Microsoft Word - 10-Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resist

Size: px

Start display at page:

Download "Microsoft Word - 10-Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resist"

이석 어
5 years ago
Views:

1 Biomedical Science Letters 2019, 25(1): 75~82 eissn : Original Article Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures Yun-Hee Baek 1,,*, Mi-Young Jo 2,3,,*, Min-Suk Song 1,*, Seung-Bok Hong 4,* and Kyeong-Seob Shin 2,,* Department of Microbiology 1, Laboratory Medicine 2, Chungbuk National University College of Medicine, Cheongju 28644, Korea 3 Department of Nursing Science, Kyungbuk College of Natural Sciences, Yeongju 36133, Korea 4 Department of Clinical Laboratory Science, Chungbuk Health & Science Unviersity, Cheongju 28150, Korea We developed the multiplex LAMP assay using 16S rrna, fema and meca genes for direct detection of the methicillin resistance in Staphylococci from positive blood culture. To simultaneously recognize Staphylococci genus, S. aureus and methicillin resistance, three sets of six primers for 16S rrna, fema and meca were designed, respectively. The performance of LAMP assay was affirmed using VITEK system for the phenotypic methods of identification and for oxacillin and cefoxitin antimicrobial susceptibility. The optimal condition for LAMP assay was obtained under 64 for 50 min. The detection limit was determined to be of 20 copies and CFU/reaction (10 4 CFU/mL). For clinical application of comparison with phenotypic methods, the sensitivity and specificity of the LAMP with fema gene for detecting S. aureus was 95.31% and 100%, respectively. The sensitivity and specificity of the LAMP with meca gene for detecting methicillin resistance was 98.46% and 100%, respectively. The multiplex LAMP assay with fema and meca gene successfully detected all of MRSA (38 isolates) isolates from 103 Staphylococci in blood cultures. The LAMP assay developed in this study is sensitive, specific, and of excellent agreement with the phenotypic methods. Key Words: Blood culture, Methicillin-resistant Staphylococcus aureus, 16S rrna, fema, meca, Loop-mediated isothermal amplification 서론 Staphylococcus aureus 는의료기관연계감염 (hospital associated infection) 뿐만아니라지역사회연계감염 (community associated infection) 의주요한원인균으로모낭염 (folliculitis) 이나봉와직염 (cellulitis) 과같은국소감염부터 심내막염 (endocarditis), 패혈증 (sepsis) (Lowy, 1998), 그리고독성쇽증후군 (toxic shock syndrome) (Shands et al., 1980) 과같은전신성감염도일으킨다. Methicillin-resistant Staphylococcus aureus (MRSA) 는 meca 유전자를획득하여 penicillin binding protein 2a (PBP2a) 를발현함으로써 methicillin 을포함한모든 β-lactam 항균제와의결합력을감소시켜내성을나타낸다 (Hartman and Tomasz, 1984). 또한 MRSA Received: February 8, 2019 / Revised: March 17, 2019 / Accepted: March 19, 2019 These authors contributed equally to this study. * Professor. Corresponding author: Kyeong-Seob Shin. Department of Laboratory Medicine, Chungbuk National University College of Medicine, Cheong-ju 28644, Korea. Tel: , Fax: , ksshin@chungbuk.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

2 는 β-lactam 항균제외에도여러종류의항균제에내성을나타나게되어사용할수있는항균제가제한되기때문에 MRSA에감염된환자의유병률 (morbidity) 과사망률 (mortality) 은 methicillin-susceptible S. aureus (MSSA) 감염환자보다높다 (Cosgrove et al., 2003). 따라서혈액배양에서 MRSA의빠르고정확한검사는이들환자의적절한치료에매우중요하다. 혈액배양양성검체에서 MRSA의검출을위해자동화기기를이용한동정이나감수성검사는검사실에서가장흔히이용하는방법이지만, 이방법은 18~36 시간이상이소요되므로치료시기가늦어질수밖에없다. 한편 PCR 과같은분자유전학적방법은빠르고정확한검출이가능하지만 thermocycler 나증폭산물을검출해야하는특별한장비가필요하며경험이있는검사자가필요하다. Notomi 등 (Notomi et al., 2000) 이 Bst polymerase 에의해가닥변위핵산자가합성 (autocycling strand displacement nucleic acid amplification) 에기초한 loop-mediated isothermal amplification (LAMP) 방법을개발한이후이방법을이용한검사가다양한균의동정에이용되고있다 (Iwamoto et al., 2003; Hara- Kudo et al., 2005; Ito et al., 2006; Curtis et al., 2008; Hara-Kudo et al., 2008; Yamazaki et al., 2008). LAMP 방법은 4~6개의 primer 를이용하여 PCR 보다특이하며, 등온조건 (isothermal condition) 에서증폭하기때문에증폭시간이더빠른장점이있다. 게다가증폭산물도매우많아증폭산물을검출하는특별한방법이필요치않으며 ph 지시자 (indicator) 나형광을이용하면눈으로증폭산물의확인이가능하다 (Li et al., 2017). 이연구에서저자들은혈액배양에서 S. aureus 와 methicillin 내성을직접검출하기위해 3가지유전자를이용한다중 (multiplex) LAMP 방법을개발하였고이방법의성능을평가하고자하였다. 재료및방법대상균주충북지역한대학병원의미생물검사실에서혈액배양양성검체를대상으로하였다. 또한 16S rrna 의특이도를평가하기위해 10개의그람양성표준균주 (Staphylococcus aureus [ATCC 25923, ATCC 25913], Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus bovis, Streptococcus pneumoniae, Streptococcus salivarius, Enterococcus faecalis, Enterococcus casseliflavus), 6개의그 람음성표준균주 (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumannii), 그리고 1개의진균 (Candida albicans) 등총 17개의표준균주를이용하였다. 혈액배양및혈액배양양성균주의전처리환자로부터 10 ml의혈액을무균적으로채혈하여 BacT /ALERT SA standard aerobic bottle (biomérieux Inc., Marcy-I Étoile, France) 과 BacT/ALERT SN standard anaerobic bottle 에각각 5 ml씩넣었다. 배양은 BacT/ALERT 3D 자동혈액배양기 (biomérieux Inc., Durham, NC, USA) 에서 5일까지시행하였다. 배양양성신호가나올경우그람염색을시행한후혈액우무배지 (Blood agar plate, Asan, Korea) 및 MacConkey 우무배지 (Asan, Korea) 에계대배양하여균의동정및항균제감수성검사에이용하였다. 그람염색에서 Staphylococcus species 가관찰될경우혈액배양병에서혈액 5 ml를취하여혈청분리관 (serum separator tube) 에옮긴후 3,000 rpm에서 10분간원심분리하여상층액을버리고겔바로위에있는균침사를면봉으로 eppendorf 튜브에옮겨시험을할때까지 -20 에보관하였다. 균의동정및 methicillin 감수성검사균의동정은생화학적검사로 catalase, coagulase, mannitol 검사를포함하였고 VITEK 2 자동미생물분석기 (biomérieux Inc., Hazelwood, MO, USA) 로확인하였다. Methicillin 항생제감수성검사는 Vitek system (biomérieux) 을이용하여 oxacillin 과 cefoxitin 감수성검사를시행하였다 (CLSI, 2018). LAMP primer 의설계및최적반응조건 Staphylococcus species, S. aureus 및 methicillin 내성유전자를검출하기위해 16S rrna, fema 및 meca 유전자를 GenBank 에서검색하였고각각의염기서열을결정하였다. 이후 LAMP assay를위한 primer 설계프로그램인 Primer Explorer V5 software ( Eiken chemical Co. Ltd, Japan) 를사용하여각유전자별로 2종의외부 primer (F3와 B3), 2종의내부 primer (FIP 와 BIP) 및 2종의 loop primer (F loop primer () 와 B loop primer ()) 등총 6종의 primer 를설계하였다 (Table 1 & Fig. 1). LAMP assay 반응은 primer (FIP & BIP, 40~80 pmol; F3 & B3, 5~10 pmol; &, 20 pmol) 3 μl, genomic DNA 2 μl, WarmStart colorimetric LAMP master mix (New England Biolabs Inc., MA, USA) 5 μl를더하여최종용량을 10 μl

3 Table 1. The sequences of primers for LAMP assay used in this study Target gene Primer Sequence (5' 3') 16S rrna F3 * TGGAATTCCATGTGTAGCGG B3 * FIP BIP AGGCGGAGTGCTTAATTGC TCGCACATCAGCGTCAGTTACA-ATGCGCAGAGATATGGAGGA AGATACCCTGGTAGTCCACGCC-CACTAAGGGGCGGAAACC CCAGAAAGTCGCCTTCGCCACT AAACGATGAGTGCTAAGTGTTAGG fema F3 * CAGAATCAAAAGCTTTTGCTG B3 * FIP BIP AAGTTATCTCGCTTGTTGTG CTAAAGGTACTAACACACGGTCTTT-TCGTGATGACAAATTTTACTACA AAGAACTAAACGAAGAGCGTGAT-CAGGACGTTTTTCAATATCCTT GTAATATTTTAAGCGAT TAAAGATTTAAATAAAGCGT meca F3 * TGATGCTAAAGTTCAAAAGAGT B3 * FIP BIP GTAATCTGGAACTTGTTGACC AGGTGTGCTTACAAGTGCTAATAAT-CAACATGAAAAATGATTATGGCT TGACGTCTATCCATTTATGTATGGC-GAGGTTCTTTTTTATCTTCGGTTA TGAGGGTGGATAGCAGTACC TGAGTAACGAAGAATAT *Two outer primers F3 and B3 were also used as primers for PCR of 16S rrna, fema and meca. Abbreviations: loop-mediated isothermal amplification; FIP, forward inner primer; BIP, backward inner primer;, forward loop primer;, backward loop primer A. 16S rrna 621 TGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGA F3 F2 F1c TCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCT B1 B2c B3 834 B. fema 635 CAGAATCAAAAGCTTTTGCTGATCGTGATGACAAATTTTACTACAATCGCTTAAAATATTACAAAGACCGTGTGTTAGTACCTTTAGCGTATATCAACTTTGATGAATATATTA 748 F3 F2 F1c 749 AAGAACTAAACGAAGAGCGTGATATTTTAAATAAAGATTTAAATAAAGCGTTAAAGGATATTGAAAAACGTCCTGAAAATAAAAAAGCACACAACAAGCGAGATAACTT 857 B1 B2c B3 C. meca 982 ATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAAC AGGTGAATTATTAGCACTTGTAAGCACACCT 1092 F3 F2 F1c 1093 TCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATA ATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCA 1204 B1 B2c B3 Fig. 1. Primers designed for 16S rrna, fema, meca loop-mediated isothermal amplification (LAMP) assays. Nucleotide sequences of 16S rrna (A), fema (B), meca (C) and the location of LAMP primers. The forward and backward inner primers are F1c-F2 and B1c-B2 sequences, respectively. The forward and backward outer primers are F3 and B3, respectively

A B Fig. 2. Visual (A) and agarose gel (B) images of the 16S rrna, meca, fema loop-mediated isothermal amplification (LAMP) product of MSSA and MRSA on various reaction times.

The electrophoresis was performed at 2% agarose gel and the amplified products typically showed the ladder like shape (B). The best reaction was obtained at 64 and 50 min.

LAMP assay 의반응최적시간및온도를확인하고자다양한시험조건으로하였으며, T100 TM Thermal cycler (Bio-Rad Laboratories Inc., Foster City, CA, USA) 를이용하여증폭을한후효소활성을제거하기위해 80 에서 5분간반응시킨후종료하였다.

4 A B Fig. 2. Visual (A) and agarose gel (B) images of the 16S rrna, meca, fema loop-mediated isothermal amplification (LAMP) product of MSSA and MRSA on various reaction times. The yellow color change of ph indicator was interpreted positive for amplification of DNA (A). The electrophoresis was performed at 2% agarose gel and the amplified products typically showed the ladder like shape (B). The best reaction was obtained at 64 and 50 min. Abbreviations: NEG, negative; MSSA, methicillin-susceptible Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; M, molecular size marker 로하여시행하였다. LAMP assay 의반응최적시간및온도를확인하고자다양한시험조건으로하였으며, T100 TM Thermal cycler (Bio-Rad Laboratories Inc., Foster City, CA, USA) 를이용하여증폭을한후효소활성을제거하기위해 80 에서 5분간반응시킨후종료하였다. 반응이끝난후튜브의색변화를육안으로관찰하여양성여부를판독하였고, 동시에 2% agarose gel에서전기영동을하여 LAMP assay에서특이적으로나타나는사다리모양의증폭유전자밴드를확인하여양성여부를판정하였다. LAMP, PCR 및 RT-PCR 의검출한계 MRSA 부유액 ( CFU/mL) 을 CFU/ ml까지 10배수단계희석하여각농도에서 16S rrna, fema 및 meca 에대한 LAMP 및 PCR을시행하였다. 검출한계실험은 3회씩반복실험하였다. LAMP assay 는본연구에서확립된표준조건대로시행하였으며, PCR은 F3 및 B3 primer 를이용하였으며 DNA 2 μl를 PCR kit (Enzymomics, Daegeon) 에섞어최종용 량 10 μl가되게한후시행하였다. PCR 반응은 T100 TM Thermal cycler (Bio-Rad) 를이용하여 denaturation (95 ) 30초, annealing (58 ) 30초, elongation (72 ) 60초로 35회시행하였고 72 에서 7분간최종반응을시행하였다. 증폭산물은 2% agarose gel에서전기영동하였다. 16S rrna LAMP assay 의특이도 17개의표준균주를이용하여 16S rrna LAMP assay 의특이도를평가하였다. 임상균주를이용한 LAMP assay 의성능평가혈액배양이의뢰된검체중균이증식하였고그람염색에서 Staphylococcus species 가관찰된 103개의임상검체를대상으로 Vitek 2 system 에의한균의동정그리고감수성검사를시행하였고동시에 16S rrna, fema 및 meca 에대한 multiplex LAMP assay를시행하여 2가지검사간의성능을비교하였다

The yellow color change of ph indicator was interpreted positive for amplification of DNA (A).

5 A B Fig. 3. Visual (A) and agarose gel (B) images of the 16S rrna, meca, fema loop-mediated isothermal amplification (LAMP) product of clinical MRSA isolate at serial diluted concentration (10 1 ~ 10 7 CFU/mL). The yellow color change of ph indicator was interpreted positive for amplification of DNA (A). The electrophoresis was performed at 2% agarose gel and the amplified products typically showed the ladder like shape at 10 4 CFU/mL except faint amplification products of 16S rrna (B). Abbreviation: Neg, negative 결과 MSSA와 MRSA 균을대상으로 60~65 와 30, 40, 50, 60분의반응시간으로 LAMP assay 를시행하여 64 50분에서가장뚜렷한결과를보였으므로이후의모든실험은 64 에서 50분으로진행하였다 (Fig. 2). LAMP assay 검출민감도측정 S. aureus 를이용한 16S rrna, fema, meca gene 에대한 LAMP과 conventional PCR에대한검출민감도는 Table 2 과같았다 (Table 2 & Fig. 3). Conventional PCR은 16S rrna 에서는 10 5 CFU/mL 이상에서증폭되었고 fema와 meca 는 10 6 CFU/mL 이상에서증폭되었지만 LAMP 방법은 3개의유전자모두 10 4 CFU/mL 농도에서증폭되어 conventional PCR에비해 10~100 배민감하였다. 16S rrna LAMP assay 의특이도 17개의표준균주 ( 그람양성알균 10주, 그람음성간균 6주, Table 2. Detection limits of LAMP assay, conventional PCR for 16S rrna, fema and meca genes of Staphylococcus aureus C. albicans 1 주 ) 를대상으로한 16S rrna LAMP 검사에서 모두일치하는결과를보였다 (Table 3). 임상검체를적용한 LAMP assay 의민감도에특이도 평가 Gene Detection limit CFU/mL (copies/run) LAMP PCR 16S rrna 10 4 (20 copies) 10 5 (200 copies) fema 10 4 (20 copies) 10 6 (2,000 copies) meca 10 4 (20 copies) 10 6 (2,000 copies) Abbreviation: CFU, colony forming unit 혈액배양에서분리된 103 개의포도알균을대상으로한 16S rrna LAMP 검사에서모두양성을보였다. fema LAMP 검사는 3 개의 MSSA 를검출하지못하였으나나머 지 100 균주는정확한결과를보여생화학적동정및감

6 Table 3. Specificity of LAMP assay for detecting the 16S rrna gene of Staphylococci in reference strains Microorganisms Gram positive bacteria Reference strains Result for 16S rrna LAMP assay Staphylococcus aureus ATCC Positive Staphylococcus aureus ATCC Positive Staphylococcus epidermidis ATCC Positive Streptococcus pyogenes ATCC Negative Streptococcus agalactiae ATCC Negative Streptococcus bovis ATCC Negative Streptococcus pneumoniae ATCC Negative Streptococcus salivarius ATCC Negative Enterococcus faecalis ATCC Negative Enterococcus casseliflavus Gram negative bacteria ATCC Negative Escherichia coli ATCC Negative Klebsiella pneumoniae ATCC Negative Enterobacter cloacae ATCC Negative Citrobacter freundii ATCC 8090 Negative Pseudomoas aeruginosa ATCC Negative Acinetobacter baumannii ATCC Negative Fungus Candida albicans TIMM 3316 Negative 수성검사결과를기준으로평가한예민도와특이도는각각 95.31% (61/64) 와 100% (39/39) 이었다. meca LAMP 검사는 1개의 MRCoNS 를검출하지못하였으나나머지 102개의검체는정확한결과를보여예민도와특이도는각각 98.46% (64/65) 와 100% (38/38) 이었다 (Tables 4 & 5). 고찰전통적으로혈액배양에서 MRSA 의검출은표현형적방법 (phenotypic method) 으로균을동정하고 methicillin 감수성검사를하는것이참고방법으로되어있지만이방법은균의계대배양, 동정및감수성검사에 36시간이상소요된다. 검사시간을단축하기위해혈액배양양성검체에서직접분자유전학적검사방법또는 MALDI-TOF 등을이용하는여러검사들에대한보고들이 (Stamper et al., 2007; Stevenson et al., 2010; Clerc et al., 2014; Verroken et al., 2015) 있다. 그렇지만분자유전학적방법은 DNA의증폭및검출장비가필요하며증폭시간이 3시간이상소요되며 batch 방식으로시행해야한다. 그리고 MALDI-TOF 방법은 mass spectrophotometry 가필요하며, methicillin 내성을검출하기위해서는특별한단계가추가로필요하다 (Opota et al., 2015). 최근에소개된 LAMP 검사방법은 6~8개특이부위를인지하는 6개의 primer 를이용하여 Bst DNA 중합효소에의한 autocycling strand displacement DNA 합성을통하여적 Organisms Table 4. Results of LAMP assay for detecting fema and meca genes in 103 clinical isolates from positive blood culture Multiplex LAMP assay fema+meca+ fema+meca- fema-meca+ fema-meca- Number of clinical isolates MRSA MSSA MRCoNS MSCoNS Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; MRCoNS, methicillin-resistant coagulase negative Staphylococci; MSCoNS, methicillin-susceptible coagulase negative Staphylococci Table 5. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP assay with fema and meca genes for detection of MRSA 103 clinical isolates from positive blood cultures Genes Sensitivity (%) Specificity (%) PPV (%) NPV (%) fema 61/64 (95.31) 39/39 (100) 61/61 (100) 38/41 (92.68) meca 64/65 (98.46) 38/38 (100) 64/64 (100) 38/39 (97.43)

7 은양의 DNA도정확하고빠르게증폭시킬수있다. 또한 60 정도의등온에서증폭하기때문에온도가변화하는데필요한시간을절약할수있으며 PCR에비해검체에의한억제효과도거의없다. 게다가증폭산물을혼탁도 (turbidity) 나색변화 (ph indicator 또는 SYBRE Green I) 를육안으로도확인할수있는장점이있다 (Li et al., 2017). 한편몇몇유전자를이용한 LAMP 검사가 S. aureus 및 MRSA를검출하는데이용되었다. spa (Misawa et al., 2007), nuc (Chen et al., 2017), fema (Xihong et al., 2013) 가 S. aureus 를검출하는데이용되었고, methicillin 내성을검출하는데 meca가이용되었다. 또한 orfx는 S. aureus 와 MRSA를검출하는데이용되기도하였다 (Su et al., 2014). 본연구에서는 16S rrna, fema 및 meca 유전자를이용한다중 LAMP 검사를이용하여혈액배양양성검체에서 Staphylococcus species, S. aureus 및 methicillin 내성을동시에검출하고자하였다. LAMP assay의검출예민도를 conventional PCR과비교한결과, Table 2과같이 LAMP assay 에서 3개의유전자에대해검출예민도는 10 4 CFU/mL (20 copies) 였다. 한편 10 4 CFU/mL 농도의 MRSA에서 fema와 meca의유전자에대한 LAMP 검사에서명확한사다리모양의증폭산물이관찰되었지만 16S rrna LAMP 검사는 10 4 CFU/mL 에서는희미하지만 3회반복실험모두에서확실하게증폭되어 3개의유전자에대한 LAMP 의검출예민도를 10 4 CFU/mL 로결정할수있었다. 결론적으로 LAMP 방법은 conventional PCR의 16S rrna는 10배, fema와 meca는 100배정도민감하였다. 이는 LAMP assay에서 strand displacement activity 가높고 DNA 손실률이적어증폭효율이높다는것을의미한다. 103개의임상검체를대상으로생화학적동정및항균제감수성시험결과와비교한 fema와 meca LAMP 방법의민감도와특이도는 Table 4와 Table 5와같다. 64개의 S. aureus 중 fema에대해음성인균이 3균주로 fema 의민감도는 95.31% (61/64) 였으며 39개의 CNS는모두 fema에음성이어서특이도는 100% 이었다. fema 음성인 S. aureus 가 3균주가존재하기때문에 S. aureus 의감별에 fema 이외에 nuc 등유전자를추가해볼필요가있을것이다. 65개의 MRSA와 MRCoNS 중 1균주를제외하고모두 meca LAMP은양성을보였고 38 MSSA와 MSCoNS 는음성을보여예민도특이도는각각 98.46% 와 100% 이었다. 위음성인 MRCoNS의경우 methicillin 내성에관여하는유전자가 meca 뿐만아니라, meca1, A2, mecb, mecc와같은 mec 유전자유사체 (homologues) 의경우통상적인 meca 증폭에의해검출되지않았을가능성이있다 (Wu et al., 2001). 한편혈액배양에서 MRSA와 non-mrsa 의감별이치료에가장중요한데, 본연구에서 fema 와 meca 를동시에검사할때 MRSA와이외의균주와감별은 100% 가능하였다 (Tables 4 & 5). LAMP assay 뿐만아니라분자검사방법의단점은혈액배양에서 MSSA 와 MRCoNS 가동시에존재하면 MRSA 의위양성 (false positive) 결과가나올수있는것이다 (Becker et al., 2006). 혈액배양에서그런경우는매우드물지만앞으로 MRSA에대한특이 marker 의개발또는보완이나 LAMP assay 이후균의배양결과를확인해야할것이다. 결론적으로위와같은분자생물학적단점에도불구하고혈액배양양성병에서 MRSA의직접검출하는데 LAMP assay의적용은 MRSA 균혈증환자의신속한치료를가능케하는데매우도움이될것으로사료된다. ACKNOWLEDGEMENT 이논문은 2018년도정부 ( 미래창조과학부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (No. NRF-2018M3A9H ). CONFLICT OF INTEREST The authors have no conflicts of interest to disclose. REFERENCES Becker K, Pagnier I, Schuhen B, Wenzelburger F, Friedrich AW, Kipp F, et al. Does nasal cocolonization by methicillinresistant coagulase-negative staphylococci and methicillinsusceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-esistant S. aureus determinations by molecular methods? J Clin Microbiol : Chen C, Zhao Q, Guo J, Li Y, Chen Q. Identification of methicillinresistant Staphylococcus aureus (MRSA) using simultaneous detection of meca, nuc and femb by loop-mediated isothermal amplification (LAMP). Curr Microbiol : Clerc O, Prod'hom G, Senn L, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry and PCRbased rapid diagnosis of Staphylococcus aureus bacteremia. Clin Microbial Infect : Cosgrove SE, Sakaoulas G, Perencevich EN, Schwaber MJ,

8 Karchmer AW, Carmeli Y. Comparison of mortality associated with methicillin-resistant and methicillin susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis : Curtis KA, Rudolph DL, Owen SM. Rapid detection of HIV-1 by reverse-transcription, lood-mediated isothermal amplification (RT-LAMP). J Virol Methods : Hara-Kudo Y, Konishi N, Ohtsuka K, Hiramatsu R, Tanaka H, Konuma H, Takatori K. Detection of verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAP method: a collaborative study. Int J Food Microbiol : Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. Loop-mediated isothermal amplification for the rapid detection of salmonella. FEMS Microbiol Lett : Hartman BJ, Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol : Ito M, Watanabe M, Nakagawa N, Ihara T, Okuno Y. Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation. J Virol Methods : Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol : Li Y, Fan P, Zhou S, Zhang L. Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathog : Lowy FD. Staphylococcus aureus infections. N Engl J Med : Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother : Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acid Res : e63. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteremia: state of the art. Clin Microbiol Infect : Shands KN, Schmid G, Dan BB, Blum D, Guidotti RJ, Hargrett T, et al. Toxic-shock syndrome in menstruating womenassociation with Tampon use and Staphylococcus aureus and clinical features in 52 cases. N Engl J Med : Stamper PD, Cai M, Howard T, Speser S, Carroll KC. Clinical validation of the molecular BD geneohm staphsr assay for direct detection of Staphylococcus aureus and methicillinresistant Staphylococcus aureus in positive blood cultures. J Clin Microbiol : Su J, Liu X, Li Y, Chen D, Li Y, Yu G. Rapid and simple detection of methicillin-resistance Staphylococcus aureus by orfx loopmediated isothermal amplification assay. BMC Biotechnology : 18. Verroken A, Defourny L, Lechgar L, Magnette A, Delmee M, Glupczynski Y. Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subcultlure. Eur J Clin Microbiol Infect Dis : Wu SW, Lencastre H, Tomaz A. Recruitment of the meca gene homologue of Staphylococcus sciuri into a resistance deter- minant and expression of the resistance phenotype in Staphylo- coccus aureus. J Bacteriol : Xihong Z, Li Y, Park M, Wang J, Zhang Y, He X, et al. Loopmediated isothermal amplification assay targeting the fema gene for rapid detection of Staphylococcus aureus from clinical and food samples. J Microbiol Biotechnol : Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing Vibrio chol- erae using a loop-mediated isothermal amplification. BMC Microbiol : Cite this article as: Baek YH, Jo MY, Song MS, Hong SB, Shin KS. Application of Loop-Mediated Isothermal Amplification (LAMP) Assay to Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures. Biomedical Science Letters :

( )Kju225.hwp

( )Kju225.hwp 비임균성비뇨생식기감염에서 의진단적효용성 Usefulness of the Mycofast Test (MYCOFAST Evolution 2) for the Diagnosis of Nongonococcal Genitourinary Infections Hang Ro Park, Yang Hyun Kim, Ho Jae Lee, Jea Sang Oh, Hyoung Jin