J. Exp. Biomed. Sci. 2010, 16(2): 127~131 Brief Communication Detection of Mycobacterium leprae by Real-time PCR Targeting Mycobacterium leprae-specific Repetitive Element Sequence Hyunwoo Jin 1, Hye-Young Wang 1, Jong-Pill Kim 2, Sang-Nae Cho 3 and Hyeyoung Lee 1, 1 Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University 234 Maeji-ri, Heungup-myun, Wonju-si, Kangwon-do 220-710; 2 Affiliated Hospital, Korean Leprosy Control Association, Euiwang-si, Kyunggi-do 437-823; 3 Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seoul 120-752, Korea Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens. Key Words: Mycobacterium leprae, Real-time PCR, Repetitive element sequence (RLEP) 서 나병은나균인 Mycobacterium leprae가피부와말초신경에주로침해하여발생하는전염성만성면역질환으로질병의발병은줄어들고있지만, 아직도세계적으로문제가되는질환이다 (Chae et al., 2002). WHO의보고에의하면, 2005년한해전세계에서발생한신규나병환자는 296,499명이었고그중 68% 인 201,635명은동남아시아에서발생하였다 (WHO, 2006). 나균은인공배지에서배양이되지않기때문에나병진단을위해서는피부도말을항산성염색을하여확인하고피부병변및신경손상등의임상소견을기초로확진한다 (Kampirapap et al., 1998). 하지만항산성염색결과는모든 Mycobacterium spp. 가양성으로나타나기때문에나균만감별할수없고, 피부조직에서균수가최소 * 접수일 : 2010년 3월 19일 / 수정일 : 2010년 6월 18일채택일 : 2010년 6월 21일 Corresponding author: Hyeyoung Lee. Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University 234 Maeji-ri, Heungup-myun, Wonju-si, Kangwon-do 220-710, Korea. Tel: 82-33-760-2740, Fax: 82-33-760-2561 e-mail: hyelee@yonsei.ac.kr 론 10 4 개이상이존재해야양성으로나타나기때문에민감도와특이도가낮은검사법이라할수있다 (Kurabachew et al., 1998 Donoghue et al., 2001; Kang et al., 2003). 따라서최근에는분자생물학적방법인중합효소연쇄반응 (polymerase chain reaction; PCR) 을이용하여나균의특정한 DNA 염기서열을증폭시켜조직내의나균을검출하는방법이많이사용되고있다 (De wit et al., 1991; Yoon et al., 1993; Donoghue et al., 2001; Kang et al., 2003; Patrocinio et al., 2005). 최근에는 real-time PCR 기술이많은병원체의진단에서검증되어 (Rondini et al., 2003; Kesanopoulos et al., 2005) 결핵균을포함한다른마이코박테리아의진단에도응용되고있다. Real-time PCR 기술은 PCR 방법의최대의단점인실험실내오염사고를예방할수있다는장점과더불어프로브를사용한다는장점에의해기존의 PCR 에비해민감도와특이도가높다는평가를받고있다 (Kramme et al., 2004; Martinez et al., 2006; Shamsi et al., 2007). 따라서본연구에서는나균검출을목적으로한 realtime PCR을개발하고이어이방법의나균검출에서의민감도및특이도를평가하여이를기존의 PCR법에의한결과와비교함으로써 real-time PCR의나병진단에서 - 127 -
의유용성을평가하였다. 재료및방법나균및생검조직에서 DNA의추출표준균주로사용된 M. leprae strain 4264는 Colorado State University에서정제된 100 mg ( 평균 2.9 10 9 bacilli/ mg 포함 ) 을제공받아 QIAamp DNA Mini Kit (Tissue protocol; Qiagen GmbH, Hilden, Germany) 를이용하여 DNA를분리하고, 최종농도를 50 ng/μl로희석하여사용하였다. 임상검체평가에이용된 DNA는한센병연구원에서나병환자들로부터채취한피부생검조직으로부터 QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) 를이용하여추출한 DNA 80개를제공받아사용하였다. 항산균에서 DNA의추출 Real-time PCR에사용되는프라이머와프로브의특이도를검사하기위해다양한마이코박테리아균주로부터 DNA를분리하여사용하였다. 실험에사용된마이코박테리아균종은 M. tuberculosis H37Rv, M. abscessus (ATCC 19977), M. africanum (KCTC 9504), M. aichiens (ATCC 27280), M. aurum (ATCC 23366), M. avium (ATCC 25291), M. avium (ATCC 35719), M. celatum (ATCC 51130), M. celatum (ATCC 51131), M. celatum sub. chelonae (ATCC 35749), M. fortuitum (ATCC 49403), M. fortuitum (ATCC 49404), M. fortuitum sub. fortuitum (KCTC 9510), M. gallinarum (KCTC 9511), M. gastri (ATCC 15754), M. genevence (ATCC 51233), M. gordonae (ATCC 14470), M. gordonae (KCTC 9513), M. hassiacum (ATCC 700660), M. intracellulare (ATCC 13950), M. intracellulare (KCTC 9514), M. kansasii (ATCC 12478), M. kansasii (KCTC 9515), M. kubicae (ATCC 700732), M. malmonense (ATCC 29571), M. marinum (ATCC 927), M. microti (ATCC 19422), M. moriokaense (KCTC 9516), M. neoaurum (ATCC 25795), M. nonchromogenicum (ATCC 19530), M. peregrinum (ATCC 14467), M. phlei (ATCC 11758), M. porcinum (KCTC 9517), M. scrofulacium (ATCC 19981), M. senegalense (ATCC 35796), M. septicum (ATCC 700731), M. smegmatis (KCTC 9108), M. szulgai (ATCC 35799), M. terrae (ATCC 15755), M. thermoreisistable (ATCC 19527), M. trivial (ATCC 23292), M. vaceae (ATCC 15483), M. xenopi (ATCC 19250) 이었다. DNA를분리하기위해배양한균체에 lysozyme을 5 mg/ml로가하여 37 에서 1시간, 1 mg/ml의 proteinase K 및 1% SDS를가하여 55 에서 24시간반응시켰다. Cetyl Trimethyl Ammonium Bromide (CTAB) 를첨가하여 65 에서 10분간반응시키고페놀처리및에탄올침전하여 DNA를얻었다. Real-time PCR National Center for Biotechnology information (NCBI) 에서제공하는 Blast search (http://www.ncbi.nlm.nih.gov/blast/) 를이용하여나균 DNA의 repetitive sequence (GenBank accession No. AL583917) 의유사염기서열을추출한후 MultAlin program을이용하여분석하였다. 이를이용하여예상되는증폭산물이 107-bp의크기가되도록 MLF (5'-GTGTCGGCGTGGTCAATGTG-3') 와 MLR (5'-CGATACCAGCGGCAGAAATGG-3') 한쌍의 primer를제작하고 5' 끝에 5-carboxyfluoroscein (FAM) 과 3' 끝에 N,N,N',N'-tetrametyl-6-carboxyrhodamine (TAMRA) 의형광염료를부착한 MLSP (5'-FAM-CCGCACCTGAA- CAGGCACGTCCC-TAMRA-3') TaqMan probe를제작하였다 (Bioneer, 대전, 한국 ). Real-time PCR 반응액은총 20 μl 내에 iq TM Supermix (Bio-Rad, USA) 10 μl, 각각 10 pmol 의 primer 1 μl와 probe 0.5 μl, template DNA 5 μl를첨가하여사용하였다. PCR 반응조건은 predenaturation을 94 에서 3분간 1회수행한후에 94 에서 20초 denaturation, 56 에서 40초 annealing 및 extension 후, 45회실시하였고형광측정을사용하였다. icycler iq TM Multicolor Realtime PCR Detection System (Bio-Rad, USA) 을사용하였고 icycler iq TM Optical System Software Version 3.1 (Bio-Rad, USA) 을사용하여결과를분석하였다. 비교실험을위해이용된 PCR은 touch-down (TD) PCR로, 예상되는증폭산물은 129-bp가되도록 F (5'- TGCATGTCATGGCCTTGAGG-3') 와 R (5'-CACCGATAC- CAGCGGCAGAA-3') 의 primer를이용하였다 (Kang et al., 2003). 반응조건으로는먼저 pre-denaturation을 94 에서 5분간 1회수행한후에, 94 에서 45초 denaturation, 64 에서 58 까지 1 씩감소시키면서 7회동안반복시행한후에다시 94 에서 45초 denaturation, 58 에서 45초 annealing, 72 에서 90초 extension으로 35회실시하였다. 결과및고찰나균은시험관내에서배양할수없기때문에감염병 - 128 -
의진단에필수적인실험실내에서균배양과동정을거치지못하는것이나병진단의가장큰취약점이다. 따라서현재까지나병의진단은임상소견과항산성균염색에주로의존하여왔다 (Talhari, 1996). 항산성균염색은적어도균체가 10 4 개이상존재하여야양성으로판정되어민감도가낮고다른 mycobacteria와구별이되지않기때문에특이도가낮다는단점이있다 (Kurabachew et al., 1998). 최근에는분자생물학적인방법을이용한나균의 DNA 나 RNA를직접증폭시켜검출하는방법이널리사용되고있다 (Karamme et al., 2003). DNA를증폭하는방법에사용되는표적유전자는 65 kda 항원유전자 gene encoding 65 kda antigen (Plikaytis et al., 1990), 18 kda 항원유전자 (Kim et al., 1996; Williams et al., 1990), 36 kda 항원유전자 (Parkash et al., 2004), repetitive sequences (Wood and Cole, 1989; Yoon et al., 1993) 등이널리사용되고있다. RNA를증폭하는방법에사용되는표적은 16S rrna 부위에나균에특이적인부분을검출하는방법이사용되고있다 (Kurabachew et al., 1998; Phetsuksiri et al., 2006). 본연구에사용된 repetitive sequence (RLEP) 는다른세균이나항산성균에는존재하지않는나균에만존재하는유전자로알려져있으며, 약 28 copy가존재하기때문에다른유전자보다검출민감도가훨씬높다는장점이있다 (Donoghue et al., 2001; Kang et al., 2003). 나균검출을위한 real-time PCR법의결과판정을위해측정된 threshold cycle (Ct) 값을분석하여 36 이하를 M. leprae 양성, 36초과하는값에대하여음성으로판단하도록 cut off value를결정하였다. 나균을진단하기위하여제작된 MLF, MLR primer 쌍이 M. leprae에만특이적으로반응하는지를알아보기위 A B Fig. 1. The sensitivity of the (A) real-time PCR and (B) standard PCR for detecting M. leprae genomic DNA. Using the diluted DNA of the M. leprae reference strain 4264, the sensitivity of the two PCR assays were compared. (A), real-time PCR with 100 pg (B1), 10 pg (B2), 1 pg (B3), 100 fg (B4), and no DNA (B8). (B) PCR with from 100 pg to 100 ag DNA. M; 100 bp DNA ladder (Bioneer, Daejeon, Korea), N; no DNA added negative control. - 129 -
Table 1. Comparison of sensitivity between the real-time PCR and the standard PCR using clinical samples PCR Real-time PCR Positive 26 (32.50%) 53 (66.25%) Negative 43 (52.80%) 27 (33.75%) Un-ID 11 (13.70%) 0 (0%) Total (80) 80 (100%) 80 (100%) 해 M. leprae strain 4264와 35종의 Mycobacterium spp. DNA를가지고 real-time PCR을수행하였다. 측정된 Ct 값을분석한결과 M. leprae strain 4264만양성으로판정되고 35종의 Mycobacterium spp. DNA는모두음성으로측정됨을확인하였다. 이런결과로보아 RLEP 부위를검출하는 real-time PCR법은나균에대한특이성이높다는것을확인하였다. 또한, 나균검출을위한 real-time PCR법의민감도를비교분석하기위해 M. leprae strain 4264 DNA를연속적으로 10배씩희석한후 real-time PCR의 template DNA로각각사용하였다. 그결과 1 pg 이상에서증폭되는것을확인할수있었고측정된 Ct값으로 1 pg에서양성으로측정됨을확인하였다 (Fig. 1). 따라서, 민감도부분에서는비교실험에이용된 single PCR과 real-time PCR이비슷한민감도를가지고있었으나, 나병으로의심되는환자의피부조직에서추출된 DNA로부터 RLEP의특이유전자검출을실시하였다. 그결과총 80개의시료중 PCR을이용한방법으로는 26개 (32.5%) 의검체에서양성결과가나온반면 real-time PCR을이용한방법으로는 53개 (66.25%) 의검체에서양성결과가나왔다 (Table 1). 따라서 real-time PCR을이용한방법이 single PCR을이용한진단법보다높은민감도를나타내어높은진단효율성을나타내었다. Real-time PCR은형광물질이부착된 probe를사용하여실시간으로증폭산물의유무를확인하는방법으로전기영동을하지않기때문에일반 PCR보다빠른결과를얻을수있고 PCR 전기영동상에서눈으로확인하는것보다정확한형광의신호를측정하여정량측정도가능하다. 그리고 PCR에서교차오염으로인한위양성결과가 real-time PCR은뚜껑을한번닫으면열지않기때문에교차오염의위험이없다는장점이있다. 따라서, RLEP를검출하는 real-time PCR법은피부생검조직에서직접균을검출할때에 PCR을대신하여나균진단에유용할것으로생각된다. REFERENCES Chae GT, Kim MJ, Kang TJ, Lee SB, Shin HK, Kim JP, Ko YH, Kim SH, Kim NH. DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium lepraeto assess the efficacy of multidrug therapy for leprosy. J Med Microbiol. 2002. 51: 417-422. De wit MYL, Feber WR, Krieg SR. Application of a polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues. J Clin Microbiol. 1991. 29: 906-910. Donoghue HD, Holton J, Spigelman M. PCR primers that can detect low levels of Mycobacterium leprae DNA. J Med Microbiol. 2001. 50: 177-182. Kampirapap K, Singtham N, Klatser PR, Wiriyawipart S. DNA amplification for detection of leprosy and assessment of efficacy of leprosy chemotherapy. Int J Lepr Other Mycobact Dis. 1998. 66: 16-21. Kang TJ, Kim SJ, Lee SB, Chae GT, Kim JP. Comparison of Two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae. Clin Exp Dermatol. 2003. 28: 420-424. Kesanopoulos K, Tzanakaki G, Levidiotou S, Blackwell C, Kremastinou J. Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples. FEMS Immunol Med Microbiol. 2005. 43: 419-424. Kramme S, Bretzel G, Panning M, Kawuma J, Drosten C. Detection and quantification of Mycobacterium lepraein tissue samples by real-time PCR. Med Microbiol Immunol. 2004. 193: 189-193. Kurabachew M, Wondimu A, Ryon JJ. Reverse transcription-pcr detection of Mycobacterium leprae in clinical specimens. J Clin Microbiol. 1998. 36: 1352-1356. Martinez AN, Britto CF, Nery JA, Sampaio EP, Jardim MR, Sarno EN, Moraes MO. Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy. J Clin Microbiol. 2006. 44: 3154-3159. Patrocinio LG, Goulart IM, Goulart LR, Patrocinio JA, Ferreira FR, Fleury RN. Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction. FEMS Immunol Med Microbiol. 2005. 44: 311-316. - 130 -
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