacter dublinensis subsp. lactaridi subsp. nov. Int. J. Syst. Evol. Microbiol. 58, 1442-1447. 13. Liu, C., Zheng, W., Zhang, H., Hou, Y., and Liu, Y. (2009) Sensitive and rapid detection of Enterobacter sakazakii in infant formula by loop-mediated isothermal amplification method. J. Food Safety 29, 83-94. 14. Liu, Y., Cai, X., Zhang, X., Gao, Q., Yang, X., Zheng, Z., Luo, M., and Huang, X. (2006) Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. J. Microbiol. Meth. 65, 21-31. 15. Mohan Nair, M. K. and Venkitanarayanan, K. (2007) Role of bacterial OmpA and host cytoskeleton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii. Pediatr. Res. 62, 664-669. 16. Mori, Y., Nagamine, K., Tomita, N., and Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150-154. 17. Nagamine, K., Hase, T., and Notomi, T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probe. 16, 223-229. 18. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63. 19. Orlandi, P. A., and Lampel, K. L. (2000) Extraction-free, filter-based template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. J. Clin. Microbiol. 38, 2271-2277. 20. Saito, R., Misawa, Y., Moriya, K., Koike, K., Ubukata, K., and Okamura, N. (2005) Development and evaluation of a loopmediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae. J. Med. Microbiol. 54, 1037-1041. 21. Singh, S. P., Williams, Y. U., Miller, S., and Nikaido, H. (2003) The C-terminal domain of Salmonella enterica serovar typhimurium ompa is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infect. Immun. 71, 3937-3946. 22. Yamazaki, W., Taguchi, M., Ishibashi, M., Kitazato, M., Nukina, M., Misawa, N., and Inoue, K. (2008) Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J. Med. Microbiol. 57, 444-451. 23. Ye, Y., Wu, Q., Yao, L., Dong, X., Wu, K., and Zhang, J. (2009) A comparison of polymerase chain reaction and international organization for standardization methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets. Foodborne Pathog. Dis. 6, 1229-1234. 24. Yoshida, A., Nagashima, S., Ansai, T., Tachibana, M., Kato, H., Watari, H., Notomi, T., and Takehara, T. (2005) Loopmediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. J. Clin. Microbiol. 43, 2418-2424. (Received 2013.4.26/Revised 2013.8.17/Accepted 2013.8.22)