주간건강과질병 제 9 권제 34 호 유전자마커를이용한생물테러병원체검출기술동향 질병관리본부국립보건연구원병원체방어연구과조수경, 전정훈, 유재연, 이기은 * 제이에스바이오테크윤현규 * 교신저자 : gerhie@korea.kr / 043-719-8270 공동교신저자 : yoon0908@naver.com / 010-8851-6559 Abstract Bioterrorism Agent Detection System Using Genetic Markers Division of High-risk Pathogen Research, NIH, CDC Jo Soo-Kyeong, Chun Jeong-hoon, Yu Jae-Yeon, Rhie Gi-eun JS Biotech Yoon Hyun Kyu Following bioterrorism using the mail containing anthrax spores in the USA in October 2001, the importance of guarding against this threat increased day by day. Bioterrorism agents may come in the form of bacteria, virus, and fungi. The toxic effects of these pathogenic microgarnisms can cause serious social and economic losses. In addition, as bioterrorism pathogens are easy to develop compared to other weapons of mass destruction, preparedness is absolutely necessary. Bioterrorism is hard to detect in the early stage since symptoms appear much later after infection and it is not easy to distinguish that diseases are caused by bioterror or natural infection. Because of this, it is very important to detect and confirm the pathogen in the early stage to adopt preventive measures and treat exposed patients rapidly. Recently, a molecular biological test, which is able to quickly detect various causes of bioterrorism, has been developed and commercialized. This text introduces the various equipments to properly detect bioterrorism infection on site. 들어가는말 2001년 10월미국에서발생한탄저우편물테러이후전세계적으로생물테러의위협이현실화될수있다는점에서이에대한대비의중요성이나날이증가되고있다. 생물테러는살아있는박테리아나바이러스, 곰팡이그리고이들이분비하는독소등을통해다양하게발생될수있으며, 검출이어려워이로인한심각한사회적, 경제적손실이발생될수있다. 또한, 생물테러병원체를생산하거나무기화하는것이다른대량살상무기를개발하는것에비해손쉽게접근할수있어, 이에대한대비가무엇보다필요하다. 미국 NIAID의분류에따르면, 위해도에따라병원체를카테고리 A, B, C로구분하고있다 (Table 1). 카테고리 A에는 국가안보와공공의건강에큰위협이될수있고, 사회적혼란을야기할수있는병원체가포함되고, 카테고리 B에는전파속도는중간정도로쉽고, 사망률은낮으나진단능력과질병감시가필요한병원체가, 카테고리 C에는높은이환율과사망률로건강에영향을미칠가능성이있는신종병원체가포함된다. 우리나라에서는 감염병의예방및관리에대한법률 제2조제19항에고위험병원체 36종을지정하여관리하고있으며, 특히탄저균 (Bacillus anthracis), 페스트균 (Yersinia pestis), 야토균 (Francisella tularensis), 두창바이러스 (Variola virus), 보툴리눔독소 (Clostridium botulinum toxin), 바이러스성출혈열 (Viral hemorrhagic fever) 중에볼라열, 라싸열, 마버그열을생물테러감염병으로지정하여 666
Vol. 9 No. 34 PUBLIC HEALTH WEEKLY REPORT, CDC Table 1. NIAID Emerging Infectious Diseases / Pathogens Category A Category B Category C Bacillus anthracis Clostridium botulinum toxin Yersinia pestis Va riola major(smallpox) and other related pox virus Francisella tularensis Viral hemorrhagic fever - Arenaviruses Junin Machupo Guanarito Chapare Lassa Lujo - Bunyaviruses Ha ntaviruses causing Hanta Pulmonary syndrome Rift Valley fever Crimean Congo Hemorrhagic fever etc. - Flaviviruses Dengue - Filoviruses Ebola Marburg Burkholderia pseudomallei Coxiella burnetii Brucella species Burkholderia mallei Chlamydia psittaci Ricin toxin Epsilon toxin Staphylococcus enterotoxin B Typhus fever Food- and Waterborne pathogens - Bacteria Diarrheagenic E. coli Pathogenic Vibrios Shigella species Salmonella Listeria monocytogenes Campylobacter jejuni Yersinia enterocolitica - Viruses caliciviruses Hepatitis A - Protozoa Cryprosporidium parvum Cyclospora cayatanensis etc. Mosquito-borne encephalitis virus - West Nile virus - Venezuelan equine encephalitis - Japanese encephalitis virus etc. Nipah and Hendra virus Hantaviruses Tickborne hemorrhagic fever viruses - Bunyaviruses SFTS virus Heartland virus - Flaviviruses Omsk hemorrhagic fever virus Alkhurma virus etc. Tickborne encephalitis complex flaviviruses - Tickborne encephalitis viruses - Euripean subtype - Far Eastern subtype Yellow fever virus Tuberculosis, including drug-resistant TB Influenza virus Other Rickettsias Rabies virus Prions Chikungunya virus Coccidioides spp. Se vere acute respiratory syndrome associated coronavirus (SARS-CoV), MERS-CoV and other highly pathogenic human coronaviruses Antimicrobial resistance 관리하고있다. 병원체를이용한생물테러는일정기간의잠복기를거쳐증상이나타나기때문에초기감지가쉽지않고, 기존의자연발생적인감염병과구별이쉽지않으나노출된환자에대한예방과치료및적절한방역대응이신속히이루어질수있도록병원체를조기에탐지하고확인하는것이무엇보다중요하다. 원인병원체규명을위한실험실진단기술은미생물학적방법과유전학적방법, 항체를이용한혈청학적방법으로구분되며, 유전학적방법은민감도, 편의성, 재현성등으로최근많이사용되고있다. 여기서는유전자를마커로이용한생물테러병원체검출기술에대하여소개하고자한다. 때문에자동화가가능하다. 카트리지의구조는핵산추출부와 PCR 증폭부분이하나로구성되어있으며, 현재. 1, 2, 4, 16개의카트리지가동시에장착이가능한모듈로구성된기기가상용화되었다. 본시스템을이용하여생물테러원인병원체를검출할수있는 Biothreat 와 Ebola Assay kit가개발되었다. Biothreat의경우 30개이상의탄저균을 45분이내검출이가능하며 Ebola Assay kit는그최소검출한계 (LOD) 가불활화바이러스의경우 73 copies/ml (95% CI: 51-97 copies/ml), 감염력이있는바이러스의경우 1 pfu/ml 이다 [1]. 2. FilmArray 시스템 BioFire 사 FilmArray 시스템은동결건조된시약을포함한 몸말 1. GeneXpert real-time 시스템 Cepheid 사의 GeneXpert real-time 시스템은장착된카트리지에서샘플의추출, 증폭, 검출과정이이루어지기 진공플라스틱파우치와검출시스템으로구성되어있다. 파우치내에서시료의추출, 증폭, 검출이진행되어 Cepheid GeneXpert system 처럼별도의조작이필요치않아사용에편리하다. 샘플이진공에의해주입되면 lysis 용액과혼합된후추출채널로이동하게되고, 핵산추출이완료되면 1차 PCR과 2차 PCR을각각진행하여해당병원체의표적유전자 www.cdc.go.kr 667
주간건강과질병 제 9 권제 34 호 Figure 1. Cepheid GeneXpert R system 1) Table 2. Composition of FilmArray BioThreat Panel Bioterrorism agents Bacillus anthracis 3 Targets Marburg virus 2 Targets Brucella melitensis 2 Targets Ricinus communis Burkholderia 2 Targets Rickettsia prowazekii 2 Targets Clostridium botulinum Variola virus Coxiella burnetii 2 Targets Venezuelan equine encephalitis virus 2 Targets Ebola virus (Zaire) Western equine encephalitis virus Eastern equine encephalitis virus Yersinia pestis 2 Targets Francisella tularensis 2 Targets Orthopox virus 2 Targets Figure 2. FilmArray BioThreat Kits work-flow 2) 존재유무를확인함으로써정확한감염원이검출된다. 현재 탄저균을포함하여총 16 종의생물테러원인체가적용된 BioThreat Kits 가개발되었으며 (Table 1), 탄저균, 야토균, 페스트균의 genomic DNA 를적용하여평가한연구에서 250 genome equivalents (GEs) 이하의민감도를보이는것으로 확인되었다 [2]. 1 ) http://www.cepheid.com 2) http://www.biomerieux-diagnostics.com/filmarray 668
Vol. 9 No. 34 PUBLIC HEALTH WEEKLY REPORT, CDC 3. RAZOR EX BioDetection 시스템현장에서최대 12개의시료를동시에검사가능한 real-time PCR시스템으로, FilmArray 시스템과마찬가지로동결건조된시약을포함한진공파우치가장착되어편리하고신속하게원인병원체를검출할수있다. 기기에부착된화면을통해증폭곡선을확인할수있어, 현장에서탄저균, 양브루셀라균, 보툴리눔독소 A, 큐열균, 장출혈성대장균 (O157), 야토균, 리신, 살모넬라균, 두창, 페스트균등의진단이가능하다. 또한공기수집장치 (air collection devices) 를추가장착할경우공기중의탄저균을확인할수도있다 [3]. 4. VerePLEX Biosystem Microarray 기술을이용한병원체검출시스템으로현재 category A에포함된탄저균, 야토균, 페스트균, 두창바이러스를검출할수있는제품을출시하고있다. 추출된핵산을 Labon-Chip에적용하여 Multiplex PCR을수행하고증폭산물을 microarray에집적된탐침과교잡화한후세척과정을거쳐스캔하여결과를확인한다. Verechip 은실리콘히터, 온도센서, 25ul 용량의 PCR 모듈에 400개의올리고스팟을집적할수있도록되어있으며, 각병원체는반응당 125 copies 를검출할수있고, 이모든과정을수행하기위해 3시간정도소요된다. 5. ArrayTube (AT) 플랫폼 Microarray기술을이용한 ArrayTube (AT) 플랫폼은일반적으로실험에사용되는 microtube와유사한형태의플라스틱튜브하단에각원인병원체에특이적인탐침을집적하고증폭산물과교잡후 precipitation staining 을통해결과를확인하는방식이다. 본검사플랫폼은핵산, 단백질기반분석모두적용이가능하고, 형광표지가아닌 colorimetric staining법이사용되어별도의광학스캐너를이용해결과를확인한다. 현재 category B에포함된브루셀라속균, 버크홀데리아속균, 큐열균등의검출및유전형분석이가능한키트가있다 [4,5]. Figure 3. BioFire RAZOR EX BioDetection System 3) Figure 4. Veredus laboratories VerePLEX Biosystem 4) 3) http://http://biofiredefense.com/razorex/ 4) http://vereduslabs.com/system-and-software/vereplex-biosystem/ www.cdc.go.kr 669
주간건강과질병 제 9 권제 34 호 AT chip incl. 14x14 oligonucleotide probes (array size: 2 x 2 mm) Figure 5. Alere ArrayTube System 5) 6. UltraFast LabChip Real-time PCR G2-4 시스템나노바이오시스사의 UltraFast LabChip Real-time PCR G2-4 시스템은현장현시진단이가능한 real-time PCR 기기로서독자적으로개발한 plastic chip 내 16개의시료 적용이가능하다. 40 cycles PCR 과정을 20분이내에수행할수있으며하나의 channel 당 2개의표적유전자검출이가능하도록 FAM, CY5 개의형광필터로구성되어있다. 기기의총무게가 5.5 kg으로현장에서병원체를검출할수있고 Figure 6. UltraFast LabChip Real-time PCR G2-4 System 6) Table 3. Comparison of instrument specifications Specifications GeneXpert system (1 modules) FilmArray BioThreat RAZOR EX System VerePLEX Biosystem ArrayTube Nanobiosys LabChip system Instrument Size 10.2 30.5 29.7 cm 25.4 39.3 16.5 cm 25.4 11.4 19 cm 50.0 30.0 14.5 cm 8.0 12.0 15.5 cm 21.2 25.6 23.2 cm Instrument weight 0.99 kg 9 kg 4.9 kg 10 kg 0.88 kg 5.5 kg Limit of Detection (LoD) 73 copies/ ml 250 GEs* 200 GEs 125 copies/rxn 100 GEs 50 copies/rxn Turnaround Time (TAT) 45 mins 1 hours 30 mins 3 hours NA** 20 mins Technology Real-time PCR Real-time PCR Real-time PCR Microarray Microarray Real-time PCR Targets 1 16 10 4 1 1 * GEs, Genome Equivalents ** NA, Not Applicable 5) http://alere-technologies.com/ 6) http://nanobiosys.co.kr/ 670
Vol. 9 No. 34 PUBLIC HEALTH WEEKLY REPORT, CDC 별도의배터리팩을장착할경우전원공급이원활하지않은환경에서도현장현시진단이가능하다. 현재본시스템을이용하여 category B에포함된식품매개또는수인성병원체인장출혈성대장균, 살모넬라속균, 콜레라균등을검출할수있는키트가개발되어있다. 있는특성을감안할때현장에서바로생물테러원인체를조기에검출하는진단기술이필요한실정이다. 이러한점을종합할때본기고문에서기술한다양한현장현시진단시스템을도입하거나국내실정에맞도록새롭게개발하여국가차원에서의효과적인생물테러대비가필요한것으로사료된다. 맺는말 참고문헌 생물테러사건현장에서는일반적인실험실환경에서수행하는진단과달리실험환경이더열악하고짧은시간내로오염된검체에서세균이나바이러스등원인병원체를확인해야하므로다수의병원체를동시에검출할수있어야하며, 비전문가도손쉽게사용할수있는검출시스템이주류를이루고있다. 이미상용화된검출시스템은기본적으로다중검사가가능하고실험실진단장비와비교하였을때상대적으로무게가가벼운이동형시스템이많이보급되고있다. 기술적으로는 microarray 와 real-time PCR 기술기반으로구분되며, Veredus laboratories VerePLEX Biosystem 과 Alere ArrayTube 플랫폼이 microarry 기술을 Cepheid GeneXpert system, Biofire FilmArray와 RAZOR EX, 나노바이오시스사의 UltraFast LabChip 시스템이 real-time PCR 기술을적용하고있다. 일부의검출시스템은모든시약을하나의카트리지또는파우치에담아시료주입만실험자가수행하면유전자추출과증폭, 검출등의진행은기기가알아서해주는방식을채택하고있고, 일부는유전자추출과증폭시실험자의간편한조작을필요로한다. 앞서소개한검출시스템은기기성능에서는큰차이를보이지않지만편의성이나신속성, 가격적인측면에서는다소부담스러운검출시스템도있다. 진단시스템의기술적인트렌드는지속적으로소형화되며분석의결과또한클라우드시스템을이용하여공유되는방향으로개발되고있어앞으로는특정장소에한정되지않고이동성이극대화된 hand-held 진단시스템이시장이개발되어보급될것으로보인다. 우리나라는남북이대치하고있고, 북한은다양한종류의생물테러원인체를보유하고있다고알려져있어무기화등에대한철저한대비가필요하다는점과대도시에인구가밀집되어 1. Benjamin A. Pinsky, Malaya K. Sahoo, Johanna Sandlund, Marika Kleman, Medha Kulkarni, Per Grufman, Malin Nygren, Robert Kwiatkowski, Ellen Jo Baron, Fred Tenover, Blake Denison, Russell Higuchi, Reuel Van Atta, Neil Reginald Beer, Alda Celena Carrillo, Pejman Naraghi- Arani, Chad E. Mire, Charlene Ranadheera, Allen Grolla, Nina Lagerqvist, David H. Persing ; Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus. PLoS ONE 10(11): e0142216. 2015 2. DR Seiner, HA Colburn, C Baird, RA Bartholomew, T Straub, K Victry, JR Hutchison, N Valentine, and CJ Bruckner-Lea ; Evaluation of the FilmArray system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. J Appl Microbiol. 114(4): 992 1000. 2013 3. Spaulding UK, Christensen CJ, Crisp RJ, Vaughn MB, Trauscht RC, Gardner JR, Thatcher SA, Clemens KM, Teng DH, Bird A, Ota IM, Hadfield T, Ryan V, Brunelle SL.; RAZOR EX anthrax air detection system. J AOAC Int. 95(3):860-91. 2012 4. Gernot Schmoock, Ralf Ehricht and Lisa D Sprague ; DNA microarray-based detection of Coxiella burnetii, the causative agent of Q fever. Acta Veterinaria Scandinavica 56:27. 2014 5. Birgit Huelseweh, Ralf Ehricht and Hans-Juergen Marschall ; A simple and rapid protein array based method for the simultaneous detection of biowarfare agents. Proteomics 6, 2972 2981. 2006 www.cdc.go.kr 671