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1 대한임상검사학회지 : 38 권제 3 호, , 2006 rpob 유전자의 PCR-RFLP 를이용한 Mycobacterium 균종동정의유용성 미육군 121 병원진단검사의학과 1, 서울보건대학임상병리과 2 유경래 1 박정오 2 Identification of Mycobacterium species by rpob Gene PCR-RFLP Kyong-Nae Yu 1 and Chung-Ho Park 2 Department Pathology 121st General Hospital U.S. Army, Seoul , Korea 1 Department of Biomedical Laboratory Science, Seoul Health College, Sungnam , Korea 2 Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rdna, rpob, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(pcr-rflp) based on the region of the rpob gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as MspⅠ in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpob gene PRA method. The PCR-RFLP method based on the rpob gene is a simple, rapid, and accurate test for the identification of Mycobacterium. Key words : PCR-restriction fragment length polymorphism analysis rpob gene, MspⅠ 1)I. 서 론 Mycobacterium 속균종중에서결핵균은전세계적으 로심각한보건상의문제를야기하는병원성이강한균종이다 ( 김등, 2002; Fitzgetrald 등. 2003). 1980년대이후 교신저자 : 유경래, ( 우 ) 미육군 121 병원진단검사의학과 Tel : ryoukr@nate.com 부터는비결핵 Mycobacteria 감염증이계속해서증가되고있다 (Guthertz 등, 1998; Ruiz 등, 2002). 이들은항산성, 호기성으로일부균주를제외하고는매우느리게성장하는균종들로써약 70여종의다른종으로구성되어있으며이중 30여종이인간에게각종심각한질환을야기한다고보고되고있다 (Cloud 등, 2002; Collins 등, 2003). 전통적인방법은균주의성장속도, 색조, 균주형태, 현미경적형태관찰및생화학적인검사를시행한다 (Shinners 158

2 등, 1999). Niacin, catalase, nitrate 등을이용한생화학적인방법은균분리이후 4~6주의시간이소요된다 (Collins 등, 1990). 그러나결핵균의신속한동정법이개발됨에따라 mycobacteria를동정하는전통적인방법은그효용이감소되고있다. 현재결핵균을동정하는모든검사실은신속한동정법을이용하도록권고되고있다. 신속한동정법으로는 DNA probe 이용, PCR, DNA sequencing, 그리고 chromatography와같이균의구성성분을물리화학적방법으로직접검출하는법이있다 ( 김등, 2002; Bannantine 등, 2002; Huard 등, 2003). BACTEC 12B system은 p-nitroacetyl-p- aminohydroxypropiophenone(nap) 을이용하여 Mycobacterium tuberculosis(mtb) complex와 nontubercuosis Mycobacterium (NTM) 를 5~6일만에감별할수있다 (Alcaide 등, 2003). 결핵균의신속한동정법이개발됨에따라전통적인동정법은그효용이감소되고있다. 현재결핵균을동정하는모든검사실은신속한동정법을이용하도록권고되고있다. BACTEC 12B system은 NAP을이용하여 TB complex와 NTM를 5~6일만에감별할수있다. 그러나일부 NTM도 NAP에의하여억제될수있으므로 probe나생화학적인방법으로확인하여야한다. 최근 DNA 증폭기술의발달로개발된분자유전검사법은, Mycobacterium 균속내여러부위의특이적인특정유전자를이용하여보다빠르고정확한진단을위한유전자증폭및 probe hybridization, 염기서열분석등과같은여러검사방법이시도되고있다 (Kim 등, 1997). 이와같은진단법에이용되는표적유전자 (target gene) 에는 hsp65, 16S rrna, 그리고 dnaj 등여러유전자들이사용되고있다 (Lee 등, 1998; Kim 등, 1999; Lee 등, 2000; Braccolo 등, 2003). 하지만이들의방법은결과가정확하고반복적이라는장점이있지만고난이도의기술과여러가지의효소처리로분석이쉽지않은단점들도있다. II. 재료및방법 1. 대상 2005년 6월부터 8월사이에삼광의료재단검사실에의뢰된임상검체로부터분리배양된 mycobacteria 13주와한국결핵연구원 (KIT) 으로부터분양한표준균주 7주 (Table 1), 총 20주에대하여분석하였다. 13주의임상분 Table 1. Reference strains of mycobacterial species used in this study Species Strain Source M. avium ATCC a KIT b M. chelonea ATCC KIT M. godonae ATCC KIT M. kansasii type Ⅰ Pasteur institute KIT M. smegmatis ATCC KIT M. celatum type Ⅰ ATCC KIT M. absessus Pasteur institute YUMC c M. tuberculosis ATCC KIT a ATCC, American type culture collection b KIT, Korea institute of tuberculosis c YUMC, Yonsei university college of medicine 리주는생화학적검사로균동정을확인하였다. 각균주는전통적인감별방법인 Niacin strip test와 Gen-Probe test을실행하여결핵균의여부를재평가하였으며, 불일치균주에대해서는 PCR을통한확인실험을실시하였다. 2. 핵산추출과중합효소연쇄반응 PCR 증폭을위한유전자준비는가열파쇄유전자추출법을사용하여 DNA를추출하였다. Lowenstein-Jensen medium에서배양된균주의집락을증류수 400 μl와 screw-cap microcentrifuge tube에넣어 5분간 boiling 하여균체를파쇄하였다. 균파쇄액은 rpm으로 5분간원심분리하고 DNA가함유된상청액 100 μl를상청액을 PCR에사용하였다. 마이코박테리아균종들의 rpob 유전자를증폭하기위한프라이머는 rpob 유전자염기서열 (GenBank accession No. P47766) 내의첫번째과변부위 (V1) 와두번째상보적부위 (conserved region2; C2) 중대장균 (Escherichia coil) 의유전적정보를바탕으로 V1의 171 bp와 C2의 189 bp 부위에해당하는부분을표적으로하여제작하였다. 한쌍의프라이머는 5'-TCAAGGAGAAGCCGTACGA-3'(RPO5') 와 5'-GGATGTTGATCAGGTCTGC-3'(RPO3') 로유전자내 902번째염기에서부터 1261번째염기까지 360 bp를증폭하였다. PCR 반응은 10 pmol의각각의 primer, 2 mm MgCl 2, 200 mm deoxynucleoside triphosphate, 1 U 의 DyNAzyme Ⅰ DNA ploymerase(finnzymesy, Espoo, Finland) 가들어있는 DNA PCR Premix(M&D, Wonju, 159

3 Korea) 를이용하여 genomic DNA 10 μl와멸균증류수 40 μl를혼합하여최종부피가 50 μl가되도록혼합물을만들었다. 첫번째 pre-denatruation 과정은 94 에서 5분실시후에, denaturation 94 20초, annealing 58 20초, elongation 72 30초로 35회반복하였으며, 최종 elongation 72 에서 10분간실시하였다. Thermocycler는 model 2700 PCR system(aplied Biosystem, Fostercity, CA) 가사용되었다. 각 PCR 과정에서 positive, negative control, PCR size maker(m&d, Wonju, Korea) 를항상사용되었다. 양성대조로는 M. bovis 표준균주를 PCR mixture에사용하였고, 음성대조로증류수를사용하였다. 증폭산물은 2% agarose gel 위에서전기영동을한후에 ethidium bromide(etbr) 염색을통해확인하였다. 3. Restriction fragment length polymorphism 마이코박테리움속분류를위하여위 PCR 증폭산물을 MspⅠ(Boehringer Manheim biochemicals, Mannheim, Germany) 을이용하여소화하고 RFLP 분석을실시하였다. 증폭된 PCR 산물 15 μl(genomic DNA 1.5 μg) 에 MspⅠ(10 U/μL) 0.5 μl, 10x MspⅠ buffer 2 μl, 멸균된증류수 2.5 μl를넣어 20 μl의혼합물을잘섞어주고, 12,000 rpm에서 3~5초간원심분리하고 37 항온수조에서 2시간반응하였다. 2 μl의 loading buffer(0.25% bromophenol blue, 40% sucrose) 를각검체에첨가한후, PCR-RFLP DNA size marker(m&d, wonju, Korea) 와함께 4% metaphore agarose gel(fmc Bioproducts, Rockland, Maine) 에잠적한후에 100 V에서 60~75분간전기영동탱크에얼음을담아전기영동하였다. Gel을 EtBr 염색한후, UV transilluminator로분절편들의결과를확인하였다. 결과판독은 Mycobacteria- Nocardia 동정알고리즘 (Fig. 4) 을참고하여균을동정하였다. III. 결과 1. rpob 유전자증폭및제한효소분절법을이용한표준균주동정마이코박테리움동정에있어서보다신속, 정확, 간편한방법을개발하기위하여여러표적유전자를이용한 유전자증폭법이연구되어지고있다. 여기서이용한표적유전자 rpob gene은유전자염기서열분석을통하여 PCR-RFLP 법으로마이코박테리움동정에적합한유전자구간으로확인되었다. rpob gene PCR을통한 Mycobacterium의표준균주에대한동정가능여부를확인하기위하여대표적인표준균주 8주 (Table 1) 에대하여 DNA 과변부위의증폭으로얻어진산물에서의다형태적특성을기초로유전자증폭효소분절분석법을사용하였다 (Fig. 1). 600 bp 500 bp 400 bp 300 bp 200 bp M N P Fig. 1. Results of PCR amplification of the rpob gene using mycobacterial reference strains. PCR products were run on a 2% agarose gel. Lanes; M, DNA size marker; 1, M. avium; 2. M. chelonea; 3, M. godonae; 4, M. kansasii Type I; 5, M. smegmatis; 6, M. celatum; 7, M. abscessus; N, negative control; P, positive control M. tuberculosis. 증폭된유전자를효소분절분석을위하여사용된 Msp Ⅰ 은이미 M. tuberculosis, M. leprae, and M. smegmatis 의 rpob gene 염기서열분석정보를통하여선정되었다. Msp Ⅰ는 DNA 염기서열중 5'-C CGG-3', 3'-GGC C-5' 에서 forward sequence의 C와 C사이와상보적인 reverse sequence의 C와 C사이를 sticky형태로분절시키는효과가있다. 효소처리를통한 RFLP 분석법에의하여표준균주 8주에대하여동정한결과 4% metaphore agarose gel 위에 Mycobacterium. avium은 PCR product가 105, 80, 50, 45-bp로각각분절되어 M. avium만의고유한분절편을형성하여동정이가능하였다 (Fig. 2, Table 2). rpob gene으로항산균동정의특이성을확인한후삼광의료재단의임상검사실에서임상검체로부터분리, 배양, 동정된임상표준균주 13주에대해서도 PCR- restriction fragment length ploymorphism analysis (PCR- RFLP) 특이도와정확도를빠르고간편하게 Mycobacterium species 를동정할수있었다 160

4 Table 2. PCR-RFLP profiles obtained from reference strains of mycobacteria used in this study Reference strain of Mycobacterium sp. Strain DNA fragment size(bp) Species identified by PCR-RFLP algorithm M. avium ATCC , 80, 50, 45 M. avium M. chelonae ATCC , 95, 80, 50, 40 M. chelonae M. gordonae ATCC , 80, 45 M. gordonae M. kansasii type Ⅰ Pasteur institute 175, 60, 45, 40 M. kansasii type Ⅰ M. smegmatis ATCC , 90 M. smegmatis M. celatum type Ⅰ ATCC , 95, 45 M. celatum type Ⅰ M. abscessus Pasteur institute 105, 95, 80 M. abscessus M. tuberculosis ATCC , 80, 60, 40 M. tuberculosis 350 bp M N M P M 250 bp 200 bp 175 bp 150 bp 125 bp 105 bp 95 bp 80 bp 60 bp 50 bp 45 bp 40 bp Fig. 2. Results of PCR-RFLP analysis of mycobacterial reference strains. A set of PCR primers, RPO5' and RPO3' was used. Amplified DNAs were digested with MspⅠand run on a 4% metaphore agarose gel. Lanes: M, PCR-RFLP size marker; 1, M. avium; 2. M. chelonea; 3, M. gordonae; N, negative control; 5, M. kansasii Type I; 6, M. smegmatis; 7, M. celatum; 8, M. abscessus; P, positive control, M. tuberculosis 이와같은결과는단일효소 MspⅠ을이용한 PCR-RFLP 법으로임상검사실에서분리되어지는거의모든항산균을분리동정할수있는것을확인하였다 (Fig. 3, Table 3). Ⅳ. 고찰 Mycobacteria의배양과동정은성장이매우느리고각균명을정확히동정하는데많은시간과노력이소요되어, mycobacteria 감염증치료에적절한검사정보를제공하지못하고있다. 특히최근에증가되는 AIDS 환자와비결핵성 mycobacteria 감염증과의연관성이밝혀짐에따라 (Guthertz 등, 1998), Mycobacterium의균속동정에더욱더많은연구가필요되고있다. 이러한이유로보다신속하고정확한방법으로 mycobacteria를동정하고자하는연구가진행되어여러가지검사법이소개되었다. 제한효 소와 DNA probe를이용한방법 (Collins 등, 1990), 각균종에특이적으로반응하는 mycobacteriophage 기법 (Alcaide 등, 2003), DNA sequencing 법 (Cloud 등, 2002), 그리고 PCR-RFLP 법 (Kim 등, 1999; 김등, 2002) 등이소개되었으며, 이중 DNA sequencing 법은표준검사법으로정착하고있고 PCR-RFLP 법은임상검사로정착하고있다. 최근에는 real-time PCR 법 (Broccolo 등, 2003) 이연구되고있어보다쉽고정확하게 mycobacteria 균속을동정할수있게될것이다. 분자유전학적인방법으로 mycobacteria 균속의동정을하기위해서는각균종간변이가심한유전자를선택하는것이중요하다. 전통적으로는 16S rdna 유전자가많이사용되었다 (Cloud 등, 2002) 그리고 rpob 유전자 (Kim 등, 1999) 등이시도되었으며 PCR-RFLP 분석을위해서는 rpob 유전자가가장많이이용되고있다 (Lee 등, 2000; 김등, 2002). RFLP 분석에서제한효소의선택은검사의특이도를결정하는가장 161

(A) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 600 500 400 300 200 (B) 350 bp 250 bp 200bp 175 bp 150bp 125 bp 105 bp 95 bp 80 bp M 1 2 3 4 M 5 6 N 7 8 M 350 bp 250 bp 200 bp 175 bp 150 bp 125 bp 105 bp

Clincal isolates of mycobacteria were used for PCR amplification using primers RPO5' and RPO3'. PCR products were run on a 2% agarose gel.

MspⅠdigestion of PCR products were run on a 4% metaphore agarose gel. Lane M, M&D PCR-RFLP DNA size marker, lanes 1-14, clinical isolates of mycobacteria, lane 15. negative control. Table 3.

5 (A) M (B) 350 bp 250 bp 200bp 175 bp 150bp 125 bp 105 bp 95 bp 80 bp M M 5 6 N 7 8 M 350 bp 250 bp 200 bp 175 bp 150 bp 125 bp 105 bp 95 bp 80 bp M M M 60bp 60 bp 50 bp 50 bp 45 bp 45 bp 40 bp 40 bp Fig. 3. PCR-RFLP analysis using rpob gene for species identification of clinical isolates of mycobacteria. (A). Clincal isolates of mycobacteria were used for PCR amplification using primers RPO5' and RPO3'. PCR products were run on a 2% agarose gel. M, DNA size marker; Lane M, M&D PCR DNA size marker, lanes 1-14, clinical isolates of mycobacteria, lane 15, negative control. (B), PCR-RFLP analysis of PCR products shown in (A). MspⅠdigestion of PCR products were run on a 4% metaphore agarose gel. Lane M, M&D PCR-RFLP DNA size marker, lanes 1-14, clinical isolates of mycobacteria, lane 15. negative control. Table 3. PCR-RFLP profiles obtained from clinical isolates of mycobacteria used in this study Species identified by biochemical test DNA fragment size(bp) M. ulcerans 105, 70, 60, 42, 40 M. ulcerans Species identified by PCR-RFLP algorithm M. kansasii typeⅡ 195, 80, 70, 60 M. kansasii typeⅡ M. malmoense 110, 75, 70, 60, 45 M. malmoense M. cleatum typeⅡ 175, 140, 42 M. cleatum typeⅡ M. intracellulare typeⅡ 150, 100, 80 M. intracellulare typeⅡ M. gordonae Ⅳ 175, 80, 45 M. gordonae Ⅳ M. tuberculosis 175, 80, 60, 40 M. tuberculosis M. avium 105, 80, 50, 45 M. avium M. godonae type Ⅲ 160, 95, 45 M. godonae type Ⅲ M. celatum type Ⅰ 150, 95, 47, 42 M. celatum type Ⅰ M. gordonae typeⅡ 150, 80, 45 M. gordonae type Ⅱ M. abscessus 190, 120, 42, 40 M. abscessus M. avium 105, 80, 50, 45 M. avium M. tuberculosis 175, 80, 60, 40 M. tuberculosis 162

6 Msp I Strains Hae III Other enzymes N. atitidiscaviarum 190 M. gastri 180/ M. smegmatis 200/90/50 R. erythropolis R. rhodochrous N. nova 250/ R. equi 200/95 70 N. nova M. kansasii type II 210/100 N.salmonicida N. vaccinii M. kansasii type III 210/100 M. kansasii type IV 180/ M. gallinarum 200/ M. celatum type II 210/95/ N. rhodnii 70 N. seriole M. fortuitum type I 180/ M. gordonae type III R. erythropolis M. intracellulare type I 180/90 70 N. violacefusca 90 M. austroafricanum 200/90 Sau 3A1 Xcm I TB/M. bovis 250/ /70 200/120/45 M. microti 250/ /70 240/120 M. africanum 250/ /90/70 200/120/45 45 M. szulgai 200/115 M. gordonae type IV M. kansasii type V 175/55/ M. kansasii type I 205/ M.leprae M. intracellulare II M. xenopi M. marinum N. brasiliense 150/90/80 Kpn I M. celatum typei 210/95/90 175/ M. gordonae type I 210/95/ M. gordonae type II N. pinensis M. malmoense 190/ M. terrae 195/ M. terrae Bst EII M. abscessus 130/100/90 145/ M. fortuitum type II 120/90/80 M. peregrinum M. simiae 180/110 M. genavense 150/100 Bst EII M. chelonae 130/100/90 225/ M. ulcerans 210/80/ M. asiaticum M. avium M. flavescens 200/85/ N. species 65 M. thermoresistibile 200/ M. phlei 210/90 M. moriokaense 200/ M. pulveris 250/ M. haemophilum M. marinum 200/ N. carnea 75 N. flavorozea N. faracinica Fig. 4. The algorithm used the species identification of mycobacteria in this study(lee, 등, 2000) 163

7 중요한변수이다. rpob 유전자의각균종간변이가많은부위에 Msp I 제한효소자리가분포하여 Mycobacterium 균종간차이를잘표현하는것으로알려졌다 (Lee 등, 2000). MspⅠ는 DNA 염기서열중 5'-C/CGG-3' 부위의 C 와 C사이를 sticky 형태로분절시킨다. 본연구에서임상검체에서가장흔하게분리되는 8종의 Mycobacterium 표준균주는상호교차반응이나해석상의어려움이없이정확하게감별되었다. 또한 13주의임상균주도동일한성적을보였다. Kim 등 (1999), Lee 등 (2000), 그리고김등 (2002) 의결과와큰차이가없었으며현재임상검사실에서결핵균동정진단에가장널리사용되고있는방법은 Gen-Probe로전통적인방법인 niacin 시험보다빠르고정확하다고평가되고있다. 그러나비용이많이들고비결핵균성 mycobacteria의감별에는적용하기가어려워 M. tuberculosis와 NTM과의감별검사로만사용되고있는실정이다. 따라서여러검사실에서 NTM의감별동정을위해검사를참고검사실 (reference laboratory) 로의뢰하는실정이다. rpob 유전자를대상으로하는 PCR-RFLP 방법은최근 Kit system으로개발되면서검체및시약조작의편이성, 결과의정확성및재현성을높여서임상검사실에서분리된결핵균집락을이용하여쉽게이용할수있도록하였다. 이방법은앞으로임상검사실에서도 NTM 동정에쉽게적용될것으로기대된다. 검체로부터직접 PCR-RFLP 를적용하여 mycobacteria를동정하는방법을적용하면, 상재균으로인하여해석상문제가생기는검체를제외하고는, 매우유용한적용방법이될것으로도기대한다. 참고문헌 1. Alcaide F, Gali N, Dominguez J, Berlanga P, Blanco S, Orus P, Martin R. Usefulenss of a New Mycobacteriophage-Based Technique for Rapid Diagnosis of Pulmonary Tuberculosis. J Clin MIcrobiol 41: , Bannantine JP, Baechler E, Qing Z, LingLing Li, Kapur V. Genome Scale Comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium Reveals Potential Diagnostic Sequence. J Clin MIcrobiol 40: , Broccolo F, Scarpellini P, Locatelli G, Zingale A, Brambilla AM, Cichero P, Sechi L A, Lazzarin A, Lusso P, Maltati MS. Rapid diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR assay. J Clin MIcrobiol 41: , Cloud JL, Neal H, Rosenberry R, Turenne CY, Jama M, Hillyard DR, Carroll KC. Identification of Mycobacterium spp. by using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries. J Clin MIcrobiol 40: Collins DM, Gabric DM, de Lisle GW. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endounclease and DNA hybridization J Clin MIcrobiol 28: , Collins DM, Zoete MD, Cavaignac SM. Mycobacterium avium subsp. paratuberculosis Strains from Cattle and Sheep can be distinguished by a PCR test based on a Novel DNA Sequence difference. J Clin. MIcrobiol 40: , Fitzgetrald SD, Zwick LS, Berry DE, Church SV, Kaneene JB, Reed WM. Experiential inoculation of pigeons(columba livia) with Mycobacterium bovis. Avian disease 47: , Guthertz LS, Damasker B, Bottone EJ, Ford EG, Midura TF, Janda JM. Mycobacterium avium and Mycobacterium intracellulare infection in patients with and without AIDS. J Infect Dis 160: , Huard RC, Lazzarini LC de O, Butler WR, Sooligen DV, Ho JL. PCR-Based method to differentiate subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions. J Clin MIcrobiol 41: , Kim BJ, Lee SH, Lyu MA, Kim SJ, Bai GH, Kim SJ, Chae GT, Kim EC, Cha CY, Kook YH. Identification of Mycobacterial Species by comparative sequence analysis of the RNA Polymerase gene(rpob). J Clin MIcrobiol. 37: , Kim BJ, Lee SH, Lee MA, Lyu SJ, Kim GH, Bai SJ, Kim GT, Chae EC, Kim CY, Cha, KooK YH. 164

8 Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpob). J Clin MIcrobiol 37: , Lee, HY, Park HJ, Cho SN, Bai GH, Kim SJ. Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the rpob Gene. J Clin MIcrobiol 38: , Lee H, Cho SH, Bang HE, Lee JH, Bae GH, Kim SJ, Kim JD. Molecular analysis of rifampin-resistant Mycobacterium tuberculosis isolated from Korea by polymerase chain reaction-single strand conformation polymorphism sequence analysis. Int J Tuber lung dis 2: , Kim bj, Kim SY, Park BH, Lyu MA, Park IK, Bai GH, KIm SJ, Cha CY, Kook YH. Mutations in the rpob gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing. J Clin Microbiology 35(2): , Ruiz. P, gutierrez J, Zerolo J. Cassl M. Geno Type Mycobacterium Assay for identification of Mycobacterial Species Isolated from Human Clinical Samples by Using Liquid Medium. J Clin MIcrobiol 40: , Shinners D, Yeager H Jr. Nontuberculous mycobacterial infection; clinical syndromes and diagnosis; overview, In Scholossberg D(ed.). Tuberculosis and nontuberculous mycobacterial infections, 4th ed., p , W.B. Sanuders Co. Philadelphia, Pa, 김범준, 변경희, 배길한, 김상재, 이근화, 황응수, 차창룡, 국윤호. DT1-DT6 PCR에의한국내분리 Mycobacterium avium complex의감별. Korean J Bacteriol Virol 32:33-38,

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untitled ORIGINAL ARTICLE Korean J Clin Lab Sci. 2015, 47(2):83-89 http://dx.doi.org/10.15324/kjcls.2015.47.2.83 pissn 1738-3544 eissn 2288-1662 Korean J Clin Lab Sci. Vol. 47, No. 2, Jun. 2015 83 Identification